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+{"text": "The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5\u2032-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes. The analytical performance of biosensors has been greatly enhanced through the development of nanomaterials by nanotechnology engineering . Due to The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)\u2013CRISPR-associated protein 9 (Cas9) genome engineering technology has facilitated editing the genome of any species . RecogniIn addition to CRISPR-dependent HDR, there are some other CRISPR-Cas-based tools that are precise in gene editing and do not depend on double-strand break repair mechanisms. For example, CRISPR base editors can introduce single base substitutions to target DNA sites in a programmable manner without double-strand break formation ,21. In aInosine-substituted probes have been used for the detection of genomic mutations ,26. The Here for the first time, electrochemical nanogenosensing based on inosine-substituted probes and disposable pencil graphite electrodes was used to verify the presence of CRISPR-Cas9-introduced mutations in murine cells. This method allows for efficient detection of the desired outcome created by HDR in CRISPR-Cas9 studies with high selectivity and specificity.The differential pulse voltammetry (DPV) technique was used for analysis with an Autolab PGSTAT-30  electrochemical analysis system and GPES 4.9 software package . A three-electrode system involving disposable graphite electrodes (PGEs) with diameters of 0.5 mm and lengths of 3 cm long as working electrodes , a platinum wire as the counter electrode and a reference electrode (Ag/AgCl).N-hydroxysulfosuccinimide sodium salt (NHS), [N-(3-dimethylamino)propyl)]\u2013N\u2032-ethylcarbodiimide (EDC) and Trizma hydrochloride were purchased from Sigma-Aldrich Chemical Company , Sodium dodecyl sulfate (SDS) and tri-sodium citrate were purchased from Merck . Other chemicals were of analytical reagent grade and supplied by Merck and Sigma. All experiments were performed at 25 \u00b0C. The synthetic oligonucleotides were provided from Invitrogen  by Thermo Fisher Scientific as a lyophilized powder. The base sequences of the oligonucleotides are as follows (I = inosine):2-C6-CICtAAITICTCTIIAIaIIT-3\u2032S-331 genome wild-type (WT) probe: 5\u2032-NHTCTCCAGAGCACTTAGCG-3\u2032Synthetic WT target: 5\u2032-ACC2-C6-CICCAAITICTCTIIAICIIT-3\u2032S-331 genome mutant-type (MT) probe: 5\u2032-NHGCTCCAGAGCACTTGGCG-3\u2032Synthetic MT target: 5\u2032-ACCSynthetic non-complementary sequence: 5\u2032-GGCAGCGGTGACTATGGCACC-3\u2032ACCGCTCCAGAGCACTTGGCGCCCCAGCAGGATGTCCTGGAG-3\u2032CRISPR-Cas9-edited S-331 gene PCR amplicon (underlined bases refer to the introduced point mutation region via CRISPR/Cas9 system): 5\u2032-TTAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGGAACTGGGTCAAAGGCCTCTGGGAAGATAGAGCTTTGGTCTTCTTGGATTTGCTGGTTTGTTTTCATTTTTGAGACAATCTTGGCTGACCTAGAACTCACTATGTAGACCAGGCTGGCCTCAACTCTTCAGAAGAGATCCGCCTGTCTCTTCCTCCCTAGGGTCAGGATCAAAGGCATAGACCACCACAACTGGCTTTTTGCTTATCTTTGGATCTTTGCTAGCTCAGAGGAGTCCACCGAGAAAGGCCCTACAGGGCAGCCACAAGCAAGGGTCCAGCCTCAGACCCAGATGACAGCACCAAAGCAGACACAGACCCCGGATCGGCTGCCTGAGCCACCAGAAGTCCAAATGCTGCCGCGTATCCAGCCACAGGCACTGCAGATCCAGACCCAGCCAAAGCTGCTTTGGCTGGGTCTGAGGCAGGCACAGACACAGE. coli): 5\u2032-AAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGACTATGGCACCACCAACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCAC-3\u2032Non complementary PCR amplicon (All oligonucleotide and primer stock solutions (1000 \u00b5g/mL) were prepared with ultrapure water  and stored at \u221220 \u00b0C until use. Immobilization of probes onto the surfaces of electrodes was achieved via carbodiimide chemistry (5 mM EDC and 8 mM NHS). Dilutions of the capture probe (CP) were prepared with an aqueous acetic acid (0.5 M) with NaCl (20 mM)  buffer. Synthetic target sequences and the PCR products and non-complementary amplicons were diluted with hybridization buffer (HB) containing 5 \u00d7 saline sodium citrate (SSC) + 0.05% solution , and 1\u00d7 SSC + 0.1% SDS solution  was used as a washing buffer unless otherwise indicated.CIZ1 gene and proximal to a protospacer adjacent motif (PAM). The guide sequence was introduced into the linearized GeneArt\u00ae CRISPR nuclease vector-containing Cas9 and the CD4 reporter using the manufacturer\u2019s instructions (Invitrogen). Amplification of the plasmid was performed by transformation of One Shot\u00ae TOP10 chemically competent E. coli, antibiotic selection and overnight cultures. DNA sequencing of the plasmid was performed to verify the construct (Eurofins). Single strand oligonucleotide-directed mutagenesis was used to introduce mutations into the CIZ1 gene using HDR with a 98 nt single-stranded oligodeoxynucleotide (ssODN): 5\u2032-GCAGATCCAGACCCAGCCAAGCTGCTGAGGCAGGCACAGACACAGACCgCTCCAGAGCACTTgGCGCCCCAGCAGGATCAGGTAGAGCCACAGGTAC-3\u2032.The following oligonucleotides were used for the construction of the guide sequence  were complementary to the region surrounding the PAM site to aid in efficient HDR. \u00ae CRISPR nuclease (CD4 reporter) Vector using Dynabeads\u00ae CD4 magnetic beads (Invitrogen). Single cells were plated into a 96-well plate, and mutated cells were identified using a restriction endonuclease specific for desired mutations.Transfection of 3T3 embryonic mouse fibroblast cells with 1 \u00b5g of CRISPR-Cas9 expression vector and 2.5 \u00b5L of 50 \u00b5M ssODN, using 100 \u00b5L of transfection reagent Kit-R , was carried out by electroporation using the Nucleofector\u2122 program U-030 (Lonza). Cell enrichment was carried out to isolate the transfected cells with the GeneArtSingle cells were cultured in a flat bottomed 96-well plate for 2 weeks, replacing media every 2\u20133 days and passaging cells on to 24 well plates. Genomic DNA (gDNA) was extracted using 50 \u03bcL of QuickExtract\u2122 DNA extraction solution (Epicentre-Lucigen). The sequences of interest were screened after digestion with BanI (S331) restriction enzymes. gDNA was PCR amplified using One Taq Quick-Load 2\u00d7 master mix , 94 \u00b0C-30 s, 30 cycles of 94 \u00b0C-30 s, 68 \u00b0C-30 s, 68 \u00b0C-60 s, the final extension was carried out at of 68 \u00b0C-5 min. PCR products were DNA sequenced to verify the presence of mutations.\u00ae TOPO\u00ae PCR cloning kit\u2014Thermo Fisher Scientific, Waltham, MA, USA) and DNA sequenced  for multiple clones to confirm the presence of desired mutations.Identification of colonies of interest was identified by restriction endonuclease digestion with BanI according to manufacturer\u2019s instructions. Clones that showed the correct restriction digest pattern were inserted into the TOPO10 Blunt cloning vector  H2. Then, the temperature was set to 1000 \u00b0C with the same ramp rate. After this, CNT synthesis was commenced by passing CH4 with a flow rate of 50 sccm and H2 with 200 sccm over the catalyst. CNT synthesis lasted 40 min under atmospheric pressure. To end the synthesis, the CH4 flow was turned off, and the CNTs obtained were left to cool under an atmosphere of H2.In this study, the carbon nanotubes (CNTs) were synthesized by thermal chemical vapor deposition (CVD)  utilizinPrior to probe immobilization, PGE electrodes were activated at 1.40 V for 30 s in acetate buffer solution . Probe immobilization was carried out on the surface of the PGEs. The probe and CNTs were allowed to interact in solution as previously described . BrieflyDenaturation of the PCR amplicons was carried out at 95 \u00b0C/8 min and 0 \u00b0C/2 min to separate the strands. Hybridization buffers containing the 5 \u00b5g/mL of complementary and non-complementary target sequences and amplicons were used for the hybridization between the probe and target sequences. Hybridizations were carried out on electrode surfaces at room temperature by placing the electrodes inside the vials containing 30 \u00b5L of the target or non-complementary solutions in a hybridization buffer for 30 min. The electrodes were then immersed in a washing buffer for 1 min to remove the unhybridized DNA.DPV was used to measure the oxidation signal of guanine after placing the electrode in ACB (pH 4.8) by scanning from +0.75 V to +1.40 V at ambient conditions (25 \u00b0C). The raw data were obtained as the analytical signal after moving average baseline fitting using a peak width of 0.01 V. All results presented in this paper are the means of at least five measurements, and the error bars show the standard deviations. The procedure for electrochemical biosensing is presented in CIZ1 protein sequence [CIZ1 is a protein that cooperates with cyclin A-CDK2 to promote the initiation of DNA replication [CIZ1 sequence by using HDR, including a point mutation that provides the S331A substitution in the CIZ1 protein and a silent point mutation that allows the formation of a BanI restriction site  to determine the selectivity of the designed genoassay. CNT-free ssDNAs were immobilized on the surface of unmodified PGEs, and guanine oxidation signals were found to be 220 nA. However, after the modification of ssDNA with CNTs, the guanine signal increased to 750 nA, resulting in a (3 times) increase over the unmodified PGEs  synthetic target sequence was obtained. To assess the efficiency of mutation detection, differences in guanine signals obtained from hybridization with a complementary synthetic target and from hybridization with a synthetic target containing two base mutations in the sequence region of interest (FM/MM = mismatch) was obtained. By varying only the probe concentrations ranging from 0.1 to 10 \u00b5g/mL, we found that the highest FM/NC and FM/MM values were obtained at 5 \u00b5g/mL probe concentration . Then weFinally, the optimized nanogenosensor has been used on real PCR amplicons . The guaCIZ1 gene with sufficient sensitivity and specificity.Electrochemical biosensing methods at the DNA level are used in a broad range of areas from disease-causing organisms to mutated genes and even detection of food contaminants, DNA/drug interactions and monitoring of the environment. The principle of the proposed detection strategy relies on a well-established method based on electrochemical genosensors. On the other hand, the emergence of the CRISPR/Cas9 system paved the way to alter the genome and control gene expression. The outcomes of any CRISPR-Cas9 assay should be investigated to perform safe and effective genome editing. This study demonstrates the possible use of inosine-substituted probes along with CNT/PGEs for label-free electrochemical genosensing of CRISPR-Cas9-mediated genomic mutations. The techniques used in this study are based on established genosensing methods capable of detecting nucleic acids with high sensitivity and sequence-specificity ,26,33,37Higher levels of guanine oxidation signals were obtained with PCR amplicons than with 21-base synthetic targets . This isThe genosensor developed here has potential applications in the evaluation of CRISPR-mediated genome editing results. For example, in any HDR-based gene editing assay, the edited clones can be precisely detected by sequencing all the clones; however, this is time-consuming and expensive. Using this genosensor, correctly edited clones can be quickly selected from among tens or hundreds of clones prior to precise verification by sequencing. Moreover, the use of nanogenosensor can be exploited for the detection of single point mutations that cause disease and for detection of CRISPR-Cas9-mediated reversion of the mutation. This method can also be further developed for the detection of other types of CRISPR-based manipulations, including small insertions and deletions and multiple base substitutions generated by HDR, base editing, or prime editing. This approach is also suitable for expansion by employing an array system of sequence-specific probes to detect multiple gene editing outcomes. Further studies could be designed to detect off-target mutations caused by HDR, NHEJ, or other CRISPR-based tools to screen genome editing outcomes more comprehensively.CIZ1 gene was edited using CRISPR-Cas9, and the introduced point mutations were detected using the genosensor as a proof-of-concept. Therefore, future work is needed to fully establish this nanogenosensor as a platform to detect CRISPR-based genome editing outcomes. To address these limitations, several other genes will be targeted, and the nanogenosensor will be used to detect other types of mutations such as multiple base substitutions, insertions, and deletions introduced via CRISPR/Cas9-HDR, base editing, and prime editing. Another limitation of this genosensor is that it may not work efficiently if the GC content of the edited DNA site is quite low. To prevent this, localization of the probe should be optimized such that it matches at least several guanines. Further investigation of this system will provide a basis for exploring other potential applications of the method.Although promising, this study includes some limitations. The results are preliminary, and only the CIZ1 gene, and the presence of the mutations was confirmed based on electrochemical nanogenosensor by monitoring guanine oxidation signals using disposable pencil graphite electrodes. Some parameters, such as probe concentration, target concentration, hybridization buffer, and washing time, were also optimized in order to improve the performance of the nanogenosensor. The presence of DNA hybridization on the pencil graphite electrodes was confirmed by the observation of an increase in guanine oxidation signal. The irreversible oxidation signal of the guanine base at +1.0 V vs. Ag/AgCl reference electrode was monitored using the DPV technique. The detection method relies on the measurement of increased oxidation currents of the guanine bases only in the presence of the hybrid due to the electrochemical inactivity of the inosine base in the probe sequence. Thus, a simple, sensitive, selective, non-time-consuming and cost-effective electrochemical-based \u201cyes/no\u201d detection system was developed. This hybridization signal was able to differentiate between non-complementary, complementary, and mismatched sequences. The hybridization signal of non-complementary DNA was relatively small because due to minimal interactions. This study has, for the first time, demonstrated direct measurement of CRISPR-Cas9-mediated mutations within a target gene using a nanogenosensor.Here, CRISPR-Cas9 was used to edit the"}
+{"text": "Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function.cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function.We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci. Noncoding RNAs have been shown to play central roles in biology. Key cellular machines such as telomerase and the ribosome are\u00a0comprised of\u00a0proteins and noncoding RNAs and serve as classic examples of RNA-based functionalities , 2. WhilTug1 for further analysis because of its high conservation [We previously reported the generation of 20 lncRNA loci knockout mouse strains, five of which displayed either viability, growth, or brain phenotypes . From thervation  as well ervation .Tug1 was first identified in a microarray screen for genes upregulated in response to taurine in a heterogenous culture of retinal cells [Tug1 is transcriptionally regulated by p53 [Tug1, including but not limited to the development of photoreceptors [Tug1 in cancer by acting as a tumor suppressor in human gliomas [Tug1 underscores the importance of this locus.al cells . In addid by p53 . A numbeeceptors  and in receptors \u201327. Ther gliomas , 29 and  gliomas . Thus, tTug1 locus using multiple genetic approaches and describe a physiological function in spermatogenesis and male fertility. We show that deletion of the Tug1 locus in mice leads to male sterility and also report an underappreciated molecular complexity at the Tug1 locus. Using several complementary genetic approaches , we provide evidence of a DNA-based repressive element within the Tug1 locus that regulates several genes in cis. We show that a gene expression program dysregulated in Tug1-knockout testes can be partially rescued by ectopic expression of Tug1 RNA in vivo. Finally, we show that the Tug1 locus contains an evolutionarily conserved ORF, which can be translated into a stable protein that impacts mitochondrial membrane potential upon overexpression. Collectively, this study implicates the Tug1 locus as essential in male fertility and provides evidence that the Tug1 locus contains at least two noncoding regulatory activities and a putative coding function.Here, we characterize the Tug1 lncRNA locus is located on chromosome 11 and has three annotated transcripts  is located approximately 680\u00a0bp upstream of the first Tug1 TSS. The Tug1 locus is enriched with hallmarks of active transcription, such as RNA polymerase II (Pol II) and histone H3 lysine 4-trimethylation (H3K4me3) at its promoter, H3K36me3 across its gene body, and abundant transcription as shown by RNA-seq  and higher than other well-characterized lncRNAs including Hottip (71%), Neat1 (69%), Xist (30%), and Firre (4%)    mouse model where the gene body of the Tug1 locus was removed and replaced with a lacZ reporter cassette downstream of the endogenous promoter, thereby preserving the act of transcription . Therefore, we sought to investigate the fertility of Tug1\u2212/\u2212 mutants in more detail. We separately mated Tug1\u2212/\u2212, Tug1+/\u2212, and wild-type males or females to C57BL/6J mice. We did not observe a difference in the mounting behavior between wild-type and Tug1\u2212/\u2212 mice, as assessed by the presence of a vaginal plug. Strikingly, matings between Tug1\u2212/\u2212 males (n\u2009=\u20098) and C57BL/6J females did not produce any offspring, whereas matings involving either Tug1+/\u2212 males (n\u2009=\u20098) or wild-type males (n\u2009=\u20098) with C57BL/6J females resulted in similar numbers of offspring  in Tug1\u2212/\u2212  compared to wild type  , which produced on average only 40% as many sperm as wild-type mice  .To further understand the underlying fertility defect in Tug1\u2212/\u2212 males. We examined the morphological features of sperm and quantified the frequency of 15 different abnormalities  compared to wild-type males   and abnormal sperm morphology (teratozoospermia).Based on these results, we investigated whether perturbations in sperm morphology could also contribute to the complete sterility observed in \u20099) Fig.\u00a0d. We obsTug1 locus leads to abnormal sperm morphology, we examined the timing of Tug1 expression at different stages of spermatogenesis. To this end, we took advantage of the knock-in lacZ reporter driven by the endogenous Tug1 promoter and assessed the expression by lacZ staining of histological sections of Tug1+/\u2212 testis and epididymis. From stages IX to XI of spermatogenesis in the testis, lacZ staining was restricted to the excess cytoplasm, known as residual bodies, which are phagocytosed toward the basement membrane by Sertoli cells \u00a0[Tug1\u2212/\u2212 model enables us to test for potential cis-regulatory activity within the Tug1 locus because the gene-ablation design removes potential cis-acting elements yet keeps the act of transcription intact   were significantly upregulated in Tug1\u2212/\u2212 testes compared to wild type  in embryonic stem cells [We next sought to investigate what, if any, molecular activities  are present at the -acting)\u00a0\u201312. The em cells , 40.Figcis-effect upon deletion of the Tug1 locus is more widespread, we performed RNA-seq on 6 additional tissues ) as well as re-analyzed an existing brain dataset from wild-type and Tug1\u2212/\u2212 mice , 9 genes were dysregulated in one or more tissues (7 upregulated and 2 downregulated) , and 8430429K09Rik (6 of 8 samples)  Fig.\u00a0a. Of theTug1 locus, we reasoned that the repressive activity could be mediated either directly by the Tug1 transcript or by regulatory DNA elements within the locus. To determine if the repressive effect of Tug1 on neighboring genes occurs on the same allele (cis-acting), we performed allele-specific RNA-seq using a hybrid mouse strain. To generate this strain, we crossed Tug1+/\u2212 C57BL/6J females with Mus castaneus (Cast/EiJ) males  and identified significant changes in the gene expression relative to wild type. Deletion of the Tug1 locus was accompanied by 2139 significantly dysregulated genes across all samples examined. We observed global changes in the gene expression clustered by tissue type, indicating tissue-specific gene dysregulation , we identified an isoform of Tug1 that lacks the 5\u2032 region, thus ensuring we would address the role of Tug1 RNA alone. To this end, we generated a doxycycline (dox)-inducible Tug1 transgenic mouse by cloning a Tug1 isoform downstream of a tet-responsive element )  in the Tug1\u2212/\u2212 background that also contained an allele that constitutively expresses the reverse tetracycline transcriptional activator gene (CAG-rtTA3)  .\u00a0Notably, 53 (including Tug1)\u00a0of the 1051 genes that were significantly\u00a0dysregulated in Tug1\u2212/\u2212 testes were found significantly and reciprocally regulated in the testes from dox-fed Tug1rescue mice  and C57BL6/J female mice (n\u2009=\u200912) did not produce any progeny , similar to the levels observed in Tug1\u2212/\u2212 males (mean\u2009=\u20094.69\u2009\u00d7\u2009105\u2009\u00b1\u20091.6\u2009\u00d7\u2009104 cells/mL) compared to wild type (mean\u2009=\u20099.32\u2009\u00d7\u2009105\u2009\u00b1\u20093.9\u2009\u00d7\u2009103 cells/mL)  .To further characterize the potential translated regions in ing data , a techning data , 50\u201352. nt snRNA , 54, is n\u2009=\u200950)    Fig.\u00a0a. NotablF1) Fig.\u00a0b. In conRNA Fig.\u00a0: Fig. S9Since CMXR is commonly used to measure mitochondrial membrane potential, we reasoned that either impaired mitochondrial integrity or impaired redox potential at the mitochondrial membrane could account for the accumulation defect of CMXR in mitochondria upon TUG1-BOAT overexpression. To address these possibilities, we immunostained for TOM20, a redox independent translocase located on the outer mitochondrial membrane . We obseTug1 lncRNA locus in vivo and also report an underappreciated molecular complexity at the Tug1 locus  cis-acting DNA repressive element, (ii) a lncRNA that acts on gene expression in trans, and (iii) a conserved putative ORF which when overexpressed affects mitochondrial membrane potential , each with the potential to exert function. In this study, we report an essential role in male fertility for the cus Fig.\u00a0. We findial Fig.\u00a0. TogetheTug1 locus has an essential role in male fertility in mice. Based on our results, we speculate that the sterility of Tug1\u2212/\u2212 males arises from a combination of oligozoospermia (low sperm count) and teratozoospermia . Notably, there is some evidence that Tug1/TUG1 may have a conserved role in male fertility in humans. Microarray profiling of sperm from sterile men  has significantly less TUG1 expression compared with sperm from fertile males  have been found to be important in spermatogenesis, but in contrast to the Tug1-knockout phenotype reported in this study, none lead to male sterility when disrupted in mouse models [Tug1, may be less common.Indeed, studies have performed systematic gene expression profiling at defined stages during spermatogenesis and have identified many developmentally regulated lncRNAs, suggesting that lncRNAs may have important roles during spermatogenesis . Four ote models \u201361. On te models . Thus, oTug1 locus harbors at least two distinct noncoding activities, and potentially a third coding one. Several lines of evidence indicate that the Tug1 locus has a cis-acting repressive DNA function. We observed that upon deletion of the Tug1 locus, several genes located downstream of Tug1 were consistently upregulated across multiple tissues, and in an allele-specific manner in the testes. This dysregulation is consistent with a previous study from our group in which genes located near the Tug1 locus were dysregulated in the brain of Tug1\u2212/\u2212 mice [cis-effect is mediated by DNA regulatory elements within the Tug1 locus. Consistent with a repressive modality of the Tug1 locus, a recent study which systematically tested for enhancer activity across lncRNA loci, reported a lack of enhancer activity across the Tug1 gene body but found enhancer activity at all other lncRNA loci examined [Tug1 locus.Second, we found that the \u2212/\u2212 mice . Collectexamined . Contrarexamined \u201366. FurtTug1 in the testes by a trans mechanism. In support of this conclusion, we found that a subset of genes dysregulated in Tug1\u2212/\u2212 testes could be rescued by ectopic expression of Tug1 RNA, even at low levels. While the Tug1 transgene was expressed at lower levels than wild-type Tug1 RNA, other lncRNAs such as Hottip and Xist have been shown to exert a biological activity at relatively low copy numbers [Tug1 RNA appears to have functional activity even at low levels. Consistent with our finding of a trans-acting role for Tug1 RNA on gene expression, a previous study found that Tug1 RNA can regulate the levels of Ppargc1a mRNA in cultured podocytes [Ppargc1a was not significantly dysregulated, but it is important to note that our dataset does not include the kidney. We also note that low levels of Tug1 transgene RNA were not sufficient to rescue the Tug1 male fertility defect. More studies will be needed to determine the direct or indirect mechanisms of Tug1 RNA on gene expression in a male germ cell context in vivo.In addition to a DNA regulatory modality, this study finds evidence for an RNA-based role for  numbers , 68; thuodocytes . In our Tug1 locus. We demonstrate that the 5\u2032 region of the Tug1 locus contains an evolutionarily conserved ORF that when overexpressed, impacts mitochondrial membrane potential. The protein product which we named TUG1-BOAT is predicted to have a high positive charge (net charge ~\u2009+\u200916.5). Thus, we speculate that the accumulation of such a positively charged protein at the mitochondria in an overexpression context could lead to depolarization of the mitochondrial membrane. Consistent with this finding, a recent study also identified a conserved ORF in the 5\u2032 region of the human TUG1 locus and showed that the ORF produces a stable protein that localizes to the mitochondria [Tug1 does not appear to be limited to the putative protein. One study found that overexpression of a Tug1 RNA isoform lacking ORF1 affects mitochondrial bioenergetics in cultured podocytes derived from a diabetic nephropathy mouse model [Tug1 RNA (lacking the ORF1 sequence) overexpression on mitochondrial membrane potential by CMXR staining in cell-based assays. Identifying endogenous and disease contexts in which Tug1 RNA and TUG1-BOAT are detected and function endogenously will be important to further explore the potential link between Tug1 and the mitochondria and determine whether the locus does contain a third coding function.A number of recent studies have identified ORFs at lncRNA loci and have also demonstrated that some ORFs can produce proteins , 50, 51.chondria . In addise model . In our Tug1 DNA, RNA, or putative protein could individually or in combination mediate the observed male fertility defect in Tug1-knockout mice. There are few biological contexts in the literature describing noncoding loci implicated in human disease with potential DNA, RNA, and protein functionalities. However, one intriguing example is the human D4Z4 locus, which is a repeat region that is reduced in copy number in humans with facioscapulohumeral dystrophy (FSHD) [D4Z4 locus, including a lncRNA, a protein that is detected in the disease context, and a DNA repressor element [Tug1-knockout mice, there is evidence to support a potential role in male fertility for each modality, thus warranting further investigation.Finally, this study opens the possibility that y (FSHD) , 70. Inty (FSHD) , 72. Mec element \u201376. Whilcis genes dysregulated upon deletion of the Tug1 locus have a role in male fertility. Loss-of-function mutations in 2 of the 6 cis genes upregulated in Tug1\u2212/\u2212 testes, Smtn and Pla2g3, are characterized to have male fertility and sperm maturation defects [cis genes upon deletion of Tug1  have not yet been reported. In addition, there is some evidence for a potential role of Tug1 RNA in male fertility. Mice with a loss-of-function mutation for Selenop, a gene that was found significantly upregulated in Tug1\u2212/\u2212 testes and significantly and reciprocally regulated in Tug1rescue testes, are reported to have reduced male fertility [There is some evidence that the  defects , 78. Howertility . As for Tug1 locus in male fertility. This study also highlights the molecular complexity embedded at lncRNA loci as our findings provide evidence that the Tug1 locus harbors two distinct noncoding activities: (i) a cis DNA repressor and (ii) a trans-acting lncRNA, and we find a potential third protein-coding activity. Therefore, going forward, it will be important to investigate the individual and/or combined roles of Tug1 DNA, RNA, and/or putative protein in male fertility as well as in disease contexts in which Tug1 is altered.In summary, this study provides genetic evidence for an essential role of the Tug1tm1.1Vlcg-knockout mice have been described previously [loxP-flanked neomycin resistance gene included in the targeting construct, we crossed Tugtm1.1Vlcg mice to C57BL6/J mice and then to a cre-recombinase strain (B6.C-Tg(CMV-cre)1Cgn/J, The Jackson Laboratory, 006054). Mice free of both the neomycin-resistance and cre-recombinase genes were selected for colony expansion and subsequently backcrossed to C57BL/6J mice. The Tug1-knockout allele was maintained by heterozygous breeding, and mutant mice were identified by genotyping for loss of the Tug1 allele and gain of the lacZ cassette .eviously , 41. To Tug1BL6-KO/Cast-WT mice by crossing inbred Mus castaneus (Cast/EiJ) males  with inbred heterozygote Tug1 females. The F1 hybrid male progeny  were used for allele-specific expression studies.For allele-specific gene expression analyses, we generated Tug1-overexpression mouse, tg(Tug1), we cloned Tug1 cDNA (see the \u201cTug1 (Ensembl ID: ENSMUST00000153313.2) was amplified from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites (see the \u201cTug1 cDNA by sequencing (see the \u201cTug1) cassette were identified by genotyping for the pTRE allele and individually mated to female C57BL/6J mice  to expand the colonies. Next, we generated quadruple allele transgenic mice to test the functionality of the Tug1 RNA by the following strategy. We mated tg(Tug1) males to Tug1tm1.1Vlcg females and identified male progeny that were Tug1+/\u2212; tg(Tug1). These mice were then mated to female rtTA mice (B6N.FVB(Cg)-Tg(CAG-rtTA3)4288Slowe/J mice ), and we identified male progeny that were Tug1+/\u2212; tg(Tug1), rtTA. Finally, we mated male Tug1+/\u2212; tg(Tug1), rtTA mice to Tug1+/\u2212 females, and at the plug date, females were put on 625\u2009mg/kg doxycycline-containing food . We genotyped progeny from the above matings  and identified male progeny that were Tug1\u2212/\u2212; tg(Tug1), rtTA, and maintained these mice on the doxycycline diet until the experimental end point.To generate an inducible see the \u201c\u201d sectionsee the \u201c\u201d sectionsee the \u201c\u201d sectionTug1\u2212/\u2212 mouse embryonic fibroblasts (MEFs) from E14.5 littermates from timed Tug1+/\u2212 intercrosses as described [l-glutamine, and non-essential amino acids. We genotyped MEFs derived from each embryo and used only male Tug1\u2212/\u2212 and wild-type littermate MEFs at passage 2 for all experiments. 3T3 , HeLa , and BJ  cell lines were purchased from ATCC and cultured as recommended.We derived primary wild-type and escribed . We mainTug1 (see the \u201cTug1 cDNA (Ensembl id: ENSMUST00000153313.2) and performed in situ hybridization on a minimum of three C57BL6/J embryos per embryonic stage. For whole-mount staining, embryos\u00a0were fixed in 4% paraformaldehyde for 18\u2009h at 4\u00a0\u00b0C, followed by three washes in PBS\u00a0for 10\u2009min. We then dehydrated embryos through a graded series of 25%, 50%, and 75% methanol/0.85% NaCl incubations and then finally stored embryos in 100% methanol at \u2212\u200920\u00a0\u00b0C. Embryos were then rehydrated through a graded series of 75%, 50%, 25%, and methanol/0.85% NaCl incubations and washed twice with PBS with 0.1% Tween-20 (PBST). Embryos were treated with 10\u2009mg/mL proteinase K in PBST for 10\u2009min  or 30\u2009min . Samples were fixed in 4% paraformaldehyde/0.2% glutaraldehyde in PBST for 20\u2009min at room temperature and washed twice with PBST. We then incubated samples in pre-hybridization solution for 1\u2009h at 68\u00a0\u00b0C and then incubated samples in 500\u2009ng/mL of Tug1 antisense or sense riboprobe at 68\u00a0\u00b0C for 16\u2009h. Post-hybridization, samples were washed in stringency washes and incubated in 100\u2009\u03bcg/mL RNaseA at 37\u00a0\u00b0C for 1\u2009h. Samples were washed in 1\u00d7 maleic acid buffer with 0.1% Tween-20 (MBST) and then incubated in Roche Blocking Reagent  with 10% heat-inactivated sheep serum  for 4\u2009h at room temperature. An anti-digoxigenin antibody  was used\u00a0at 1:5000 and incubated for 18\u2009h at 4\u00a0\u00b0C. Samples were washed 8 times with MBST for 15\u2009min, 5 times in MBST for 1\u2009h, and then once in MBST for 16\u2009h at 4\u00a0\u00b0C. Prior to developing, the samples were washed three times with NTMT , 50\u2009mM MgCl2, 0.1% Tween-20, 2\u2009mM levamisole). The in situ hybridization signal was developed by adding BM Purple  for 4, 6, 8, and 12\u2009h. After the colorimetric development, samples were fixed in 4% paraformaldehyde and cleared through a graded series of glycerol/1\u00d7 PBS and stored in 80% glycerol. Imaging was performed on a Leica M216FA stereomicroscope (Leica Microsystems) equipped with a DFC300 FX digital imaging camera.We generated an antisense riboprobe against see the \u201c\u201d sectionTug1 single-molecule RNA FISH as described previously [Tug1 transcripts were designed with LGC Biosearch Technologies\u2019 Stellaris probe designer (Stellaris Probe Designer version 4.2) and manufactured by LGC Biosearch Technologies.We performed eviously . Brieflyl-lysine (10\u2009\u03bcg/mL) diluted in PBS. Prior to hybridization, coverslips were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 10\u2009min at room temperature, and washed twice more with PBS. Coverslips were immersed in ice-cold 70% EtOH and incubated at 4\u00a0\u00b0C for a minimum of 1\u2009h. We then washed the coverslips with 2\u2009mL of Wash Buffer A (LGC Biosearch Technologies) at room temperature for 5\u2009min. Next, we hybridized cells with 80\u2009\u03bcL hybridization buffer (LGC Biosearch Technologies) containing Tug1 probes (1:100) overnight at 37\u00a0\u00b0C in a humid chamber. The following day, we washed the cells with 1\u2009mL of Wash Buffer A for 30\u2009min at 37\u00a0\u00b0C, followed by another wash with Wash Buffer A containing Hoechst DNA stain  for 30\u2009min at 37\u00a0\u00b0C. Coverslips were washed with 1\u2009mL of Wash Buffer B (LGC Biosearch Technologies) for 5\u2009min at room temperature, mounted with ProlongGold (Life Technologies) on glass slides, and left to curate overnight at 4\u00a0\u00b0C before proceeding to image acquisition (see below).Human foreskin fibroblasts (ATCC\u00ae CRL-2522\u2122) and mouse 3T3 fibroblasts  were seeded on glass coverslips previously coated with poly-Tug1\u2212/\u2212 (n\u2009=\u20098) and wild-type (n\u2009=\u20099) males between 8 and 41\u2009weeks of age were sacrificed and weighed. We then dissected the entire male reproductive tract in phosphate-buffered saline (PBS). One testis was removed, weighed, and fixed in 4% paraformaldehyde (PFA) for histology (see below). Sperm were collected from one cauda epididymis by bisecting and suspending the tissue in a solution of Biggers-Whitten-Whittingham (BWW) sperm media at 37\u00a0\u00b0C. After a 15-min incubation, we used the collected sperm solutions to analyze sperm morphology and counts.We characterized sperm morphology by fixing sperm in 2% PFA in PBS, mounting 20\u2009\u03bcL of suspended sperm in Fluoromount-G media (Southern Biotech) on Superfrost glass slides (Thermo Fisher Scientific) and scanning each slide in a linear transect, recording the morphology as normal or abnormal for each sperm cell encountered (between 30 and 120 sperm). When abnormal, we also recorded the type of morphological defects: headless, head angle aberrant, head bent back to midpiece, debris on the head, debris on the hook, head misshapen, midpiece curled, midpiece kinked, midpiece stripped, debris on the midpiece, tailless, tail curled, tail kinked, broken tail, or multiple cells annealed together.Tug1\u2212/\u2212 (n\u2009=\u20097) and wild-type (n\u2009=\u20099) mice were determined using a Countess Automated Cell Counter according to the manufacturer\u2019s protocol . For the Tug1rescue experiment, sperm counts for control (WT and Tug1+/\u2212) (n\u2009=\u20092), Tug1\u2212/\u2212 (n\u2009=\u20092), and Tug1\u2212/\u2212; tg(Tug1); rtTA mice (n\u2009=\u20093) were determined by manual counts using a hemocytometer. For all analyses, statistical comparisons between Tug1\u2212/\u2212 and wild type were performed using the two-tailed Wilcoxon rank-sum tests with an a\u2009=\u20090.05. The results for testis, sperm count, and morphological parameters are presented in Additional\u00a0file\u00a0Tug1\u2212/\u2212 versus wild type for relative testis size, sperm morphology, and sperm counts were performed using R ).Sperm counts for each lacZ reporter and histological staining for morphological analysis of male reproductive tissues was conducted on the testes and epididymis from Tug1\u2212/\u2212 (n\u2009=\u20092) and wild-type (n\u2009=\u20092) mice. We fixed the testis and epididymis in 4% paraformaldehyde in PBS overnight at 4\u00a0\u00b0C and washed the tissues three times in PBS. For lacZ staining, we rinsed Tug1+/\u2212 and wild-type tissues three times at room temperature in PBS with 2\u2009mM MgCl2, 0.01% deoxycholic acid, and 0.02% NP-40. We performed X-gal staining by incubating the tissues for up to 16\u2009h at 37\u00a0\u00b0C in the same buffer supplemented with 5\u2009mM potassium ferrocyanide and 1\u2009mg/mL X-gal. The staining reaction was stopped by washing three times in PBS at room temperature, followed by 2\u2009h post-fixation in 4% paraformaldehyde at 4\u00a0\u00b0C.Expression of the knock-in We then embedded the organs in paraffin, sectioned the organs at 6\u2009\u03bcm thickness, and then mounted the sectioned samples onto glass microscope slides. The testis sections were additionally stained with Mayer\u2019s hematoxylin, periodic acid, and Schiff\u2019s reagent , and the epididymis sections were stained with eosin . Images were collected using a Zeiss AxioImager.A1 upright microscope or on an Axio Scan Z.1 (Zeiss).Tug1 mutant and wild-type samples, on the Illumina HiSeq platform using the rapid-full flow cell with the 101-bp paired-end reads sequencing protocol .We isolated total RNA from mouse tissues, mouse embryonic fibroblasts (MEFs), and blood cells using TRIzol  by chloroform extraction followed by spin-column purification  according to the manufacturer\u2019s instructions. RNA concentration and purity were determined using a Nanodrop. We assessed RNA integrity on a Bioanalyzer (Agilent) using the RNA 6000 chip. High-quality RNA samples (RNA integrity number \u2265\u20098) were used for library preparation. We then constructed mRNA-seq libraries using the TruSeq RNA Sample Preparation Kit (Illumina) as previously described . The libWe mapped sequencing reads to the reference mouse genome (GRCm38) by STAR  with theTug1-/- and\u00a0 Tug1-/-\u00a0vs. Tug1rescue)\u00a0using the generalized linear model. Statistical significance was calculated with the assumption of the negative binomial distribution of the read counts and with the empirical estimation of variance by using the R packages DESeq2 [p values by the false discovery rate (FDR) method were smaller than 0.05.We identified differentially expressed genes by comparing the mean read counts of biological replicates between the groups\u00a0 to evaluate the enrichment of the gene sets available from MSigDB  after ma, CAMERA .C57BL/6J, Cast/EiJ diploid genome by incorporating single nucleotide polymorphisms and indels (obtained from the Mouse Genome Project: ftp://ftp-mouse.sanger.ac.uk/REL-1303-SNPs_Indels-GRCm38) from both strains into the M. musculus GRCm38 reference genome sequence. We created a transcriptome annotation set as follows. The gencode.vM2.annotation GTF file was downloaded, and Mt_rRNA, Mt_tRNA, miRNA, rRNA, snRNA, snoRNA, Mt_tRNA_pseudogene, tRNA_pseudogene, snoRNA_pseudogene, snRNA_pseudogene, scRNA_pseudogene, rRNA_pseudogene, and miRNA_pseudogene were removed (not enriched in our RNA-seq libraries). To create an extensive set of transcripts, we added to the gencode.vM2.annotation all transcripts from the UCSC knownGene mm10 annotation file, which are not represented in the gencode.vM2.annotation set. We also added all functional RNAs from the Functional RNA database (fRNAdb) [C57BL/6J, Cast/EiJ diploid transcriptome set.We performed allele-specific expression analysis as previously described . For mou(fRNAdb) , which dhttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore) with stringency level 3. We then mapped each of the C57BL/6J::Cast/EiJ hybrid RNA-seq libraries to the C57BL/6J and Cast/EiJ diploid genome and transcriptome splice junctions using STAR RNA-seq aligner [C57BL/6J and Cast/EiJ diploid transcriptome using MMSEQ [Each RNA-seq library was first subjected to quality and adapter trimming using the Trim Galore utility  from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites  (see below).We amplified the full-length see the \u201c\u201d sectionTug1\u2212/\u2212 mice (112\u2009days old) and were homogenized in TRIzol  with a gentle MACS Dissociator . RNA was isolated by column purification  using a QiaCube (Qiagen). RNA was assessed and quantified on a Bioanalyzer (Agilent). cDNA was synthesized using 500\u2009ng of total RNA as input with SuperScript IV VILO Master Mix with and without RT . PCR was performed on the synthesized cDNA (RT and no RT samples) using MyTaq Red Mix  with the cycling parameters: 95\u00a0\u00b0C for 1\u2009min, followed by 35\u2009cycles of 95\u00a0\u00b0C for 15\u2009s, 60\u00a0\u00b0C for 15\u2009s, and 72\u00a0\u00b0C for 10\u2009s. PCR products were run a 1% agarose gel and verified by Sanger sequencing (GeneWiz). The following are the primers used for RT PCR: 7SK F1: GACATCTGTCACCCCATTGA and R1: TCCTCTATTCGGGGAAGGTC; Tug1 F1: CGGAGGAGCCATCTTGTCTTGTC and R1: GCTTCCAATTCCATACACACACTG; Tug1 F2: CTCTGGAGGTGGACGTTTTGT and R2: GTGAGTCGTGTCTCTCTTTTCTC.The testes were collected from wild-type and Tug1 cDNA sequences with CLC Genomics Workbench (Qiagen) for open reading frames (ORFs), allowing both canonical and non-canonical start codons . After, sequences with annotated ORFs were aligned using MUSCLE alignment. All further sequence and amino acid alignments were performed with CLC Genomics Workbench.We analyzed human and mouse Tug1 ORF1 that contained an in-frame 3xFLAG epitope tag prior to the stop codon, with and without the 5\u2032 leader sequence (GeneWiz). We also synthesized a construct containing mouse ORF1 with an HA tag after the 3xFLAG before the stop codon, with and without the 5\u2032 leader sequence (GeneWiz).We generated a synthesized construct for human Tug1 cDNA sequence with primers (see the \u201cTug1 vector plasmid using Q5 polymerase (Roche) and under the following conditions: 96\u00a0\u00b0C for 2\u2009min, 35\u2009cycles of , 72\u00a0\u00b0C for 4\u2009min, and gel purified the amplicon. We digested the inserts and pcDNA3.1(+) plasmid with proper restriction enzymes according to the manufacturer\u2019s instructions. After digestion, the plasmid was dephosphorylated using alkaline phosphatase. We then ligated the plasmid and inserts using T4 ligase (NEB) in a 1:3 ratio, respectively, followed by bacterial transformation, culture growth, and plasmid isolation (Qiagen Mini-Prep Kit).We amplified the see the \u201c\u201d sectionl-lysine-coated 18-mm glass coverslips. Next, we transfected the cells with 14\u2009\u03bcg of plasmid (pcDNA3.1(+) containing each of the inserts) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) per manufacturer\u2019s recommendations. Forty-eight hours post-transfection, cell pellets were harvested for protein extraction (see below) and coverslips were processed for RNA FISH and/or immunofluorescence (see below).We seeded 3T3 and HeLa cells in 10-cm plates containing poly-We resuspended 3T3 and HeLa cell pellets in RIPA Lysis and Extraction Buffer 48\u2009h post-transfection (Thermo Fisher Scientific). Total protein was quantified with Pierce\u2122 BCA Protein Assay Kit (Thermo Fisher Scientific). We then separated a total of 20\u201325\u2009\u03bcg of denatured protein on a 12.5% SDS polyacrylamide gel for 100\u2009min at 120\u2009V. We transferred proteins to an Immobilon-PSQ PVDF membrane  at 400\u2009mA for 75\u2009min. After blocking in 5% dried milk in TBST, the membrane was incubated with properly diluted primary antibody  in 5% dried milk/TBST overnight at 4\u00a0\u00b0C. The next day, we washed the membrane three times for 5\u2009min each in TBST (0.5% Tween-20). We then incubated the membrane with horse radish peroxidase-conjugated secondary antibody , diluted in 5% dried milk/TBST for 1\u2009h at room temperature. Following three 5-min washes in TBST, SuperSignal\u2122 West Pico PLUS chemiluminescent substrate  was added and chemiluminescence was detected using ImageQuant\u2122 LAS 4000 imager. Original, uncropped images of western blots are provided in Additional\u00a0file\u00a0l-lysine-coated coverslips. Forty-eight hours post-transfection, we rinsed the coverslips twice with PBS and fixed the cells with 3.7% formaldehyde in PBS for 10\u2009min at room temperature. After 2 washes with PBS, we permeabilized the cells with PBT  for 15\u2009min at room temperature. Next, we blocked the coverslips with 5% BSA in PBT for 1\u2009h at room temperature and then incubated the coverslips with properly diluted primary antibody  in 5% BSA in PBT for 3\u2009h at 37\u00a0\u00b0C in a humid chamber. Coverslips were washed three times for 5\u2009min each with PBT and incubated with diluted secondary antibody  in 5% BSA in PBT for 1\u2009h at room temperature. Cells were then washed twice for 5\u2009min with PBS, once for 20\u2009min with PBS containing Hoechst DNA stain , rinsed in PBS, and then mounted on glass slides with ProLong Gold (Thermo Fisher Scientific).We plated HeLa and 3T3 cells on poly-l-lysisne-coated coverslips and transfected as described in the previous sections. Forty-eight hours post-transfection, cells were incubated with 200\u2009nM MitoTracker red chloromethyl-X-rosamine  in 1\u2009mL FBS-free growth media for 40\u2009min. We then washed the cells twice with PBS, fixed with 3.7% formaldehyde for 10\u2009min at room temperature, and processed for immunofluorescence and/or RNA FISH (as described previously).We plated cells on poly-We acquired z-stacks (200\u2009nm z-step) capturing the entire cell volume for single-molecule RNA FISH, single-molecule RNA FISH/CMXR staining, 3xFLAG tag immunofluorescence/CMXR staining, and/or Tom20 immunofluorescence with a GE wide-field DeltaVision Elite microscope with an Olympus UPlanSApo 100x/1.40-NA Oil Objective lens and a PCO Edge sCMOS camera using corresponding filters. 3D stacks were deconvolved using the built-in DeltaVision SoftWoRx Imaging software. Maximum intensity projections of each image were subjected for quantification using Fiji.Age- and sex-matched adult mice were used in all flow cytometry experiments. We obtained peripheral blood by cardiac puncture and collected blood into a 1.5-mL Eppendorf tube containing 4% citrate solution. Next, we added the blood-citrate mixture to 3\u2009mL of 2% dextran/1\u00d7 PBS solution and incubated for 30\u2009min at 37\u00a0\u00b0C. The upper layer was transferred to a new 5-mL polystyrene FACS tube  and centrifuged at 1200\u2009rpm for 5\u2009min at 4\u00a0\u00b0C. We then lysed red blood cells for 15\u2009min at room temperature using BD Pharm Lyse . Cells were washed twice with staining media  containing 2% FBS and 2\u2009mM EDTA). The following antibodies were added (1:100) to each sample and incubated for 30\u2009min at room temperature: Alexa Fluor 700 anti-mouse CD8a , PE/Dazzle-594 anti-mouse CD4 , APC anti-mouse CD19 , Alexa Fluor 488 anti-mouse NK-1.1 , and PE anti-mouse CD3 , and Zombie Aqua Fixable Viability Kit  was used as a live-dead stain. We washed samples twice with staining media and sorted directly into TRIzol LS using a BD Aria FACS.We isolated and quantified RNA from sorted blood populations as described in the RNA Isolation and RNA-Seq Library Preparation. One hundred nanograms of total RNA was used as input to generate cDNA using SuperScript IV VILO Master Mix , according to the manufacturer\u2019s protocol. cDNA was diluted 1:3 with DNase- and RNase-free water, and 1\u2009\u03bcL was used per each reaction. We performed qRT-PCR using FastStart Universal SYBR Green Master Mix with ROX  on a ViiA 7 Real-Time PCR System (Thermo Fisher). Analysis was performed using the \u2206\u2206Ct method . PrimersAll primary RNA-seq data are available at the Gene Expression Omnibus (GSE124745 and GSE88819).GAGACACGACUCACCAAGCACUGCCACCAGCACUGUCACUGGGAACUUGAAGAUCCAAGUUUCUGUCCAGAACCUCAGUGCAAACUGACAACACUCCAUCCAAAGUGAACUACGUCCCGUGCCUCCUGAUUGCUGAAUGUUCACCUGGACCUGCCAAUGACCUUCCUUCUGCUACUCCAUCAGCCUACAGACCUGGUACUUGGAUUUUUGUCCAUGGUGAUUCCUUCCACCUUACUACUGAAGAAGACACCAUUCCAGUGGACCACUGUGACCCAAGAAGCAUUCAGCCAUCAUGAUGUGGCCUUUACCUCCACUCCUGUCCUACUCUGCCCAGAUUCAGCACAGCCCUUUAUAGUGCAGUCAAGAGUCUUCAAGCCAAAUAACUGAAGCUAUUUUAUCACAACAAAGGCCAGGUUUAUUCCAUAAAUGUACAGUUCAUUUCUGCAGUUUAUUCUUCAGAGACACAUAGUAAAUUUGGACCAGGGGAUUUUGTug1_5190-5166TD_Forward:TGACTGGCCCAGAAGTTGTAAGTug1_5190-5166TD_Reverse:GCAAGCAGGTCTGTGAGACTATTClacZ_5_Forward:TTGAAAATGGTCTGCTGCTGlacZ_5_Reverse:TATTGGCTTCATCCACCACAYchr_Forward:TCTTAAACTCTGAAGAAGAGACYchr_Reverse:GTCTTGCCTGTATGTGATGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGGGGTTTTTTGTTTTGTTTTTTAAATTGAAGGCTAAAGTTTTTGAAAAAACTTTGTTGGACTCTGGCTGGGACACAAAATCAGATATTTGGAATCATTTTGAAGCTTAACTTTTTCCTAACCAGCCTTGTATTCTAATTGCTTGCAAATGTGAGACTGAATGGCCAAAATGCCGTTTGTTTGTTTGTTTATTGTCAGCTGCTTTTATCAAATTCCAGGCCATTATCCAGCAAACACTATTAAAATGTTTGAACAGTTGGGTTTCAAACATTTTTGTTTTGTGGAGTGGTGCTTATTAAGTGGTACAGCTCTCTAAGCAAGTGAACACAAACATATTTAAGTGTATTTTGTATGATTAGATGTTACCAATTCTGATATTTTATTCAAATGTCTAAAAAAATAAGTTGACTTATTCCCTTTACCAAAGGGCCAGAGACAAATGGTTTCCTTTCAAGAGAAATGACTGTTTTGAAGAAAAACTCTGTTGGTCTTAGCTCTTTTGTAATTAAATCTGGATGTACCTCAAAAGACTCTTTAAGACTGTGGTGTTAAAAGGCTTTCCTCTGGAGAAGGAGAAAAAATAAAATCAACTGGAACTTAAAAGCTTGAAATTCCATGACAAAACACAGATGTCCAGGATTGGAGGTTCATAAAGTACATGCAGTAGTTGGAGTGGATTCCATTTTCAGTGTAGCTGCCACCATGGACTCCAGGCTCCCAGATTTTCAAGAACTGGACCTGTGACCCAGAAGAGCTTGTCAAGATATGACAGGAACTCTGGAGGTGGACGTTTTGTATTCAATTTTGGAACTGTTGATCTTGCCGTGAGAAAAGAGAGACACGACTCACCAAGCACTGCCACCAGCACTGTCACTGGGAACTTGAAGATCCAAGTTTCTGTCCAGAACCTCAGTGCAAACTGACAACACTCCATCCAAAGTGAACTACGTCCCGTGCCTCCTGATTGCTGAATGTTCACCTGGACCTGCCAATGACCTTCCTTCTGCTACTCCATCAGCCTACAGACCTGGTACTTGGATTTTTGTCCATGGTGATTCCTTCCACCTTACTACTGAAGAAGACACCATTCCAGTGGACCACTGTGACCCAAGAAGCATTCAGCCATCATGATGTGGCCTTTACCTCCACTCCTGTCCTACTCTGCCCAGATTCAGCACAGCCCTTTATAGTGCAGTCAAGAGTCTTCAAGCCAAATAACTGAAGCTATTTTATCACAACAAAGGCCAGGTTTATTCCATAAATGTACAGTTCATTTCTGCAGTTTATTCTTCAGAGACACATAGTAAATTTGGACCAGGGGATTTTGTTTTGTTTATATTGTCAACACTGTCTGAAGAAAGGCATCTCTGAGAACAGCATTGGACCCTACTCCACAATCTCAAATGATTGAAGTTTCATAAACTGCCTAGGATCCTGTCAAGGCCACTGGACTCTTGTTCTTTTCCTACTTCAAAATCTGTAGCTGTCTACTAAATGACAAAGCAGATATTCTGACCCATTGGGATCAAAACCAAGGCATTTTGAATTCCTCATAGTATCATCTTCGGGTTACTCAGGAACCAAAACTTTTCACACCAATTTAAGAAATTCTACTGAGGAATCCCTTTACCTAACCATCTCACAAGGCTTCAACCAGATTCCTGAAAAGGCCTCTTGATATATCAAGATAGAACCTACATGCATTTTGTGAACAACTTATCACTGATTTTCCAAAGGCTTTGTGCTCTTGAAGTTCTTTGAAGGAAAGCTGTGTGGAAGTCCAGAGTAAAGTGAAGCTGCTCTGGATGAAGTAGTGAAGTGGGAGTTGAGGTCTACAACCTGCCACAACCATCTTCCTTTACCACCATGGTGATGCCAAAAGGGACTTCCTTAAAGCTCTTCAGAAAATCCTGCTTGAAACCACTACCCTAGACAACATGTTTGACCTGGATGGCATTCTCTTCAAAACAATTCATATTCAGTTGATGCTCAACATGTTTGGAGATGCTTTATTCAGAGAATGATGATAATTACAGCATTGTCTAATGAAGTTTTATTAATAGCATTCCATCCAAGGTGGACTTCCTGGAGTTGGATATAACCAGAGAGCAATTCATATGTATCCTACACTGAAGAACACCATTAACTTTCAGCAACCTATAGCTAGTGGTACTAGAAGTACGTGTCTTGGAAGTCTATGAGAGCTGGTATTGAAGCTGATGCCTCCTTAAGGCCATCTTAGACCAAGTTGTTTGTTTGACCTCTCCTCATTAACTATGGAGCAGAATTGAAATACAAATTTTTCCTAAAGGGACTTGCAACCTGGTTATCATTCATTATCTCAAGTTTCAAGTCATGTTGATGCAACCAGTAGTTATTAAACTGCTCCATGGTTTTTTGTTATTTAATACTTTTTCCAGGGCTTAAAAAAACAAAATTAAATTTCTCCAACACGTCTATACTTGTCTGTTCAAAAGTAACTACTCACCACTATATGGAACAGATGATTCTGAAGACACTCTGAGCATCCTTTATGATATTTGTGACTTAAAATGTGGCTGGAAATTTTCCTTCTACCCAGTGAAATATTTAATGATTAGTCTTCATGCCTGATACCATCAACTGTATATGCGTGATAGGCAAAGTTTGACATAGGCATTTGACTCTAGGCTATGATAGCTTGCTAGTAACTTCAAGTAGCATATTGTCAACCTGTTTGCTGGAAAAGTAGAGTAACTTGGAAAAAAAACTAAATGGCAGCTAAGGATTTTTTTCAGTATTCCTGAGTTTCTGTCCTTGGGATATTTCAATGAAATTTTCACCTGTCTCTTCACTTAACAGAGTGACTGACTCCTTACTATGAAGTATTCTTAAGACATTAAGATTACTTTTGTAGAAAGGATAAAATTCCTGACCATCCAAATCATCATAGTGAACAAGACTTCAATTTGTGACCTGAGAAAATCTCATTTCTCTACTTCGTAGTCAATGTAAGGGCCAATGCTATCAGCTACTCTGAGTGCACTGGGTAAACGTTGGAACTGCCTTCTTTATATCATTACTTTTTATCCTCTAAATTAATCATGGTTATGTAATTCTCGCCACAAATCAGCAAATCAGACTCAGATCTGGTTATTCTAGACTGCTCACAGTTAACAAATCAAACTCTGGATGACTTCTGCTTGTATATGCAACTACTATTTGTAAAGAAATTGCAAATTCACTTTTCTATTACCTCTACATTGCTAGCTCTTTCTTTTGTGTTTGTATTAAAAACAAAAATAAGCTACACTGCCAGCTATTCCCTCCTGCCATACTCAGTTAAAATGAAGAATCGGGAATCTAACCAGTGAATGGATAAGTAGAAAAAACTAAAACTTAAGGCAAAAGCCTTAATCTAGGGCCTTTTCTACTATCTTCATGTCTTGGATTTCATCTAAAATCAACAGTGCCACCCAACCAGTCTGAGGTCTTGACTTGCTTTTAAGATGATTCTTAGAGATGGGCTGTATTACAGAAGGTGAAGACTTGATTACCAAAGAAAGTAGAGCCAACTTTGACAAACCTGGCTCTACAATCCTATTGCTTCCAGATGTAGCATAGACTCATAACTAGAACCTCAAGTCTGCATTGAGGATATAGCCTTCTAAGCTGACAGTTCTTGCAACAGGTGAGCAAGAAAATGAAAGCTGTTATACCCAACTGGCCCTTTAAGATCCAAAAATAATGTCTGGACTAAACCCTATGGAGTACCCAGGACAAAAACTAATTTACAGAGCTTCATTATTAATCTGCCTGTTCTTCTAGCTTAATTATTGGTATGGCTGGCCCTACTGAAGTAGTTTGTCTGTTTACCTGTCTTCAGCTCTTAACCTGGCTATTTTGACATGCTACTGCAATTAGACTAACTGGCTTTGAGAAGACTACAATCAGTTTCAGCCTCTCCTTTGCCCAATTTCACCAAGGAATTTTGATAAGAGGAACCCATACCTCACCCCACCAGAACAGAAAGGACCATGCTGCATATTCCTTGACCAGCAACTTTAAGTAGAGAACAACCCTGCTTGTTTTCAACATCTGAAACACCATTTGATCTAATAGGAGTATAGAAGGTTGACAGCAGAGTACACTACTTACTTCTTTCATAACTCAGAAATGAATATGACTGGCCCAGAAGTTGTAAGTTCACCTTGACAAGAAACAGCAACACCAGAAGTTTACTGCTGAACTTAACTTGCCACTTACTCGAATAGTCTCACAGACCTGCTTGCCAAGTAGGAGGCTAGTTTTCCTGCTTCATATCACCATTGGAGTGGGGCTCAATGGGGTCAATGTTAATACTGACTTGAATGGGGACCTTATGGTGAATCCTAGACTATGAGGCTAATGGAAATTATTGTCTATTCAAGTGGATTATAGATTTCCTGAGGACAGAACAGACATCACTCCTGGTGATTTTTAGAACTTGATTACCAAGGAAGAAATACCAGCTGCTAACAGTCAACTTCATGGGCAAAGATTAAGCTCTCTATATCTGGTCGTATCCTGGATGCTAGTTTTTTATTGCCCAGTGACCATTTCCATCTCACGCTTAACTTCCTGATGTTTTTTGGAACCATCTCTTCCAATTTTCAGTCCTGGTGATTTAGACAGTCTTTTCATGCTGGACATTTTGTTGCAACCTCATCAATCACAGCAAAGTCCATCTTGACTTTAGTGATTAGTTCAGGAATGGATGCATGATTCAAGTTTGTCCAATGATAATCAACCCTAGGTGTTTTCTCAGTTGTGGAGAAGTTCTCTTAGATGCTTTAGCTTTGTAGGAGAAAACTCAAACCAACAGGGCCTACCTACTATGTTGAATGATTGTAGGAGAAAACTCAAACCAACCAGGCCTACCTACTATGTTGAATGAGCCAGGCAGAAAATGAAGCCAGTACAGAGGGAAATGGAGCCAAAAGAGGAAGAGACTTGAGTTCTGATGATCACATTTATGCCCCTGTATCCAACTGTGCCTGAAGCTAATAGTACATCACCTGGACTTTTCAGTTATGTGAACCAATAAATTCCCCTTTTTGTTTAAGTTACTTTGAGTTTug1_MluI_cDNA_Fwd gagaacgcgtTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGGGGTTTTTTGTTTTGTTTTTTAAATTGAAGGCTAAAGTTTTTGAAAAAACTTTGTTGGACTCTGGCTGGTug1_EcoRV_cDNA_RevgagagatatcAACTCAAAGTAACTTAAACAAAAAGGGLNCXAGCTCGTTTAGTGAACCGTCAGATCTUG1_76TTTAAATTGAAGGCTAAAGTTTTTGAATUG1_266GGCCATTATCCAGCAAACACTUG1_755ACTCCAGGCTCCCAGATTTTTUG1_1254TCTTCAAGCCAAATAACTGAAGCTUG1_1741AGAACCTACATGCATTTTGTGAATUG1_2268ATGCCTCCTTAAGGCCATCTTUG1_2754TGTCAACCTGTTTGCTGGAATUG1_3267TTGCAAATTCACTTTTCTATTACCTCTUG1_3746CCCAACTGGCCCTTTAAGATTUG1_4241TGACAAGAAACAGCAACACCATUG1_4740TCACAGCAAAGTCCATCTTGATug1_Tg F/KpnIataggtaccGCCCCGAATTCACGCGTTTug1_Tg R/NotIatagcggccgcACCTGAGGAGTGAAGATug1_FwdCTCTGGAGGTGGACGTTTTGTTug1_RevGTGAGTCGTGTCTCTCTTTTCTCGapdh_Fwd GGTGAAGGTCGGTGTGAACGGapdh_Rev CTCGCTCCTGGAAGATGGTGHuman ORF1:Human ORF1+UTR:Mouse ORF1:Mouse ORF1+UTR:Additional file 1:Fig. S1. Mouse and human Tug1 locus and chromatin context in different cell types. (A)Tug1 mouse and (B) human genomic loci. Evolutionary nucleotide conservation (PhyloP) of the locus are presented along with the chromatin context  and protein binding ChIP-seq peaks  from ENCODE  datasets in the indicated cell types.Additional file 2: Fig. S2. In vivo expression pattern of Tug1 during murine embryogenesis. RNA in situ hybridization of Tug1 RNA using a digoxigenin-labeled antisense RNA probe in mouse embryos at different developmental stages. Embryonic day (E)8.5, E9.5, E10.5, E11.5, and E12.5 are shown.Additional file 3: Fig. S3. Overview of the Tug1 locus in mouse. UCSC genome browser showing the murineTug1 locus. The three predominate Tug1 isoforms are depicted (black) and the Tug1 transgene (tg(Tug1)) is shown (blue). For Tug1 knockout, the longest annotated Tug1 isoform was replaced by a lacZ reporter cassette, leaving the promotor and first exon intact. The deleted region is indicated by red dashed lines. The open reading frame (ORF) encoding the TUG1-BOAT protein and PhyloCSF scores for the (-2) frame across the locus are depicted (grey). Chromosomal coordinates (mm10) are shown.Additional file 4: Fig. S4. Morphology analysis of Tug1-/- mice and sperm (A) Body mass (g) measurements over 11 weeks of male and female Tug1-/- mice compared to wild type littermates. Males: Tug1-/- (n = 7); WT (n = 8). Females: Tug1-/- (n = 3), WT (n = 7). Significant p values at specific time points are indicated (*). (B) Representative images from adult male mice (12 weeks old) show normal physiological appearance of external genitalia and reproductive tracks in Tug1-/- compared to WT. Seminal vesicles (SV), vas deferens (VD), bladder (B), testicle (T), epididymis (E), anterior prostate (AP). (C) Box plots of body mass (g) (left panel), relative testis mass  and total sperm count for wild type (n = 9) and Tug1-/- males. (D) Box plots of the percentage of different sperm morphological abnormalities for wild type (n = 9) and Tug1-/- (n = 8) males. Significant (*) p value (Wilcoxon rank sum test) is indicated.Additional file 5: Table S1.Tug1-/- and wild type sperm morphological defects.Additional file 6: Table S2. Testes RNA-seq and Tug1rescue RNA-seq in testes.Additional file 7: Table S3. Prostate, spleen, eyes, brain, heart, liver, and MEF RNA-seq.Additional file 8: Table S4. Allele-specific RNA-seq in testes, expression data.Additional file 9: Table S5. Allele-specific RNA-seq in testes, differentially expressed genes.Additional file 10: Fig. S5. Gene expression of multiple tissues in Tug1 WT and KO mice. Gene expression of multiple samples (columns) were described with log scale of TPM: Log2(TPM+1). Genes (rows) are clustered by hierarchical clustering with Ward\u2019s method based on Euclidean distance. Annotation of samples are provided in the top panel in terms of genotypes and tissues.Additional file 11: Fig. S6.Tug1 transgene expression and fertility assessment. (A) qRT-PCR for Tug1 RNA expression in testes and sorted peripheral blood populations: WT (n = 1), Tug1+/- (n = 1), Tug1-/- (n = 1), and Tug1rescue (n = 1) and sorted peripheral blood populations. Error bars indicate the relative quantification minimum and maximum confidence interval at 98%. Not detected (n.d.). (B) Representative flow cytometry gating strategy for NK, CD4, and CD8 cells in peripheral blood from WT, Tug1+/-, Tug1-/-, and Tug1rescue mice . (C) Scatter dot plot (mean with standard error of the mean shown) of the number of pups at birth per copulatory plug for matings using male wild type, Tug1+/-; tg(Tug1); rtTA, Tug1-/-, or Tug1rescue (on dox diet) with wild type C57BL/6J females. Each dot represents a litter from a different mouse. (D) Sperm count from control , Tug1-/- (n = 2), and Tug1resuce (n = 3) mice. Each dot represents a different mouse and the error bars indicate the standard error of the mean. (E) Hematoxylin and eosin staining in Tug1+/-, Tug1-/-, and Tug1rescue testes and epididymis. (F) Morphological analysis of sperm from Tug1-/- (n = 2), and Tug1rescue (n = 3) mice.Additional file 12: Fig. S7. Changes of gene expression in two comparisons of Tug1-/- (KO) vs. WT and Tug1rescue (Rescue) vs. Tug1-/- (KO). Each dot represents a gene whose x-axis value is the fold change of gene expression between KO and WT and y-axis value shows the fold change in Rescue vs. KO. Color describes statistical significance of fold change : no statistical significance in either comparison ; significance in KO vs. WT ; significance in Rescue vs. KO ; significance in both comparisons, KO vs. WT and Rescue vs. KO .Additional file 13: Fig. S8. The 5\u2019 region of human TUG1 contains a conserved ORF. (A) GWIPS-viz tracks for human TUG1 genomic locus (hg38) is shown. Global aggregate of ribosome occupancy (ribosome profile), RNA-seq (mRNA coverage), and evolutionary protein-coding potential (PhyloCSF) across the TUG1 locus is shown. ORF1 and ORF2 are outlined with red and gray boxes, respectively. Tracks surrounding both ORFs are zoomed in for clarity (bottom). (B) Scheme showing human ORF1 construct design. hORF1 (labeled with a 3xFLAG epitope tag prior the stop codon) with the 5\u2019UTR was inserted into pcDNA3.1(+) and transfected into HeLa cells. 48 hours post-transfection, TUG1-BOAT-3xFLAG localization was analyzed by immunofluorescence (IF) (shown in C). (C) Maximum intensity projection of HeLa cells expressing human 5\u2019UTR-hORF1-3xFLAG. Localization of 3xFLAG tagged TUG1-BOAT was assessed by immunostaining against the 3xFLAG (green). Nucleus was monitored by DAPI (blue) and mitochondria was monitored by immunostaining against mitochondrial membrane translocase TOM20 (gray). Bar plot shows localization analysis of TUG1-BOAT. Scale bar is 5 \u03bcm.Additional file 14: Fig. S9. GFP over-expression does not compromise mitochondrial membrane potential. Maximum intensity projections of z-stacks acquired 48\u2009h post-transfection of 3T3 cells with GFP cloned into pcDNA3.1(+) under CMV promoter and staining with Chloromethyl-X-rosamine (CMXR). GFP (green) was used as control. CMXR is shown in red, DAPI in blue. On the right, quantification of cells expressing GFP and mitochondria membrane potential by CMXR (n = 50). Scale bar is 5\u2009\u03bcm.Additional file 15: Fig. S10. Loss of TUG1 expression in infertile human males. Heatmap of microarray data from three different probe sets showing decreased expression of TUG1 in sperm from infertile teratozoospermic men (T) compared to fertile (normospermic) individuals (ND). In all cases, p < 4.39 x10-4.Additional file 16: Fig. S11. Uncropped western blot images from Figure\u00a0(A) Western blot of constructs overexpressed in 3T3 cells targeting the 3xFLAG tag (top). GAPDH is used as a loading control (bottom). (B) Western blot of constructs overexpressed in HeLa cells targeting the 3xFLAG tag (top). GAPDH is used as a loading control (bottom).Additional file 17. Review history."}
+{"text": "In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647\u2009N fluorophores in 90% glycerol solution with an average duration of ~16\u2009s at count rates of ~10\u2009kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385\u2009bp) of single DNA molecules at rates up to 10\u2009\u00b5m Single molecule observation has been limited to tethered molecules to ensure that the target remains in the field of view (FOV). Here, the authors develop a real-time tracking method that locks onto rapidly diffusing targets and tracks them in a 3D volume, enabling single molecules to remain in the FOV for minutes at a time. Valuable insights have been gained in a wide range of sub-disciplines, ranging from DNA transcription5 to enzyme catalysis8, among many others. To date, the vast majority of single-molecule investigations have been restricted either to short times (using a tightly focused laser spot to monitor single molecules which transiently diffuse through the observation volume) or rely on tethering the molecule to a surface . The former solution-phase measurements acquire limited information due to the rapid diffusion of molecules through the focal spot. The latter tethered methods require isolation of the molecule from its environment, precluding observation of the native functions of molecules such as enzymes, which may critically rely on the crowded and non-equilibrium surroundings of the cellular interior. Several methods have sought to overcome this need to tether molecules, including liposomes9, convex lens induced confinement (CLIC)11 and the anti-Brownian electrokinetic (ABEL) trap16. Although each of these methods remove the tether, they still restrict the molecule in an isolated environment and cannot continuously monitor unbound diffusing molecules, such as an enzyme freely diffusing through the cellular interior.Single-molecule spectroscopy has been a critical tool in investigating molecular dynamics in biological systems17. RT-3D-SPT acquires high-speed position measurement of a diffusing particle and implements a closed feedback loop to effectively lock the rapidly moving target in the observation volume. Feedback is applied using either piezoelectric nanopositioners or galvo mirrors18, meaning the target molecule or particle is followed at very high speed, rather than being physically confined. Pioneering work has shown that RT-3D-SPT can lock-on to a wide range of targets, from gold nanoparticles20 and single quantum dots23 all the way to viruses26, and is easily extended to live cell tracking28. This is because the \u201cconfinement\u201d is not a physical impediment to the target, but rather a high-speed chase which follows the particle without perturbation.A group of methods with the promise to break this tether is real-time 3D single particle tracking (RT-3D-SPT)27. While truly a groundbreaking achievement for RT-3D-SMT, the trajectories were limited to several 100\u2009ms. The same group followed-up 2 years later, showing the ability to disentangle the oligomerization states of azami green, though still in 90\u2009wt% glycerol and still for only several 100\u2009ms29. Further experiments with the same method have shown an expansion of the target scope (FRET), but with no increase in the trajectory duration despite still working in highly viscous solutions30. Similar results were observed by Liu and coworkers for single-labeled DNA31.While the pioneering work on RT-3D-SPT by several groups occurred more than a decade ago, the extension of this promising method to real-time 3D single-molecule tracking (RT-3D-SMT) has been limited  or the bleaching rate . Starting with the latter case, it is certainly possible that these methods suffered from bleaching, leading to the short trajectories. However, it is also possible that the reduced emission rate of single molecules, compared to multiply labeled fluorescent beads, could lead to an inability to lock-on to single molecules for any appreciable period of time.What has limited RT-3D-SPT methods from achieving long and stable RT-3D-SMT trajectories? A commonality among the methods introduced above is the need to slow down molecular diffusion by increasing the solution viscosity for successful tracking. This is not surprising and would presumably be necessary, given that a piezoelectric nanopositioner is typically bandwidth limited to about 1\u2009kHz . Photon arrival times detected by an avalanche photodiode (APD) are converted to particle positions in real-time on a field-programmable gate array (FPGA) using a Kalman update filter with assumed Gaussian density of all likelihoods34. The results of 3D-SMART can be seen in Fig.\u00a037. As a result, avoiding the excitation and allowing relaxation of the triplet state can reduce the rate of photobleaching. Pulse optimization to allow for dark state relaxation has led to increased photon yields in both confocal laser scanning39 and STED microscopies41. The fast, periodic illumination pattern of 3D-SMART likely reduces photobleaching in a similar way, where the relatively long time intervals between illumination of the tracked molecule permits triplet relaxation. The high concentration of glycerol may also play a role, as has been demonstrated in fluorescent proteins42.3D single-molecule active real-time tracking (3D-SMART) was achieved by applying these optimized parameters to the previously reported 3D-DyPLoT2/s, corresponding to a hydrodynamic diameter of 0.98\u2009nm, well in line with what is expected for a single molecule . Applying the Stokes\u2013Einstein relation reveals a measured diffusion coefficient of 2.44\u2009\u03bcmule Fig.\u00a0. A furthule Fig.\u00a0\u20137. PhotoAn important consideration in any single-molecule study is the throughput. For a method which collects serial single-molecule traces to compile statistics, the microscope must be capable of capturing many molecules in a row, with minimal down time between trajectories. This is true of the 3D-SMART method. Figure\u00a0The data presented above demonstrate that 3D-SMART only requires a single fluorophore for continuous observation of a molecule diffusion three dimensions. As such, the application possibilities are expansive. Here we demonstrate the applicability of this method to proteins, DNA, RNA, and even multicolor dynamic processes such as DNA transcription. Figure\u00a044 as compared to a more sophisticated wormlike chain model which takes chain stiffness into account45. We found that the 3D diffusion of the various lengths of dsDNA, as measured by 3D-SMART, did not conform to the simple model of a flexible polymer put forth by Rouse and Zimm, but agreed almost exactly with the WLC model developed by Yamakawa and Fujii  in solution at high temporal resolution for long times enables examination of different models for the diffusion of semiflexible polymers. By tracking different lengths of dsDNA , longer dsDNA (547\u2009bp) still shows good agreement with the WLC model, indicating that no change in the dsDNA conformation as function of glycerol concentration can be detected within the resolution of these measurements. This demonstrates that single fluorophore tracking is achievable in low viscosity environments at high diffusive speeds.Furthermore, dsDNA tracking is useful for extrapolating the speed and size limits of 3D-SMART. For single dye molecules, the required glycerol concentration for successful active feedback tracking is 90\u2009wt% glycerol. For single proteins (BSA), it is 73.5\u2009wt% glycerol. For dsDNA at a length of 1385\u2009bp, with a single fluorophore labeled at the terminus, active feedback tracking is achieved in PBS . Upon binding, the chromophores are stabilized by the aptamer and go from nonfluorescent to fluorescent. By tracking individual aptamers, the number of bound chromophores can be extracted  pair to act as a fluorescence in situ hybridization (FISH) marker. Upon transcription of the DNA by the RNA polymerases, the FISH probe complements to the nascently transcribed RNA, separating the fluorophore from the quencher and resulting in a burst of fluorescence  to reduce fluorescence background, resulting in a low transcription detection rate in these experiments (6 of 628 trajectories) since hybridization rate is only 1.2\u2009\u00d7\u200910\u22123\u2009s\u22121 without the presence of glycerol. Moreover, the presence of glycerol will further decrease the hybridization kinetics55. Considering the low probe binding rate in the experiment, the start points of the transcription signal in Fig.\u00a054 the binding of the fastFISH probe after the transcription has already started. On the other hand, the Alexa 488 bleaching time , so the disappearance of the signal should indicate dissociation of the transcribed RNA from the diffusing dsDNA. The results of this assay can be seen in Fig.\u00a02/s to D ~2.5\u2009\u00b5m2/s, Fig.\u00a0E.coli RNAP resulted with no transcription bursts associated with probe binding observed.As a final demonstration of the power of 3D-SMART, we show the observation of a compete biochemical reaction at the single-molecule level in solution Fig.\u00a0. To do skept low 0\u2009pM to r2/s, all with photon-limited temporal resolution. 3D-SMART has the power to completely untether single-molecule experiments. This opens the path towards measuring the fast internal molecular motions of single proteins in the complex cytoplasmic milieu, though this represents only a tiny portion of what 3D-SMART can enable. One reason single-molecule observation has been so powerful in biological systems is that molecules are intrinsically \u201cconfined\u201d to the cellular container, making observation over long times more feasible. 3D-SMART can extend single-molecule spectroscopy to completely unconfined systems, going beyond molecular biology to synthetic chemistry  and condensed matter physics . Untethering single-molecule spectroscopy in this way is critical towards increasing the application scope of these powerful methods.The above demonstration of 3D-SMART shows a dramatic improvement in tracking speed and information acquired from single molecules in solution, enabling continuous observation of a single, freely diffusing dye molecule for up to minutes at a time and diffusion coefficients up to 10\u2009\u00b5mf\u2009=\u2009150\u2009mm  and f\u2009=\u2009200\u2009mm , and a pinhole with 75\u2009\u00b5m diameters  are used to collimate and purify the 488\u2009nm beam. The polarization state of 488\u2009nm beam is cleaned by a Glan-Thompson Polarizer  and tuned by a half-wave plate . For 640\u2009nm laser beam, two lenses with focal length f\u2009=\u2009100\u2009mm  and f\u2009=\u2009150\u2009mm  and a pinhole with 100\u2009\u00b5m diameters  are used for collimation. The polarization state of the 640\u2009nm beam was cleaned by a polarizer  and tuned by a half-wave plate . A dichroic mirror  is used to combine the 488-nm beam and 640-nm beam. The beams then travel through two electro-optic deflectors . The EODs are used to produce the designed scanning pattern. A lens pair  is placed after the EOD to act as beam expander. The TAG lens  is used to modulate the beams focal position dynamically. The TAG lens has a dynamically changing focus with low aberration performance and up to megahertz frequencies. Here we run the TAG lens at the ~70\u2009kHz resonance. The beam from the TAG lens is relayed to the microscope objective lens  by two f\u2009=\u2009200\u2009mm focal length lenses . A dichroic mirror  reflects the beam towards the back aperture of the objective lens. A bandpass filter  separates the fluorescence emission from the excitation lasers. For two color experiments, a dichroic filter  separates the fluorescence onto different detection paths. Two APD are used to measure fluorescence signal with a lens with focal length 50\u2009mm  installed before each APD. A micropositioning system  enables coarse sample positioning. A 3D piezoelectric stage system  is used to move the sample and objective lens for active feedback 3D tracking. In photon antibunching experiment, a 50/50 beamsplitter  was used to split Atto 647N fluorescence onto the two APDs and a time-correlated single-photon counting model  tags the arrival times of fluorescence photons in each APD. An IR blocking filter  is placed in front of one APD for preventing optical cross talk between two detectors.The 3D-SMART setup uses a 488\u2009nm frequency-doubled solid-state laser  and 640\u2009nm diode laser  for excitation. A telecentric system, consisting of two lenses with focal length A field-programmable gate array  based data-processing unit controls the EOD scanning, collects photon counts from the APD, interpolates the TAG lens signal, calculates the particle position estimates, applies position feedback with the piezoelectric stage, and records the particle position information. The system operates in custom software implemented with LabView 15 .For Alexa 488 tracking, the laser power was ~1.7\u2009\u00b5W at the objective focus. For Atto 647\u2009N tracking, the laser power was ~2\u2009\u00b5W. For mRNA-DFHBI-1T tracking, the laser power was 1.9\u2009\u00b5W. All the tracking experiments were performed at room temperature (~23\u2009\u00b0C).Atto 647\u2009N NHS ester  or Alexa 488  was first dissolved in Dimethyl Sulfoxide  at 5\u2009mg/ml and aliquoted into 5\u2009\u00b5l for each vial and stored in \u221280\u2009\u00b0C. Then the dye was diluted into 10\u2009pM with distilled water. Then 1\u2009mL dye solution was mixed with 9\u2009g glycerol  and added 400\u2009\u00b5l solution to a home-made sample holder for 3D-SMART experiments  was dissolved in DPBS  at 2.6\u2009mg/ml (40\u2009\u03bcM). Then 40\u2009\u03bcl BSA was mixed with 1\u2009\u03bcL Atto 647\u2009N NHS ester or Alexa 488 NHS ester (5\u2009mg/ml). The mixture was incubated for 2\u2009h (25\u2009\u00b0C) on shaking stage. The labeled BSA solution was then purified by Zeba spin desalting columns  according to the manufacturer\u2019s procedure. The purified BSA solution was diluted to 2\u20135\u2009pM in 73.5\u2009wt% glycerol-PBS solution. 400\u2009\u00b5L of the solution was added to the home-made sample holder for 3D-SMART experiments.To tag the DNAs with fluorescent molecules, DNAs were modified with amino (synthesized by Integrated DNA Technologies). To label the amino modified DNA with Atto 647\u2009N NHS or Alexa 488 NHS, the amino modified DNA was dissolved in PBS at a concentration of 100\u2009\u00b5M, then incubated with dye at a molar ratio of ~1:5 for 2\u2009h (25\u2009\u00b0C) while shaking. The labeled dsDNA was then purified with NAP-5 G25 column  according to the product manual. All the amino modified DNA premers and single DNA strand were labeled with fluorescent molecules before the PCR amplification and hybridization.For 99\u2009bp dsDNA tracking the following sequence was used (synthesized by Integrated DNA Technologies): 5\u2032-TAT GTA ATT GGA GTG GTT AAG ATA AGG GAT AGG GTG AAA TTG TTA T /amino/ C CGC TCT CAC AAT TCC ACA CAT TAT ACG AGC CGA AGC ATA AAG TGT CAA GCC T-3\u2032. Two complementary DNAs were mixed in PBS buffer with a concentration of 1\u2009\u03bcM and then went through the following annealing procedure: (1) heat to 95\u2009\u00b0C and maintain the temperature for 5\u2009min; (2) Cool to 4\u2009\u00b0C maintain the temperature for temporary storage.42), except for a small alteration in the initial transcribed region (ITR). Region \u2212200 to +325 containing the T7A1 promoter region and the probe binding site was synthesized by Integrated DNA Technologies (IDT) as double-stranded DNA delivered on a pUCIDT plasmid. The sequence was then excised with XbaI and HindIII and inserted into pT7-7 plasmid (Addgene #36046) between the XbaI and HindIII restriction sites to generate a plasmid template construct (pT7-fastFISH (pT7-fF)).For 547\u2009bp dsDNA tracking, the following sequence was used: 5\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccatgtagtaaggaggttctaatagccatcccaatcgacaCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACCTTTTAGATACCTGATACAAAGTCCATTATGATTTTTAGATTTCGTATATTTACACTTGCACCATACGCATGTAAAATTAGAAGCAAAGTACGATTCTTAGACCGATGTATAATATAATTATGTAGATGTGATGAGTTTCTTTTATATGCTTCACCTGTCGGATCGGTCTGCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGAA-3\u2032. Here, lower case letters indicate the T7A1 promoter region (\u2212163 to +38 from the transcription start site represented by the bold typed a). Region \u2212221 to +91 is the same template sequence as previously reported . The underlined portion indicates the \u201cSpinach 2\u201d aptamer sequence40. Region +95 to +374 containing (linker-Spinach 2-linker-Spinach 2-linker) was synthesized by GenScript as double-stranded DNA delivered on a pUC57 plasmid. This construct was inserted between the probe-binding site and HindIII restriction site of pT7-fF to create pT7-Spi2-2R. The final plasmid template construct pT7-Spi2-8R, containing eight \u201cSpinach 2\u201d aptamers, was created using the repeat expansion method57. The 1385\u2009bp DNA transcription template was amplified by PCR from pT7-Spi2-8R using the Primer 1 and Primer 2.For 1385\u2009bp dsDNA tracking and DFHBI-1T-mRNA tracking experiments, the following sequencing containing eight spinach2For ensemble and single-molecule diffusion fastFISH experiments, the following sequence containing the single probe-binding site was used:atgtagtaaggaggttctaatagccatcccaatcgacaCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAA-3\u2032. The 1218\u2009bp DNA transcription template was amplified by PCR from pT7-fF using the following primers:5\u2032-GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGATCTGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCCCAGCGATTCGAACTTCTGATAGACTTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccPrimer 3: 5\u2032-GCTAATCCTGTTACCAGTGG-3\u2032Primer 4: 5\u2032-/amino/TTTGAAGTTTTGAAAGGTGTATG-3\u2032a). Here, the underlined portion indicates the oligo probe-binding site. The self-quenched oligo probe is 5\u2032-/Alexa488/GTTAAGATAAGGGATAGGG/RQ/-3\u2032. RQ indicates Iowa Black RQ.As above, lower case letters indicate the T7A1 promoter region .For 1385 base pairs DNA tracking in PBS, the DNA was firstly dissolved in PBS and incubated for 30\u2009min. For 99 and 546\u2009bp DNA tracking, 70, 40 or 25 glycerol (by weight) was added after incubating the dsDNA in PBS for 30\u2009min or by going through the annealing procedure as described above. The sample was then further diluted for tracking via 3D-SMART. The final concentration of DNA used in this work was 1\u20135\u2009pM.2). The tracking solution was made by adding 30\u2009\u00b5L of the mixture to 750\u2009mg glycerol, 200\u2009\u00b5L 5X binding buffer and 20\u2009\u00b5L H2O.DFHBI-1T  was dissolved in DMSO at 31\u2009mM concentration. Before mixing the DFHBI-1T with mRNA, the DFHBI-1T was diluted to 0.31\u2009mM with DI water and titrated to neutral pH with KOH. Then 1.2\u2009k bps mRNA (200\u2009nM) was incubated with DFHBI-1T (10\u2009\u00b5M) for 1\u2009h in binding buffer (40\u2009mM HEPES (PH 7.2\u20137.5), 125\u2009mM KCl, 10\u2009mM MgCln\u2009=\u2009(ImRNA \u2013 background)\u2009\u00d7\u2009IDFHBI\u22121, in which ImRNAis the mean intensity of first 100\u2009ms trajectory and IDFHBI is the single DFHBI-1T intensity. Single DFHBI-1T fluorescence intensity is derived from the step-wise bleaching intensity. The mean of three stepwise bleaching steps from three trajectories was measured to be 1323\u2009\u00b1\u200993\u2009Hz.The number is calculated by 2, 2.5\u2009mM rNTPs , 20% (v/v) glycerol, 2.5\u2009mM spermidine, 0.01% Triton X-100, 0.05 units YIPP (NEB #M2403S), 1 unit RNaseOUT , 1\u2009mM DTT, and 200\u2009ng of the 1218\u2009bp DNA template. 2.5 units of T7 RNA Polymerase (New England Biolabs #M0251S) was added to initiate the reaction, which was then allowed to proceed at 37\u2009\u00b0C for 2\u2009h. To this reaction mixture the self-quenched oligo probe was added at a final concentration of 1.5\u2009\u00b5M. The mixture was incubated at 37\u2009\u00b0C for 30\u2009min prior to acquiring fluorescent emission spectra.To validate transcription and oligo probe binding, bulk experiments were performed in buffer containing 40\u2009mM Tris-HCl pH\u2009=\u20098.0, 25\u2009mM MgClE.coli RNAP , 200 pM probe DNA, 80 U RNase Ribonuclease Inhibitor  and 12 pM template DNA. Before the transcription experiment, the double-strand template DNA was diluted into PBS with a concentration of 6\u2009nM and incubated for 30\u2009min in room temperature. Hundred and ten milligrams of glycerol was mixed with 314\u2009\u00b5L DI water and 50\u2009\u00b5L 10x transcription buffer. The sequentially added NTPs, RNase Ribonuclease Inhibitor, template DNA, RNAP and probe DNA. For in vitro transcription control experiment, all the experiment conditions were same as transcription experiments except without E.coli RNAP. All these experiments were performed in room temperature (23\u2009\u00b0C).All the in vitro transcription experiments were performed in NEB\u2019s transcription buffer  with 1\u2009mM NTPs , 22\u2009wt% glycerol, 100 U The transcription bursts are determined by inspection. As can be seen Fig.\u00a0The 1388\u2009bp dsDNA labeled with Alexa 488 was used to measure the photo-bleaching time of Alexa 488. The 1388\u2009bp dsDNA was synthesized by PCR using Primer 2 and Primer 5, and pT7-Spi2-8R as the template.Primer 5: 5\u2032-/Amino/ TTT CCA CAA CGG TTT CCC TCT AG.Primer 5 was labeled with Alexa Fluor 488 NHS using the same labeling procedure as described above, prior to PCR. 12 pM DNA was dissolved in PBS solution and then 500\u2009\u03bcL of\u00a0solution was applied to a quartz coverslip before the experiment. The DNA can bind to the coverslip non-specificity. In order to locate the dye in the center of laser volume, the feedback tracking was turned on at the beginning of measurement. Once the dye was captured, the feedback tracking was turned off and the laser will continually illuminate on the dye molecule. The used laser power is the same with that in the transcription tracking experiment.1388\u2009bp dsDNA labeled with Atto 647\u2009N was used to measure the tracking precision (Supplementary Fig.\u00a0Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Reporting Summary"}
+{"text": "The authors would like to draw the reader\u2019s attention to the errors in the following article.J Diabetes Investig 2020; 11: 699\u2013706.Sakurai K, Kato T, Tanabe H, Naoko Taguchi\u2010Atarashi N, Sato Y, Eguchi A, Watanabe M, Ohno H, Mori C. Association between gut microbiota composition and glycoalbumin level during pregnancy in Japanese women: Pilot study from Chiba Study of Mother and Child Health. On page 700, the primers used in the 16S rRNA sequencing V1\u20102 region were incorrectly stated. The correct primers should have been as follows:27Fmod: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTTGATYMTGGCTCAG338R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTGCCTCCCGTAGGAGTThe authors apologize for these errors and any confusion they may have caused."}
+{"text": "C.\u00a0elegans to stringently deplete embryos of Microprocessor activity. Using a combination of auxin-inducible degradation (AID) and RNA interference (RNAi), we achieved Drosha and Pasha/DGCR8 depletion starting in the maternal germline, resulting in Microprocessor and miRNA-depleted embryos, which fail to undergo morphogenesis or form organs. Using a Microprocessor-bypass strategy, we show that this early embryonic arrest is rescued by the addition of just two miRNAs, one miR-35 and one miR-51 family member, resulting in morphologically normal larvae. Thus, just two out of \u223c150 canonical miRNAs are sufficient for morphogenesis and organogenesis, and the processing of these miRNAs accounts for the essential requirement for Drosha and Pasha/DGCR8 during the early stages of C.\u00a0elegans embryonic development.MicroRNAs (miRNAs) are a class of post-transcriptional repressors with diverse roles in animal development and physiology . The Mic  \u2022Depletion of Drosha and Pasha results in embryos that fail to undergo morphogenesis\u2022The mirtron pathway enables expression of miRNAs in the absence of Drosha and Pasha\u2022Two miRNAs are sufficient to rescue embryogenesis in the absence of Drosha and Pasha\u2022miR-35 and miR-51 play an unexplored, likely conserved role in animal development C.\u00a0elegans embryos causes failure to undergo morphogenesis. This can be overcome by re-introducing two miRNAs, miR-35 and miR-51, resulting in morphologically normal larvae. These conserved miRNAs account for the essential embryonic function of Drosha and Pasha.Dexheimer et\u00a0al. show that depletion of maternal and zygotic Drosha and Pasha in  MicroRNAs (miRNAs) are short non-coding RNAs that act with Argonaute proteins to elicit translational repression and decay of specific target mRNAs . miRNAs C.\u00a0elegans, we developed a conditional approach for stringent removal of Drosha and Pasha/DGCR8 and thus miRNAs. Microprocessor depletion in embryos is generally challenging, given the maternal contribution of mRNA and protein. In C.\u00a0elegans in particular, the function of the Microprocessor has not been studied in embryos because homozygous Drosha (drsh-1) or Pasha/DGCR8 (pash-1) mutants derived from heterozygous mothers carry enough maternal RNA and/or protein to develop into adults, albeit sterile . B. BC.\u00a0eleormation , we thusormation , yieldinpash-1 and drsh-1, animals were subjected to Cas-9 mediated genome engineering via RNP-microinjection following the Co-CRISPR approach described by [S.\u00a0aureus, purified in-house), 500ng/\u03bcl TracerRNA, 100ng/\u03bcl crRNA targeting the Co-CRISPR marker gene dpy-10, and 15ng/\u03bcl dpy-10- > rol-6 repair template oligo. Additionally, 100\u00a0ng/\u03bcl crRNA against the locus of interest as well as 100-200ng/\u03bcl dsDNA repair template encoding the desired modification with around 50bp flanking homology arms were added to the mix. Note that for efficient degradation of DRSH-1 we needed to insert AID degrons at the N- and C-termini simultaneously as either degron alone was insufficient for depletion. Guide RNAs used and sequences inserted (in bold) were as follows:drsh-1 \u2013 N-terminal AID-tag \u2013 guide sequence used\u00a0= 5\u2032- TTAGATTTTCATTTAGATGT-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGGAGGAAGCGGAGGAGGAAGCGGTGGAGGAAGCGGAGGAGGAAGCGGAATGGAACAGAAACTCATCTCTGAAGAGGATCTGATGTCGGACGAAAAGATTTCAATGACGCTTAACTTCCCGAAACACAAGCGTAGTTAACGTTTTCATCTGAAATTGTAGACAGATTTAGATTTTCATTTAGdrsh-1 \u2013 C-terminal AID-tag - guide sequence used\u00a0= 5\u2032-GATACCAGCGACTAATTACG-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGTTACGGGGTTATAATTATACTATGTCTGTTTGAATGTGATTCGGTTCTCAAGTGGTTTCAGAACATGCGCCGTCGTCTTGAACAAGATACCAGCGACGGAGGCAGCGGTGGTGGAAGCGGCGGGGGAAGCGGCGGTGGAAGCGGTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGpash-1 \u2013 C-terminal AID-tag \u2013 guide sequence used\u00a0= 5\u2032-AGGTGAATATACTATTTGTG-3\u2032 \u2013 locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGGGAGGAAGCGGAGGAGGAAGCGGAGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTATATTCACCTCATATGTTTGTTGTTTTGTTGGTAGTTTTAATTTTTGGGGAAGACATGACGATTCATCATCCCCATCACATCAGAAACCACACAAAInserted sequences contained the TIR-1 recognition peptide , a FLAG (GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG) and a MYC (GAACAGAAACTCATCTCTGAAGAGGATCTG) tag, as well as GGSG linkers in between.To generate AID-tagged copies of ribed by , with moribed by . Brieflypash-1(ts) allele mj100 were kept constantly at the permissive temperature of 16\u00b0C. pash-1(ts) animals expressing the mirtron-51 transgene were selected every few generations for high levels of the co-expression marker elt-2p::dsRed, as this transgene was prone to undergo silencing. For experiments, L4 stage larvae were transferred to a fresh plate and shifted to 25\u00b0C approximately 16h later. In order to achieve maximal depletion of maternal miRNAs, mothers were kept at the restrictive temperature an additional 7h before harvesting early embryos (1-4 cell stage). Extending the time at the restrictive temperature was prohibited by the rapidly deteriorating health of gravid pash-1(ts) adults expressing mirtrons at 25\u00b0C. For backshift-experiments with mirtron-rescued pash-1(ts) animals, hatched L1s that developed under restrictive conditions were transferred to a fresh plate within 1h after hatching, allowed to develop at 16\u00b0C, and scored for their ability to reach adulthood within 7\u00a0days.Animals bearing the E.\u00a0coli OP50 at 20\u00b0C. For combined AID and RNAi treatment, NGM plates were supplemented with 1mM Carbenicillin, 1mM IPTG and 4mM Auxin . RNAiD plates were seeded with E.\u00a0coli HT115 expressing dsRNA which elicits an RNAi response against pash-1 mRNA  library, ORF-ID T22A3.5). To ensure efficient mRNA knockdown, RNAi bacteria were grown freshly for every experiment in liquid LB culture, the medium was brought to 1mM IPTG around 1h before pelleting bacteria, and plates were seeded with a 20X concentrate . For experiments, L4s were transferred to RNAiD plates, embryos were harvested 24h later, and assayed as described below. While the AID-system rapidly depletes proteins within minutes to hours [pash-1(ts) animals, eventually owed to the temperature difference and/or deleterious side-effects of PASH-1(TS) at 25\u00b0C. Efficiency of RNAi-mediated pash-1 mRNA knockdown was assessed via RT-qPCR. Briefly, 5-10 embryos in the 2-cell stage were transferred to about 1\u00a0\u03bcl of Lysis Buffer  using a thin glass needle. These samples were subjected to 10\u00a0min digestion at 65\u00b0C before heat inactivation of proteinase K for 1\u00a0min at 85\u00b0C. Next, crude lysates were reverse transcribed before performing qPCR using the GoTAQ qPCR Mastermix (Promega) according to the manufacturer\u2019s instructions. Relative expression was calculated according to the \u0394\u0394Cq-method using cdc-42 as a reference gene, the following primer sequences were employed:pash-1-F: 5\u2032-GCCTTCGAGAAAACGGGGAA-3\u2032; pash-1-R: 5\u2032-TGGCTCCCATTTCGGAGATT-3\u2032;drsh-1-F: 5\u2032-TGAGCTGGCTTTGGCTAATCT-3\u2032; drsh-1-R: 5\u2032-ACCCCGTAATTAGTCGCTGG-3\u2032cdc-42-F: 5\u2032-TGGGTGCCTGAAATTTCGC-3\u2032; cdc-42-R: 5\u2032-CTTCTCCTGTTGTGGTGGG-3\u2032Animals were kept constantly on standard NGM plates seeded with To assess the extent of protein degradation of PASH-1:AID:MYC and DRSH-1:AID:MYC upon Auxin treatment, L4s were transferred to Auxin plates prepared as described above. 24h later, gravid adults were collected in L\u00e4mmli-buffer containing 2.5% \u03b2-Mercaptoethanol (v/v), and subjected to multiple cycles of snap-freezing followed by boiling until embryos were disrupted. Proteins were separated via SDS-PAGE and transferred onto a Nitrocellulose membrane. As primary antibodies either monoclonal mouse anti-myc  or a polyclonal rabbit anti-gamma tubulin  were used. As secondary antibody, anti-mouse IgG HRP-linked Antibody  or anti-rabbit IgG HRP-linked Antibody  was applied followed by visualization using ECL reagent (Thermo Scientific).Embryos were obtained from day 1 gravid adults reared as described above, by slicing mothers in a drop of M9 buffer on a microscopic slide. 2-cell embryos of normal size were transferred to standard NGM plates or RNAiD plates and allowed to develop at the respective temperatures. Hatched animals were collected for subsequent analysis  from plates within 1h after hatching. The final hatching rate was assessed > 24h after embryo collection by scoring the fraction of animals that successfully escaped the eggshell. To obtain samples at specific developmental stages for small RNA sequencing, 2-cell embryos were harvested via slicing, collected or allowed to develop on NGM plates before being selected manually by stage at given time points .myo-2prom::mCherry. Mean and range are plotted for each genotype; unpaired t test was used for statistical comparison.For phenotypic analysis of embryonic arrest or hatched L1 larvae, animals were mounted on a thin agar-pad on a microscopic slide sealed with a coverslip. Images were recorded at 400x magnification using an AxioImager Z2 (Zeiss) equipped with DIC and fluorescence optics, and analyzed via ImageJ. Arrest phenotypes were scored as follows: cell mass\u00a0= embryo fails to initiate bean stage; morphogenesis\u00a0= embryo begins to elongate but fails to reach 2-fold stage; elongation\u00a0= embryo completes 2-fold stage but arrests before hatching. Body measurements are based on DIC micrographs. Body length was measured as the length of a segmented path running through the mid body axis from head to tail. Body width was assessed as the arithmetic mean of 3 independent width measurements in the anterior, middle, and posterior part of each animal. Pharynx length was determined by the length of a segmented path running through the pharynx middle axis in animals expressing To profile miRNA levels, a modified version of the small RNA sequencing protocol described in  was perf18-30nt-NNNNNNXXXXXAGATCGGAAGAGCAC-ACGTCT/3ddC/-3\u2032. After sufficient PCR-amplification as observed by SYBR green derived qPCR signal (requiring around 16-20 cycles), libraries were size-selected on an agarose gel to remove adaptor dimers and sequenced on a HiSeqV4 platform (Illumina). Small RNA reads were mapped and processed as in [https://github.com/lengfei5/smallRNA_nf/tree/master/dev_sRBC. To accurately quantify sRNAs, both mapped sequences and associated random nucleotides (functioning as UMIs) within the raw reads were quantified for miRNAs and piRNAs as well as spike-ins. To alleviate PCR amplification bias, UMI counts were used and normalized to spike-in as described in [https://github.com/lengfei5/smallRNA_analysis_philipp/tree/master/scripts. Every round of library preparation included one sample of a comparable amount of Arabidopsis thaliana total RNA to assess the extent of potential contamination. The average counts for reads mapping to C.\u00a0elegans miRNAs in contamination control samples (\u223c1% of the wild-type miRNA content) across four independent library preparations were subtracted from all samples as a background correction. For background-corrected, spike-in normalized, miRNA counts in molecules per embryo, see (XXXXX\u00a0= barcode): 5\u2032-ACACUCUUUCCCUACACGACGCUCUUCCGAUCUNNNN-(sRNA)ed as in . Modificed as in . The modribed in , allowinPlots were created using Prism, statistical analysis was also performed in Prism as described in the figure legends. For sRNaseq data statistical analysis was performed using DESeq2 . Figures"}
+{"text": "Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-\u03b3) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. Chronic hepatitis B virus (CHB) infection affects roughly 250 million people worldwide  and is aImmunotherapy had already emerged in the 1990s as a promising option to treat CHB. T cell responses are considered essential for viral clearance but are scarce or exhausted in CHB patients \u20137. Still\u20138\u2013in vivo. Others have explored strategies to predict HLA-I epitopes from Pol but focused exclusively on a single HLA type or assessed only a limited number of HBV sequences  or core antigen (HBcAg). However, the proteins X (HBx) and polymerase (Pol) also pose interesting targets, as both are vital for viral persistence \u201327 and i\u201330\u2013equences \u201340. TakeIn addition to a limited epitope repertoire, there is another hurdle in the development of HBV-directed immunotherapy. Established epitopes might not all be equally effective, as they can be subjected to different levels of viral mutagenesis and subsequent immune escape. Indeed, previous reports clearly demonstrate that HBV is subject to immune pressure and that mutation of epitope sequences leads to immune evasion \u201343 or ev\u201346\u2013Here, we have taken an effort to tackle the above-mentioned issues by integrating viral indispensability, genomic variation, HLA binding, and immunogenicity to identify the best HBx- and Pol-derived T cell epitopes for immunotherapy across 6 of the most prevalent HLA supertypes within the HBV-infected population. The results of this study pave the way for the development of globally effective HBV antigen-specific immunotherapies.n\u2009=\u200914) and Pol (n\u2009=\u200950)  , left, w , left, (n\u2009=\u200950)  Fig. 1,,n\u2009=\u200914 a\u2009=\u200950) 4648, 52\u201369We argued that epitopes in which the least conserved amino acid was still present in 80% of all sequences tested would be targetable in the majority of the population. Combining this criterion with the preference for functional association, we found 7 HBx-derived and 26 Pol-derived sequences reported as epitopes across HLA types to be preferential targets for global immunotherapy . We first predicted binders spanning 8 to 14 amino acids for supertype-representative HLA types using the established in silico prediction tool NetMHCpan to make a frequency distribution of predicted binders (P\u2009=\u20090.57 by a Mann-Whitney test). Predicted binders spanning 9 to 11 amino acids were subsequently aligned to our maps outlining conservation and function .The prediction yielded totals of 251 potential novel HLA binders for HBx and 1,655 for Pol , right, ed assay . For praed assay . For HLAin vitro binding assay. Peptides were classified as HLA binders when their binding capacity was higher than 25% of that of a known high-affinity peptide (Table S4). HLA-A*11:01 and HLA-A*03:01 were both tested as members of the HLA-A*03 supertype since many HBV-infected patients are Asian and HLA-A*11:01 is more prevalent in this population than the supertype representative HLA-A*03:01, which is more prevalent in Caucasians  from blood donors who had previously resolved an HBV infection were expanded in the presence of peptide pools, followed by single-peptide restimulation and an interferon gamma (IFN-\u03b3) enzyme-linked immunosorbent assay (ELISA). As expected, IFN-\u03b3 production was detected in response to the well-established epitopes c18-27 and p549-557 . FurtherThe aim of this study was to rationally address two major hurdles in developing a generic antigen-based immunotherapy for HBV: (i) the lack of prioritization of epitopes in further studies toward clinical implementation and (ii) the shortage of non-HLA*02-restricted epitopes.To address the first issue, we ranked all currently described HBx- and Pol-derived epitopes according to conservation and association with viral indispensability. Conservation patterns were similar to those previously reported, with the most conspicuous observation being that the spacer domain of Pol is extremely variable , 42, 73.Four Pol-derived epitopes were more frequently described in acute or resolved infection than in chronic infection. Of these, p756-764 and p573-581 scored the highest in conservation. p756-764 contains at least 1 amino acid important for viral persistence, but no functional relevance has been described for p573-581. Since the DNA sequence coding for p573-581 completely overlaps that of HBsAg, we interrogated the literature for the functional relevance of amino acids in this overlapping part of HBsAg. However, no functional association was reported 7375. Altho76\u2013Although we rationalized to prioritize conserved peptide sequences, we reckon that regions containing prevalent sequence variation could still be interesting if immunogenicity is preserved. Novel T cell responses may arise due to cross-reaction between the variant and the original sequence , 81. Thi+ epitopes, while HBx seemed more subject to immune-pressure-induced deletion of epitopes  vaccines. SLPs can be designed to harbor both HLA-I and HLA-II epitopes and are processed more efficiently by dendritic cells than whole proteins types covering the vast majority of the HBV-infected population that target the virus where it is most vulnerable. Collectively, the results of this study provide a valuable resource to guide future development of HBV-specific immunotherapies.n\u2009=\u20098,127) and Pol . Positions where a gap (indicated by \u201c\u2212\u201d) was most frequent were deleted, after which the dominating amino acid at each position was determined. Percentages of sequences containing the dominant amino acid were calculated as the conservation score. Combining all dominant amino acids for Pol led to the consensus sequence MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPDIHLQEDIINRCQQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLVFQTSKRHGDESFCSQSSGILSRSPVGPCIQSQLKQSRLGLQPQQGSLARRQQGRSGSIRARVHPTTRRSFGVEPSGSGHIDNSASSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAVELHNIPPSSARSQSEGPVFSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCTEHGEHHIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNSRIINNQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLYTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPDRVHFASPLHVAWRPP.A frequency table was downloaded from HBVdb V42.0 , 51 baseThe resulting consensus sequence for HBx was determined to be MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLPSPSPSAVPADHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKLVCSPAPCNFFTSA.in vitro binding assay as described below.These sequences were loaded into NetMHCpan3.0  to prediin vitro binding assay as described previously (Synthetic peptides (Peptide 2.0 Inc.) of selected potential HLA binders were used in an eviously . In briein vitro HLA binding assay were assessed for immunogenicity. Briefly, PBMCs were isolated by Ficoll  density centrifugation from buffy coats of 9 donors who had previously resolved HBV infection. Buffy coats were provided by the local blood bank with corresponding 2-digit HLA types. Four-digit HLA typing was performed for 7 out of 9 donors using the global screening array (GSA)  (Peptides with >25% binding in the tterdam) . All donP value was <0.05. For the few epitopes with a response in at least 25% of HCC patients, patient groups could not be compared due to the lack of data for CHB and acute hepatitis patients.The Hepitopes initiative previously performed an extensive literature search of the Medline and Embase databases to collect all HBV-derived HLA-I epitopes from 112 papers , 49. All55\u2013"}
+{"text": "Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused \u03b1-bungarotoxin, a neurotoxin derived from the snake  During synaptic transmission, the synaptic vesicle fuses to the presynaptic membrane and its content is released into the synaptic cleft. During this event, vesicular pH rises and falls again after endocytosis1, which is consistent with proton release into the synaptic cleft. Taking into account the very small volume of a synaptic vesicle (2\u2009\u00d7\u200910 \u221220 L) and the estimated pH before exocytosis (around 5.5), it has been calculated that there is less than one free proton per vesicle3. However, upon vesicle opening a process which includes deprotonation of proteins and neurotransmitters takes place and over 50 protons are released per vesicle3. Therefore, the synaptic cleft, which is a space 50\u00a0nM wide, is exposed to changes in pH due to the release of vesicle content and also to transmembrane fluxes owing to Ca2+/H+ exchange by the plasma membrane Ca2+ ATPase4. A transient drop in extracellular pH has been reported during synaptic activity in hippocampal sections5. It has been proposed that in photoreceptor terminals the liberation of vesicular protons results in an acidification of the synaptic cleft from 7.5 to 6.9, depending on the feedback inhibition of Ca2+ channels6. This study also showed that pH oscillations depend on the concentration of protons in the extracellular solution and that acidification is insignificant in acutely dissociated terminals, demonstrating that an intact synaptic cleft is essential.The content of synaptic vesicles has been shown to be acid1, a pH-sensitive green fluorescent protein, was fused to the extracellular domain of a postsynaptic membrane protein and used in lateral amygdala pyramidal neurons to report changes in extracellular pH at dendrite synapses7. After stimulation, the transfected pyramidal neurons transiently acidified neighboring dendrites and spines, a phenomenon which was followed by a slower increase in pH. The extent of pH fluctuations depended on stimulus frequency although quantification of the changes in proton concentration was not reported. In contrast to the established dogma, Stawarski et al. recently used genetically encoded fluorescent pH indicators to demonstrate that glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission at both the mouse calyx of Held and Drosophila neuromuscular junction (NMJ)8. Therefore, the exact changes in pH during synaptic transmission are not fully resolved.Recently, Super Ecliptic pHluorin (SEP)Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to SEP1 and efficiently expressed this fusion protein in Pichia pastoris. Using this tool, we were able to detect a drop in pH in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation.Changes in pH at the synaptic cleft at the mammalian NMJ have not been documented. With this aim, we developed a probe by fusing \u03b1 bungarotoxin (BTX), a neurotoxin derived from the snake P. pastoris and purified, resulting in an active protein of the expected size for de fusion ) was fitted to the data, where y0 represents the offset, ymax the dynamic range, and pK the pH where fluorescence is half maximal, a value that corresponds to minus the logarithm of the equilibrium constant for protonation9. The pK from the fit was 7.36.The sensitivity to pH of the SEP-BTX fusion protein probe was investigated by measuring the fluorescence intensity of the NMJ incubated at different pHs in a high capacity buffer (HEPES buffer 10\u00a0mM). A fluorescence vs pH curve was constructed taken the value at pH 9 as the maximal fluorescent output Fig.\u00a0A,B and t+ channel blocker diaminopiridine , known to increase the amplitude and prolong the time course of transmitter release10. As a result of DAP addition, fluorescence changes were significatively stronger  on neuromuscular transmission17. Activation of ASICs requires changes in pH of 0.5 units or more, a value not reached in our measurements. One possibility is that the sensor also binds to other acetylcholine receptors located outside the synaptic cleft. Since these regions would not be exposed to the release of synaptic vesicle content, this event could mask the changes in pH reported by our SEP-BTX sensor in the synaptic cleft. However, it has been established that extra synaptic acetylcholine receptors are not expressed in normal adult mouse muscle both, by iontophoretic application of acetylcholine19 and by using labelled bungarotoxin20. Moreover, we focused the detection of fluorescent changes at the synaptic spot where only synaptic acetylcholine receptors are located. Secondly, it is worth mentioning that our measurements are highly dependent on the distance of the probe in relation with the source of the protons, which in our case is the synaptic cleft width. The high mobility of protons21 and the presence of buffers in the media could result in a very fast transient non accumulative increase in proton concentration not resolved with our detection system. Furthermore, the rapid dissipating proton concentration during the intervals between evoked transmitter release (10\u00a0ms at 100\u00a0Hz and 3.3\u00a0ms at 300\u00a0Hz) will strongly down average the fluorescent signal.Our experimental results using SEP-BTX have shown that acidification takes place at the NMJ synaptic cleft, confirming previous reports in other synapses23, we were able to show for the first time an acidification process due to transmitter release at the mouse NMJ. Synaptic cleft acidification and alkalization are probably not mutually exclusive processes. While they could operate in different types of synapses, it is also feasible that they occur at the same synapsis but with different kinetics. For example, following a rapid drop in pH, an over compensating mechanism driven by the Ca2+/H+ antiporter activity of plasma membrane Ca2+ ATPase4 and aimed at maintaining proton homeostasis could result in transient alkalinization8. Further experimental development of sensors located at the presynaptic membrane close to the vesicle release site and a fast image recording system are needed to obtain more realistic measurements of changes in the pH at the synaptic cleft.In summary, using a novel approach, in contrast to other reports showing an alkalization at CNS synapses9 in frame with BTX and followed by a TEV protease site and a 6XHis tag was subjected to codon optimization for expression in P. pastoris and synthetized by Integrated DNA Technologies (IDT). The sequence of the cloned DNA fragment, including EcoRI sites, is as follows:The DNA fragment containing SEPEcoRI site of pPic9 (Invitrogen Life Technologies Inc.) in frame with Saccharomyces cerevisiae \u03b1-factor signal sequence.5\u2032GAATTCTCTAAGGGTGAAGAGCTTTTCACCGGCGTGGTTCCTATTCTTGTGGAATTGGATGGTGACGTCAATGGTCACAAATTTTCAGTGTCAGGTGAAGGGGAGGGTGACGCCACTTACGGTAAATTGACTTTGAAGTTTATATGCACTACCGGCAAATTGCCAGTTCCATGGCCTACCTTGGTTACTACCTTAACTTACGGTGTTCAATGCTTTTCTAGATATCCTGACCATATGAAGAGACATGATTTTTTCAAGTCAGCCATGCCAGAGGGATATGTACAAGAACGTACAATTTTTTTTAAGGATGACGGTAATTACAAGACTCGTGCAGAAGTTAAGTTCGAAGGCGACACGCTGGTCAATCGTATTGAACTGAAGGGTATTGACTTCAAGGAGGATGGGAACATACTAGGACACAAATTAGAATATAATTATAACGATCACCAGGTTTACATTATGGCTGACAAACAAAAGAACGGCATTAAAGCTAACTTTAAAATTCGTCATAATATTGAGGATGGGGGAGTCCAATTAGCAGATCATTACCAACAGAATACACCAATAGGAGATGGTCCAGTGCTGTTGCCTGATAATCACTATCTTTTCACTACTTCTACTTTATCTAAAGACCCAAACGAGAAGAGGGACCATATGGTTTTGCTGGAATTTGTGACTGCAGCCGGGATTACTCACGGTATGGACGAGCTTTATAAAGGGCACGTCGGGATAGTTTGCCATACGACAGCCACCTCCCCCATTTCTGCTGTTACCTGTCCTCCAGGCGAAAACCTGTGCTACAGAAAAATGTGGTGTGATGCCTTCTGTTCTTCACGTGGTAAAGTTGTCGAGCTGGGATGCGCTGCCACCTGTCCTAGTAAGAAACCTTATGAAGAAGTAACATGTTGCTCTACAGATAAATGTAATCCACATCCTAAACAACGTCCTGGTTCGCGAGAGAACCTGTACTTTCAAGGACCCGGGCATCATCATCACCATCATTAAATTTAAATGAATTC3\u2032. This fragment was cloned into the P. pastoris was previously described24. Basically, pPic9-SEP-BTX-TEV-6XHis vector was linearized with DraI and used for transformation of P. pastoris strain GS115 (Invitrogen Life Technologies) by electroporation, and recombinant clones reverting histidine auxotrophy were selected on minimal MD plates 2SO4, 2% dextrose and 2% agar). Selection of clones expressing and secreting SEP-BTX was performed by transferring colonies to a nitrocellulose membrane placed on plates of MM minimal medium 2SO4, and 2% agar) with methanol as sole carbon source, for induction of the AOX1 promoter . After 4\u00a0days of growth at 30\u00a0\u00b0C, the nitrocellulose membranes were washed with distilled water and the secreted protein was revealed by western blot using a polyclonal rabbit anti-HIS antibody . For SEP-BTX production, recombinant clones were grown in 50\u00a0mL of BMGY medium 2SO4, 400\u00a0mg/L biotin, 1% glycerol, 100\u00a0mM potassium phosphate buffer, pH 6.0) for 48\u00a0h at 30\u00a0\u00b0C and 220\u00a0rpm. Cells were harvested by centrifugation for 5\u00a0min at 1500\u00a0g and resuspended in BMMY medium 2SO4, 400\u00a0mg/L biotin, 200\u00a0\u00b5M CuSO4 and 3% sorbitol) to a final OD600 nm\u2009=\u200910 and cultivated in 1000\u00a0mL shake flasks at 28\u00a0\u00b0C and 220\u00a0rpm. Sterile methanol  was added every 24\u00a0h to maintain induction conditions.The protocol for protein expression in P. pastoris was previously described24. Briefly, P. pastoris cultures were harvested after 4\u00a0days of induction in BMMY and centrifuged at 1500\u00a0g for 10\u00a0min. The extracellular supernatant was concentrated by ultrafiltration  and buffer exchanged to 300\u00a0mM NaCl, 50\u00a0mM sodium phosphate buffer, pH 8, containing 1\u00a0mM PMSF. SEP-BTX was purified by gravity flow Ni\u2013NTA affinity chromatography using a \u201cHis select\u201d nickel affinity gel , eluted with 300\u00a0mM NaCl, 50\u00a0mM sodium phosphate buffer, pH 6.5, 250\u00a0mM imidazole, 1\u00a0mM PMSF. Fractions containing SEP-BTX were pooled and buffer exchanged to 1\u00d7 PBS by ultrafiltration . Recombinant SEP-BTX was revealed by western blot. Culture supernatants and purification fractions were separated by reducing 12% SDS-PAGE and transferred to 0.45\u00a0\u03bcm nitrocellulose membranes . Western blot was performed by probing the membranes with 0.1\u00a0\u03bcg/mL of polyclonal rabbit anti-HIS antibody  or a 1:1000 dilution of mouse anti-GFP antibody (Abcam) followed by 1:15,000 dilution of alkaline phosphatase-linked goat anti-rabbit antibody or goat anti-mouse antibody . Phosphatase activity was revealed by a chromogenic reaction using 5 bromo-4 chloro-3 indolyl phosphate and nitroblue tetrazolium as substrates .The protocol for protein purification from Levator auris longus (LAL) muscle of male C57BL/6\u00a0J mice (https://www.jax.org/strain/000664) as previously described25. Briefly, animals were supplied by the animal house of the FCEN-UBA. Animals were cared for in accordance with national guidelines for the human treatment of laboratory animals, similar to those of the US National Institutes of Health. All experimental protocols were approved by the Institutional Commission for the Care and Use of Laboratory Animals (CICUAL), FCEN-UBA. Animals were anesthetized with an overdose of 2% tribromoethanol (0.15\u00a0mL/10\u00a0g body weight) injected in the peritoneal cavity and exsanguinated immediately.Experiments were carried out on the left 2, 1 MgSO4, 12 NaHCO3, 1 Na2HPO4 and 11 glucose; continuously bubbled with 95% O2/5% CO2; pH 7.3 as previously described25.The muscle with its nerve supply was excised and dissected on a Sylgard-coated Petri dish containing physiological saline solution of the following composition (in mM): 137 NaCl, 5 KCl, 2 CaClLAL muscles were incubated in Ringer\u00b4s saline solution with the SEP-BTX sensor for 2\u00a0h and washed for 30\u00a0min. For each batch of SEP-BTX produced, containing 30\u201350\u00a0ng/\u00b5L of purified recombinant protein, preliminary experiments were done to identify the dilution (1/5 to 1/20) necessary to obtain a low background strong labelled NMJ. The preparation was then transferred to a 1.5\u00a0mL recording chamber. Experiments were performed at room temperature (20\u201323\u00baC). NMJs were visualized with Nomarsky interference optics and illuminated with a 488\u00a0nM light source .\u20136\u00a0g/mL  was used during the experiment.The nerve was stimulated via two platinum electrodes isolated with vacuum grease  coupled to a pulse generator . Supra threshold high frequency  nerve stimulation was applied during 250\u00a0ms. To avoid using high concentrations of the reporter and to prevent any remaining muscle contraction during stimulation, curare up to 5\u2009\u00d7\u200910For fluorescent image acquisition, we used a BX51WI upright microscope with a 60X objective lens (Olympus) and an electron multiplying CCD camera , together with cell^M System Coordinator/cell^R real time controller software.The only exception is the image shown in Fig.\u00a0t-test.Data analysis was done using Clampfit 10.6 , Sigma Plot 10.0, SigmaStat 3.5 and Excel 2007 (Microsoft). Average data are expressed and plotted as mean\u2009\u00b1\u2009sem. Statistical significance was determined using paired or unpaired Student\u2019s"}
+{"text": "FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 invitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.Mutations in the transcription factor  The lymphatic vasculature has a critical role in maintaining tissue homeostasis by returning interstitial fluid to the venous circulation, absorbing lipids from the digestive tract, and providing a network for immune surveillance and response . MutatioFOXC1 have primarily been dominantly associated with eye anterior segment defects, cerebellar malformation, and cerebral small vessel disease. In contrast, mutations in FOXC2 have been dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes (FOXC2 mutations (FOXC1 and FOXC2 are closely related members of the forkhead box (FOX) transcription factor family with nearly identical DNA binding domains, similar expression patterns in mesenchymal tissues during development, and essential roles in cardiovascular developmental processes . Mutatioyelashes . Work fryelashes . Additioyelashes . Furtherutations . HoweverFoxc1 postnatally impairs valve maturation, while Foxc2 deletion impairs maturation and induces valve degeneration, as previously described  in vitro, which also improves LEC barrier integrity in vivo, while valve degeneration is partially rescued in only Foxc2 mutants. Finally, via generation of transgenic mice that express Foxc2 within the Foxc1 locus, we show Foxc2 is capable of functionally substituting for Foxc1 in lymphatic development and maturation. Together, our data show a complementary role for FOXC1 in addition to FOXC2 as key mediators of mechanotransduction in the postnatal lymphatic valves and implicate new mechanistic targets for therapeutics in the treatment of lymphatic-associated diseases.Here, we report an essential role for FOXC1 during lymphatic valve maturation and maintenance. Detailed comparison of FOXC1 and FOXC2 expression and roles in lymphatic valves suggests some overlap with a broader importance for FOXC2, but more subtle, key contribution for FOXC1. In mice, endothelial cell (EC)-specific deletion of escribed . HoweverOur group previously reported that FOXC1 and FOXC2 expression co-localizes with PROX1 in lymphatic valve-forming cells at E17 and later at P3 . HoweverFoxc1 during murine embryonic development impairs lymphatic valve maturation (flFoxc1 mice (ERT2Chd5-Cre mice (Foxc1 mutant (EC-Foxc1-KO) mice. Tamoxifen was administered from P1-P5 to induce Cre-mediated recombination and we confirmed deletion of Foxc1 via qPCR analysis of isolated CD31-positive cells from hearts of P6 individuals and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  protein ligand fibronectin-EIIIA to regulate the formation of the ECM core of valve leaflets  mice and administered tamoxifen postnatally. PROX1 immunostaining showed no significant differences in total valve number of LEC-Foxc1-KO mice  mice and administered tamoxifen from P1-P5 with inactivation confirmed by qPCR in ECs isolated from P6 hearts and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  mice and administered tamoxifen from P1 to P5 with inactivation in compound mutant individuals verified by qPCR and immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  developed severe chylous ascites  compared to controls   , indicatFOXC1 knockdown in LECs impairs cell adhesion, we slightly modified our protocol: LECs were first seeded on fibronectin-coated surfaces, then transfected with scramble control, FOXC1, FOXC2, or combined FOXC1/FOXC2 (50%:50%) siRNAs and kept under static conditions for 2 days. Given that FOXC2 inactivation and exposure to OSS in LECs results in increased actomyosin contractility  suite of tools (https://ecrbrowser.dcode.org) -phosphorylated myosin light chain (pMLC) signaling pathway has been well established as a regulator of cytoskeletal contractility mechanisms . FurtherDKO mice . Recent DKO mice . Our RNADKO mice  revealed embryos . Further embryos  were notpression . Becausepression  and our of tools  was usedof tools  in activof tools . Then, wode.org)  tool to PRICKLE1 , ARHGAP2ARHGAP23  loci. ToPRICKLE1 , ARHGAP2P23 loci . We founin ECR-1 . Additio ECR-1\u20132 . Finally23 locus .ICAM1 that was not predicted to bind FOX transcription factors by in silico analysis  allele, in which the Foxc1 coding region has been replaced with the cDNA coding (from the start codon to the stop codon) for Foxc2    and FoxCrostomes . Althougnditions . TherefoPecam1 or Sdc4 and LEC-specific deletion of Cdh5 in vivo resulted in impaired valve morphogenesis phenotypes and poor orientation and elongation of LECs with the direction of flow in mesentery collecting vessels  for Foxc2 by using restriction enzyme digestion and PCR cloning. To facilitate the elimination of the selectable marker gene rNeo in mice, we introduced the ACN cassette flanked by two flox sites  mice , c2/+Foxc1 , and c2/c2Foxc1  individuals.SalI-linearized targeting vector (100 \u03bcg) was then electroporated into TL1 ES cells (129S6) as described . The cel/+) mice  were theGenotyping of mice for use in analysis was performed by Transnetyx Inc  using real-time PCR.Dissection, immunostaining, and imaging of the lymphatic vasculature in mesentery tissue from 4 week old C57Bl6 mice for whole-mounts was performed as previously described . BrieflyThe lymphatic collecting vessel vasculature of neonates was analyzed by whole mount immunostaining of mesentery tissue harvested from pups at the indicated time. Briefly, mesentery tissue was dissected from the intestinal tract, laid out in plastic dish and left until it was firmly attached. Following fixation with 2% PFA in PBS, tissues were washed with PBS, then permeabilized and blocked in PBS solution containing 0.5% BSA, 5% serum, 0.3% Triton X-100, and 0.1% Sodium Azide. Tissues permeabilized/incubated with blocking buffer were then incubated with primary antibodies listed in Imaging was performed using a Zeiss AxioVision fluorescence microscope and Zeiss Axiovision software, Zeiss LSM-510 Meta, LSM-800 and LSM 880 confocal microscopes and Zeiss Zen Blue acquisition software, or using a Nikon A1 Confocal Laser Microscope System NIS-Elements software. Images were processed with Imaris and Adobe Photoshop software. Imaris colocalization function was used to produce pictures showing p-MLC2 , vinculiPpia) or 18S was used as an internal standard for mRNA expression. Primer sequences are provided in Hearts from neonatal mice were digested in collagenase Type I solution (2 mg/mL) for 40 min at 37\u00b0C with gentle agitation. Cells were then filtered through a 70 \u03bcm cell strainer and the pellet was resuspended in Buffer 1 . The cell suspension was then incubated with magnetic Dynabeads (Invitrogen) pre-coated with CD31 antibody to isolate the endothelial cell population. After several washes with Buffer 1, RNA was extracted from endothelial cells using RNA STAT solution (Tel-Test) followed by phenol-chloroform treatment. Extracted RNA was subjected to DNAse I treatment and concentration was determined using a NanoDrop machine (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio-Rad) according to the manufacturer\u2019s instructions. Triplicates of cDNA samples for qPCR analysis were performed using a Fast qPCR machine (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene specific primer sets. Peptidylprolyl isomerase A  coated with 40 \u00b5g/ml human fibronectin, cultured for 24 hr and then subjected to LSS (4 dyn/cm2), OSS (4 dyn/cm2 and flow direction change every 4 s) using Ibidi Pump system, or kept in static conditions for an additional 24 hr prior to fixation, immunostaining, and mounting using Ibidi Mounting Medium (ibidi GmbH).Flow experiments with cultured LECs were performed as described previously . Brieflyhttps://genome.ucsc.edu/ENCODE/)(2012). Putative sites in the human genome were then searched against the mm10 mouse genome using the ECR Browser (https://ecrbrowser.dcode.org)\u00a0(Putative FOX-binding sites were determined first by using the Hypergeometric Optimization of Motif EnRichment (HOMER)  suite ofode.org)\u00a0 and rVishttps://www.promocell.com/product/human-dermal-lymphatic-endothelial-cells-hdlec/) were cultured and used according to the manufacturer's protocol. The cells were cross-linked with 1% formaldehyde, followed by sonication. The sheared chromatin was immunoprecipitated with dynabeads  conjugated with anti-FOXC2 antibody , anti-FOXC1 antibodies , or control IgG . DNA extraction and PCR were performed as previously described  from juvenile foreskin  (escribed  with priPRICKLE1 ECR-1.>hg19 chr12:42876176\u201342876523TGTTGGCTCCTGAACAACACTCCTCTTTGTGAAACTTACAGCACCCATTTGAAACAAGTTTCCAGAGAAAACACTTCAAGAAAGTGTTTGAGAAGTCACAAGACTCGAGTTGTAAAAACAAATTCCACACATAGCCTGGCTTATAAGGACACAGACTAACACCACACAAGGCGGTGCTCTAATGAGCCCATTATTTTCCATAATGGGGGATGCAGATATTTTCTCAAAATCGTGTTCTCCTCAGTCTTCTATTGATTTTTTGGATTTCTATTTTCAACAGTGGCCCGAGGAAACGGCAGCCAGACTTGACTCCAATGTACACACAGACTCAGGTTTCGCCCCGTCACCPRICKLE1 ECR-2.>hg19 chr12:42878066\u201342878454GAGCACCTACCGCCGCCCGCCCGCTCCATTCTCCCGAGCCCAGTGAGTGAAGCCGCTAAGATGCAAATACCCTAGGACGCTTATGTAAACTTCCCCCTCCCGCAGGTGCACGCGCGGGCCACGAAACGCTGGGAGAATATGAAAGGCCACCTCTTAAAGAAATCATCTCCACTCTGCCCATAACAATGATGTCAGCAAATGGCACATTTAAGCAAGTTCTCACTTAGAAGGGCTCATTAGCATATGAATTCTCTTAGGACTTTCCCTGCATTTCGGAGTGATTCCTACTGCTTAGCGCAGGAGATTTATTTTTATCAGTAAATAACAGCAAAGAAAAGGAGCCAGGTCACGCGATGTACTAACTCAAGTACACTACTGAGAGTTTTTACPRICKLE1 ECR-3.>hg19 chr12:42879624\u201342879943AGTTACTTGTAAATACGTTTTGTTATATTTTCAACAGGTACTGTCATGGTTTATTACCCATTGTAAGCGTTTTTAGTGATGAAGCAGTGTGGAGACAGTTGCAAGCTTCCTACAAAGGTTCAATCTCAATGAAACAGACTTTAGTCTGGTCTGAAAGGGGTTGTTATTGTCAAGGGTGAACTTATGCAGAATGGAGAGCAAGGCCCCCAACCAGCATCCTTTTGTTTCAGCCAGGTGGAATATTCATGCTTGCAGATCACACTTTAGGGGCCAGTTGAGAAAGGAAGCCCAAAAATTCACAGGGCTTGGTTCCCTCGCTCPRICKLE1 ECR-4 and ECR-5.>hg19 chr12:42981201\u201342982292TACATTAGCCATGACTTATTAAACTTGGAGATTTTCAAGTTCATCAGCAAGTGTGCAAACCCTTAACTGTGGGTAACATCCATTTATTTGTAGCACCTTTCGGTTTTAATATGTAGAGCACATGCATTGTTAACCTCTAAATCCTTTGT(ATAAACA)TTTCTGGAAGAGCTGGTAAAATATCTCCTTCTGTGTTTCCTGACTCGCCAGTTGATGGCATTTAGAAACCCTCTGGTACCAGCAGGTGCTGTATTTTGCTTTCTTCAGGCTCCAGCTGGGCTACAATGACAGATTCCTGTCCCAGGCCAAGCCTAGCCACCAAGGCTAGGACCACATTGGAGGCAAACTGAACCAGGCTCCACCAGACAGCAAGATGAATGGCTGCTGTTTAAGTTTAAAATCCCCTGGTGGGA(GTAAATATTGTTCCAGAGAAAAGCCTTGACAAATA)CTGCGTCATCCTTACAGAACTGTCTTGATTAAAGCAGAATCTTTGGATTAAGTTGATGCTCAATTCAAAATGTATCTATCTTGCTGTCATGGGATTTTTTTTTGTTTTTTTTTTCCTTTCTAGAGTCTGAAAACAGACATAATGTTGGGGACGGTCAAACAAGGCTGCCGGCTCCCAAGGGGCTAGAGTCCACTCCTGATAATAGAAGGCGGCTGAACACTGACACTTCACTGAGGATAATGGAGACAGCAAAGGCTTAGTGGGAAAGGGCCAGTTGTCACCTAAGTGACAGGCAACAGCTGAGCTCACACATCTGGAGCCGGACTACGGCAAACATTAGCAACCCTCACCAGTCTACACCTTGGGCCTGTCTGAAAAGACAGATGGAAGTTCCCTCTACTCCTAAAGTACATTAAAAAATGTCTGATGGTGAACCACATCAATTATATAACATCAACTGCAGGCACAGCCTTCCAAAGTACTGATTAAGACGAGGCAGTAGACAACACTGTATGCATGAACAGATACAAGATACCATTTCAGTGATTTGTCATTCATAAAACTTATCCTAAAAGACACATATACATGCATCCATTTGATAGCACAAATGCATGTTAACTCTGCAGGAGAGGCAGATTTTTACATGTPRICKLE1 ECR-6.>hg19 chr12:42982328\u201342982743CCTTTAGGCTGGTTCCCGTGGTGTGTTTGCCTTGTTCATGGTCTCAGTTCTGCCGCTGATACCCTTTTAAAAATCAGCAACCAAACGCGTTCGGCTTGTGATCCTGAACCCCCTTAGGCAAGCTGGAACTAAGCGTGATGCAGCCGTCCTCCCTCTCTCCCAACCCCCAACCTCGTTCTTCAGCCTCCTGAAGACAATCTGTGAACAATTTTCCCAAAGTCCCAAGAATAACACAGCACTGCCAATAGTCACTGGCGATGCCGTTTGTTTTTCTTAGAGGGTAATGAAAATTTAACAGCTTTCTGCTGCATCCTGAGTCCCGCTCCTAATAACTATTAACATGCCTAGTTTCTTCAACTTTTTCTACCTCAAGAGAGGAAGACGCTCCCATTTTTTCCCTATATCTGTGCTACATARHGAP21 ECR-1 and ECR-2.>hg19 chr10:25009076\u201325009514CTGTCATTGTTAATAAAAGCCAAGTGTGCAACAAACTGGAAATACTGCTTGCTAGCAAGGACAAGATGTGTCTAAATTCTTGGTTTCAGGACATCTCTTAAATGACCAAAAAAAAAAAAAAAAATCAAATAACATTAGTTCTT(GCAAACAGAATGCAAATACAATGCTAATTAAAGTATTCACAAGACAATGACAAATA)AGGCTTCAGGACCACAATACATATTATTATGTAACTGCAATACACATTAAGCAATCACCAGGTTGCAGGTAGGCCTTCCAAAAGGAGTTATTATGGTTTACCGTGATCAGAGGATTGTGGTGTTCCACTTAATCATGCTTTTGCCTGCAAGCAGGTGTTTACAGATGTCAAAAAGTAAAACACTGATTCTAAATGTAAACCTACAGTTCTGCCTAATAAATTGTACAGTAATAGCACACARHGAP21 ECR-3.>hg19 chr10:25017295\u201325017667CTATATTAATAAAAACTACAAGAAAGCTTTATACACTAAATCTAGGCAAGACATTTATGAAGATGAGAACTGTATCCTTAAAAGGTAAGTGTTGGCTTTGCTAATGACATAATATTGTTTTGGTGAACCAATATCAAGGGAAAAAATGTCAAAGCCAAAAATAGAGGCAAAGTATCCCAGCCCCTGGTGTGGAAGGCCATTCTATGATAATCTATGAATGATTTCTACTCTGAATATGTTAACAGAAGCTGGCACATCTGAGAAGCACAAGTGTTTGCTAGTGAATCCACAAATGAAATTTGCAATTTGGGTTGAAGCCTTTGCAAAACTACGTTAAGACCAATAGCCCTCAGAAGAGTAAGGGGTTTTGTTTARHGAP23 ECR-1.>hg19 chr17:36585363\u201336585767TTCTAGACACAGGCCCAGGACCCCGGGCTCTGCCGGCGAGGCTGCCCTCCCCTCTGCCCTCTCCGACCGGCTGTGGGTGGGTCAGAGCGCGGGGTGCCAGGGGCATTACTCAGCGCTGGGCTGCTCTGCCTGGGTTCTTTCATCTGCCAGCTGCTGAGGCTGGGGAGGGGCCAGCAGGGGCCTCCCAGCCCCATCCCCCCATCAGGGCCATTCCCTTACCTCTGAGCCTGGCTGCCCGCCCTGCAGGAGCCCCCCAGCAGGCCTCCCTGCTCCTAAGTTGAAGGGTTGAACACTGTCAGGCCAACAGTTTCCCTGAGCTCGGAAAAGAAATTCCCCGGGGTCCAGGTTGAGGTCAAGGCCAGGGCTGAGGCCTGTTCCTCTTTAGACAGGGCTGAAAGACTTGGGTo identify lymphatic valves, mesentery tissue was stained with PROX1 antibody and areas of high expression were quantified as mature (visible leaflets) or immature (no visible leaflets) on four lymphatic collecting vessels per individual. Total number of valves were determined and percent of mature valves was normalized to the total counted per individual. For assessment of apoptosis in lymphatic collecting vessels, mesentery tissue was immunostained with PROX1 and active caspase-3 antibody and the percentage of PROX1/caspase 3-positive LECs was quantified from 20X high-power fields generated from confocal z-stacks using Fiji software to determine the total number of LECs per field using thresholded PROX1 immunostaining. Quantification was completed from three biological replicates.in vitro quantifications, relative nuclear intensity levels were measured using Fiji software. Nuclei were considered as regions of interest from thresholded Hoechst staining pictures. FOXC1 or FOXC2 intensity was\u00a0then measured in each nuclei using RawIntDen function. For quantification of F-actin and p-MLC2 area per cell, LECs were fist manually segmented and defined as individual regions of interest, then F-actin (or p-MLC2) staining was thresholded using similar parameters for all pictures and F-actin+ (or p-MLC2+) area was measured per each region of interest. Quantification was completed from three independent experiments.For in vivo experiments was performed using GraphPad Prism v5 or v8. P values were obtained by performing a 2-tailed Student\u2019s t test or one-way ANOVA and Tukey\u2019s test. Data are presented as mean\u00a0\u00b1\u00a0standard deviation of representative experiments from at least three biological replicates. For quantification of apoptosis in LECs of compound Foxc1; Foxc2 mutant mice, the ROUT method with Q set to 1% was used to identify outliers in the data set that were then excluded from statistical analysis using student\u2019s t-test. P values less than 0.05 were considered statistically significant. Statistical analysis for in vitro experiments was performed using GraphPad Prism v8 to perform mixed-effects analysis and calculation of correlation coefficients. P values less than 0.05 were considered statistically significant for mixed-effects analysis.Statistical analysis for All procedures and animal studies were approved by Northwestern University\u2019s IACUC or by the Animal Ethics Committee of Vaud, Switzerland. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study provides new insight into the mechanically regulated activation of, and distinct roles for, FOXC1 and FOXC2 in lymphatic vessel valve development. The identification of unique roles for these closely related transcription factors is intriguing and will pave the way to a deeper understanding of the transcriptional mechanisms regulating their distinct activities. The study also implicates FOXC1 as a gene that may potentially underlie primary lymphoedema.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Natasha L Harvey as the Reviewing Editor and Reviewer #2, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mark Kahn (Reviewer #3).Thank you for submitting your article \"Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Here, Norden, Sabine and colleagues dissect the roles of transcription factors FOXC1 and FOXC2 in lymphatic vessel valve development, revealing that FOXC1 primarily controls focal adhesions in lymphatic endothelial cells, while FOXC2 primarily controls adherens junction integrity. The work builds on previous studies by the authors that have demonstrated (1) the importance of FOXC2 and shear stress for lymphatic vessel valve development and , (2) important roles for both FOXC1 and FOXC2 in embryonic lymphangiogenesis via regulation of RAS/ERK signalling . Aspects of this current study overlap with some of the authors' prior work and this should be clearly articulated in the manuscript where relevant. Distinctions and extensions of the work should also be clearly articulated. There are key novel and interesting findings described in this manuscript that address important questions regarding the distinct mechanisms by which FOXC1 and FOXC2 activity are regulated, together with the mechanisms via which these transcription factors regulate cytoskeletal architecture.Essential revisions:Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90\u00b0) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as \u03b15-laminin and/or \u03b19-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3\u2014figure supplement 1?1) The phenotype caused by early postnatal deletion of Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: \"Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration.\" The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1;Foxc2 double mutants. Figure 3\u2014figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3\u2014figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3\u2014figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example \u2013 Figure 3\u2014figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3\u2014figure supplement 3H show low PROX1 levels overall in Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If 5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?6) Figure 1\u2014figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.7) Figure 3\u2014figure supplement 2: A lower power image depicting the broader profile of \u03b19-integrin expression throughout multiple valves would enable the reader to determine how \u03b19-integrin deposition is affected more globally in EC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.Foxc1c2/c2 mice. What is the remaining signal in FOXC1 stained c2/c2Foxc1 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in  Essential revisions:1) The phenotype caused by early postnatal deletion of Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90 degrees) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as \u03b15-laminin and/or \u03b19-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3\u2014figure supplement 1?Foxc1, Foxc2, and compound Foxc1; Foxc2 mutant mice. To address the first part of this comment, we have included new representative images of \u03b19-integrin/VE-Cadherin immunostaining of lymphatic valve regions side-by-side with PROX1/CD31 immunostained lymphatic valves in Figure 2. Inset images of only the \u03b19-integrin channel more clearly show the presence of mature, intraluminal bi-leaflet valve structures in Control and EC-Foxc1-KO mice as well as valve regions that are considered immature that are not characterized by a distinct intraluminal leaflet structure by \u03b19-integrin immunostaining. In EC-Foxc2-KO mice, we have observed that overall, \u03b19-integrin expression is reduced compared to littermate control mice. However, there are regions characterized by the presence of intraluminal leaflets, as depicted by PROX1, CD31, and VE-Cadherin immunostaining, that are shortened in length. We describe these observations in the subsection \u201cFOXC1 and FOXC2 are required for postnatal lymphatic valve maturation and maintenance\u201d.The authors thank the reviewers for this comment and careful assessment of our described phenotypes in our Foxc2-KO mice and EC-Foxc1; Foxc2-DKO mice. Figure 2H and N depict representative images of a mesentery collecting vessel immunostained with PROX1/CD31 and Figure 2R and T depict 10X power images of collecting vessels immunostained with PROX1 to show the difference in phenotypes more clearly.To address the second part of this comment, we have also updated Figure 2 to include representative images showing side-by-side comparison of EC-Foxc1-KO, EC-Foxc2-KO, EC-Foxc1; Foxc2-DKO mice and respective littermate controls and included representative images in Figure 2\u2014figure supplement 1. In Figure 2\u2014figure supplement 1G, we show that FOXC2 expression is markedly reduced in EC-Foxc2-KO mice and FOXC1 expression is reduced compared to littermate controls, likely as a result of perturbed flow contributing to the reduced induction of FOXC1 expression. In contrast, Figure 2\u2014figure supplement 1E shows strong reduction of FOXC1 expression but no discernible difference in FOXC2 expression. Thus, the maintenance of FOXC2 expression in EC-Foxc1-KO mice may in part explain the differences in phenotype severity observed in Foxc2 mutant mice compared to Foxc1 mutant mice. We have revised our Discussion section (second paragraph) to note this important difference. The difference between our qPCR data, depicted in Figure 2\u2014figure supplement 1B, and our immunostaining evidence is likely attributable to the fact that CD31-positive cells were isolated from cardiac tissue to use for qPCR analysis. It is likely that FOXC2 expression levels are different between lymphatic endothelium in mesentery collecting vessels and the cardiac vasculature. Thus, this may explain the differences in deletion efficiency. The repeated immunostaining of collecting vessels with FOXC1, FOXC2, and VEGFR-3 antibodies also now shows clear expression of FOXC1 in the nucleus of both lymphatic endothelial cells and smooth muscle cells of arterioles. As shown in Figure 2\u2014figure supplement 1E, G, and I, there is no membrane signal detected. We suspect that there may be some cross-reactivity with our secondary antibodies used for the immunostaining combination of FOXC1 and VE-Cadherin, thus warranting our use of VEGFR-3 antibody to address this issue and verify the reduction of FOXC1 expression in the endothelium of collecting vessels.To address the third part of this comment, we performed immunostaining of FOXC1, FOXC2, and VEGFR-3 in mesentery collecting vessels of EC-2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: \"Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration.\" The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels\u2019 as the previous statement was not accurate. Our group previously reported that valves of LEC-Foxc2-KO mutant mice were characterized by increased apoptosis and apoptotic cells were often arranged in doublets with symmetrically organized PROX1-high apoptotic bodies, suggesting that apoptosis may occur in dividing cells. Additional evidence identified that half of the dying cells in LEC-Foxc2-KO mice were PROX1/Ki67-positive, demonstrating that valves of LEC-Foxc2-KO mice were characterized by abnormal activation of cell proliferation and increased cell death. This phenotype was associated with improper activation of TAZ signaling in the absence of FOXC2 . We have included a summary of this finding in the subsection \u201cCell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels\u201d. We then sought to characterize junctional integrity and apoptosis in compound Foxc1; Foxc2 mutant mice to assess if there was a synergistic effect of inactivation of both Foxc1 and Foxc2 in the lymphatic endothelium.We thank the reviewers for this helpful comment and careful critique of our data. We have revised our conclusion to more accurately state, \u2018Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutant littermates that show apoptotic bodies are in closer proximity to branched regions.To further address this reviewer comment, we carefully re-evaluated our caspase-3 immunostaining and repeated analysis to characterize the percent of PROX1/cleaved caspase-3 positive lymphatic endothelial cells in 20X high-power field images. Figure 2\u2014figure supplement 4I-K has been updated to include new representative images and quantitative analysis. In addition to representative images of maximum intensity projections of PROX1/cleaved caspase-3 immunostained collecting vessels, we also show panels of z-sections from selected regions in higher magnification where PROX1/cleaved caspase-3 positive cells were identified. We also now show images from two compound 3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example \u2013 Figure 3\u2014figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3\u2014figure supplement 3H show low PROX1 levels overall in Foxc1;Foxc2 double mutants. Figure 3\u2014figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3\u2014figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3\u2014figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.Foxc1; Foxc2-DKO mice more clearly depict that collecting vessels in these mice are characterized not only by discontinuous junctions, but the increased presence of overlapping junctions as well. We have also generated videos of 3D reconstructions of the collecting vessels depicted in Figure 2\u2014figure supplement 4A-H (Videos 1 \u2013 4), characterizing this observation.We thank the reviewers for this helpful comment and careful critique of our representative images. To address the first part of the reviewer comment, we have repeated immunostaining of tissues and re-evaluated the post-processing of our representative images for several figures to address issues with inconsistencies. This includes new images for PROX1/CD31 immunostaining in Figure 2C, E, I, and K and new images for PROX1/VE-Cadherin immunostaining in Figure 2\u2014figure supplement 4A-H that now show consistent PROX1 expression levels with representative images in Figure 2. Additionally, our new representative images for Figure 2\u2014figure supplement 3E-H and Figure 2\u2014figure supplement 4A-H show consistent VE-Cadherin expression levels among different tissue samples. Furthermore, our new images of VE-Cadherin immunostaining in EC-4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.Foxc2-KO and EC-Foxc1; Foxc2-DKO mutants in Figure 2\u2014figure supplement 4A-H that shows differences in the presence of discontinuous junctions in collecting vessels of EC-Foxc2-KO mice, but both discontinuous and overlapping junctions present in collecting vessels of EC-Foxc1; Foxc2-DKO mice. This new data recapitulates our in vitro evidence depicting the same observations in FOXC2-KD and FOXC1; FOXC2-KD cultured lymphatic endothelial cells as represented in Figure 4\u2014figure supplement 1, thus providing additional support for the relevancy of our in vivo and in vitro observations. In regard to the second part of the reviewer comment, although FOXC1 and FOXC2 are functionally similar, as characterized by their similar DNA-binding capacity, their transcriptional targets are likely to be different under oscillatory or laminar shear stress. For example, our group previously reported that loss of FOXC2 leads to distinct phenotypes in lymphatic endothelial cells under oscillatory shear stress and laminar shear stress . Thus, as FOXC1 is induced only by laminar shear, but FOXC2 is induced by both laminar and oscillatory shear, their downstream targets are likely different within the collecting vessels of lymphatic endothelial cells. We have also updated the fourth paragraph of the Discussion section to elaborate on these differences.We have now included new representative images of VE-Cadherin immunostaining in EC-5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.As indicated in the Materials and methods section, the flow experiment presented in Figure 3  was run for only 24 hours (subsection \u201cCell transfection and immunostaining\u201d). This shorter time \u2013 24-hour flow instead of 48-hour flow presented in our previous publications  \u2013 is not sufficient to drive an important cell elongation in the direction of flow. In In addition, we also provide the reviewer with the quantification of length:width ratio for static, LSS and OSS conditions presented in Figure 3, which shows that there is a significant elongation of cells under LSS in average . To clarThe observation that FOXC1 and FOXC2 expression are already different in LECs after only 24 hours under laminar shear stress is interesting as it suggests that FOXC1 upregulation by laminar flow is an early response of LECs to directional flow. How expression levels of FOXC1 and FOXC2 are distinctly regulated by the directionality of flow is a very interesting question that addresses the more general one of how LECs sense flow direction and are able to respond differently to LSS and OSS. Answering such a question would require further investigation and, in our opinion, is beyond the scope of this paper in which the central message is that each factor is differentially regulated by flow and plays slightly distinct but complementary roles in regulating the cell cytoskeleton in response to shear stress.6) Figure 1\u2014figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?Foxc2-KO mice compared to littermate controls. We also suggested that the differences observed in reduction of mRNA levels compared to what we observe in regard to protein expression may be attributable to the different vascular beds analyzed. The difference in the penetrance of the phenotype of chylous ascites in the current study may be attributable to both the timeline of experimental analysis as well as the differences in Cre-drivers used. In our group\u2019s previous study, tamoxifen was administered to ERT2; Foxc2fl/flProx1-Cre mice beginning at P4 with approximately 50% of mutants developing chylous ascites 4 days after tamoxifen administration and nearly all mutants presenting chylous ascites 6-8 days after tamoxifen administration . In the current study, tamoxifen administration started at P1 and we primarily investigated tissues at P6. We believe it is likely that our ERT2; Foxc2fl/flCdh5-Cre mice would present chylous ascites at later time points.As previously described in our response to reviewer comment #1, Figure 2\u2014figure supplement 1F and G show a marked reduction of FOXC2 protein within collecting vessels of EC-7) Figure 3\u2014figure supplement 2: A lower power image depicting the broader profile of \u03b19-integrin expression throughout multiple valves would enable the reader to determine how \u03b19-integrin deposition is affected more globally in EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.Foxc2-KO mice at P6, but the collecting vessels of EC-Foxc1; Foxc2-DKO mice are absent of \u03b19-integrin-positive valve regions.We thank the reviewers for this suggestion. Figure 2\u2014figure supplement 3A-D now depicts 10X low power images of \u03b19-integrin/VE-Cadherin immunostaining to show that a few, \u03b19-integrin-positive degenerating valve regions are detected in collecting vessels of EC-8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.Foxc1; Foxc2-DKO mice and littermate controls in panel K. Figure 5\u2014figure supplement 1K now shows the changes in expression for RhoA, Rock1, and Rock2, and several GTPase activating proteins associated with regulation of RhoA signaling and endothelial barrier function and lumen maintenance . Here, we see that there is a modest, yet significant increase in Arhgap18 expression and a significant reduction of Arghap20, although its expression was generally lower compared to other GAPs. Because of the previously reported evidence of the formation of a physical complex between Prickle1 and Arhgap21/23  and our observation that all three genes were significantly downregulated in our RNA-seq analysis, we focused our investigation on these putative downstream targets. In regard to changes in RAS/ERK signaling, we attempted to perform whole-mount immunostaining of pERK using the same antibody utilized in our group\u2019s previous study  but we were not successful in identifying a positive signal in control or mutant lymphatic valves at P6, testing the antibody at various concentrations. While we are interested in characterizing whether RAS-ERK signaling may be affected in our mutants during postnatal development in other lymphatic vascular beds, the authors believe that this is not of focus for the mechanism described in this study and is beyond its scope.We agree with the reviewers that providing additional details regarding the choice to focus on Prickle1, Arhgap21, and Arhgap23 would be helpful. We have now updated Figure 5\u2014figure supplement 1 in the revised manuscript to include additional data from our RNA-seq analysis for LECs isolated from the dorsal skin of embryonic LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.ICAM1 promoter that is not predicted to bind to FOX transcription factors. Panel J then shows data from three separate experiments showing no detection of band signals from ChIP with our FOXC1 or FOXC2 antibodies. Within the text of the manuscript, we have also included relevant literature that has identified variations in immunohistochemistry related work for antibodies recognizing the same immunogen peptide that are dependent on the source of the manufacturer. This information is included in the second paragraph of the subsection \u201cFOXC1 and FOXC2 regulate LEC expression of negative RhoA signaling regulators PRICKLE1, ARHGAP21, and ARHGAP23\u201d.We thank the reviewers for this important suggestion. We have now included Figure 5\u2014figure supplement 1I and J to directly address this comment. Panel I depicts the location of primers used to amplify a transcriptionally active region of the c2/c2 mice. What is the remaining signal in FOXC1 stained Foxc1c2/c2 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in Foxc1c2/c2Foxc1 mice, we have also included new representative images of FOXC1/VEGFR-3 immunostaining in Figure 9D and E. As previously stated in our reply to reviewer comment #1, we suspect that the remaining signal in the FOXC1 channel of Figure 9H is non-specific and may be related to a cross-reactivity issue of secondary antibodies. However, we believe that Figures 9F-H clearly depict a reduction in nuclear FOXC1 expression as expected.We agree with the reviewers that it is likely not accurate to describe that FOXC2 levels are modestly increased. Thus, this statement has now been removed from the revised manuscript. To verify that FOXC1 protein expression is absent in collecting vessels of"}
+{"text": "Serratia marcescens TKU011 with the highest yield of 4.62 mg/mL at the optimal conditions of liquid medium with initial pH of 5.65\u20136.15 containing 1% \u03b1-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4. Fermentation was kept at 25 \u00b0C for 2 d. Notably, \u03b1-chitin was newly investigated as the major potential material for PG production via fermentation; the salt CaSO4 was also found to play the key role in the enhancement of PG yield of Serratia marcescens fermentation for the first time. PG was qualified and identified based on specific UV, MALDI-TOF MS analysis. In the biological activity tests, purified PG demonstrated potent anticancer activities against A549, Hep G2, MCF-7, and WiDr with the IC50 values of 0.06, 0.04, 0.04, and 0.2 \u00b5g/mL, respectively. Mytomycin C, a commercial anti-cancer compound was also tested for comparison purpose, showing weaker activity with the IC50 values of 0.11, 0.1, 0.14, and 0.15 \u00b5g/mL, respectively. As such, purified PG displayed higher 2.75-fold, 1.67-fold, and 3.25-fold efficacy than Mytomycin C against MCF-7, A549, and Hep G2, respectively. The results suggest that marine chitins are valuable sources for production of prodigiosin, a potential candidate for cancer drugs.Marine chitins (MC) have been utilized for the production of vast array of bioactive products, including chitooligomers, chitinase, chitosanase, antioxidants, anti-NO, and antidiabetic compounds. The aim of this study is the bioprocessing of MC into a potent anticancer compound, prodigiosin (PG), via microbial fermentation. This bioactive compound was produced by  Chitin, an abundant material, has been widely produced from fishery processing byproducts. Of the natural chitin-containing materials, shrimp shells, squid pens, and crab shells have the highest chitin content , and as Serratia marcescens and some other Gram-negative bacterial strains [Prodigiosin (PG), a red pigment is a typical alkaloid constituent produced by several bacterial genus,  strains . PGs hav strains ,20,21,22 strains  activiti strains . S. marcescens. However, numerous scientific parameters were not investigated in our previous studies, such as the kind of marine chitin (\u03b1 or \u03b2), protein sources, chitin/protein ratio, and supplementary minerals for the best PG productivity production by S. marcescens. All those previously unknown items were newly investigated in this study, and the PG produce from the medium containing marine chitin was also evaluated for its effect on four cancerous cell lines\u2014A549, Hep G2, MCF-7, and WIDR\u2014in this report.Due to the wild range of unique applications of PG, the production studies on this bioactive compound have been received with great interest ,24, and Serratia marcescens TKU011, compared to other materials; SPP was reported to contain approximately 60% chitin and 40% protein [S. marcescens, the chitins obtained from SPP (\u03b2-chitin) and shrimp shells (\u03b1-chitin) by using the method reported by Wang et al., 2006 [w/w) and used as the sole carbon and nitrogen source for fermentation by S. marcescens TKU011; SPP was also used as the control for comparison purpose. The results in w/w) give higher PG yield production (2.73 mg/mL) than that of SPP (2.45 mg/mL) fermented by S. marcescens TKU011, while \u03b1-chitin mixed with free protein at the ratio of 5/3 (w/w) reach the greatest PG yield production of 3.23 mg/mL. In addition to the use of \u03b1-chitin providing higher PG yield, and \u03b1-chitin could be more abundantly obtained from vast resources  than \u03b2-chitin (mainly obtained from squid pens); thus, \u03b1-chitin was chosen for our further investigation. Based on the recent literature review, PG has been produced by S. marcescens with various types of carbon/nitrogen sources [Carbon source has been proven to play an important role in PG production via microbial fermentation . In prev protein . Thus, cl., 2006  were mix sources ,31,32,33 sources  as the cS. marcescens strains, a total four strains including S. marcescens TKU011, S. marcescens CC17, S. marcescens TNU01, and S. marcescens TNU02 were conducted for fermentation. As shown in the S. marcescens TKU01, S. marcescens TNU01, and S. marcescens TNU02 showed their same level in PG production with the PG yield of 325\u2013335 mg/100mL, and 236\u2013243 mg/100mL when the medium contained newly designed C/N source (0.94% \u03b1-chitin and 0.56% Casein), and 1.5% squid pens, respectively. S. marcescens CC17 demonstrated the lowest production of PG yield.For the comparison of the PG producing by different S. marcescens TKU011 strain reached 335 (mg/100mL). However, two previous studies reported that with 2.0% sesame seed [S. marcescens TKU011 in this study. In these above cited reports [To date, PG has been produced from many carbon sources with multiple designed media ,31,32,33ame seed  and the  reports ,33, the S. marcescens TKU011, some chitinous materials, including chitosan (a derivative of chitin), N-acetyl-glucosamine (monomer of chitin), glucosamine (mono of chitosan), and some other carbon sources, such as cellulose and starch, were used for fermentation. As shown in S. marcescens with the greatest yield of 3.21 mg/mL, followed by its monomer N-acetyl-glucosamine with the PG yield production of 1.81 mg/mL, and all other tested carbon source give low yield PG production (\u2264 0.96 mg/mL). Thus, \u03b1-chitin was chosen as an excellent substrate for further investigation. To further investigate the effect of the combination of \u03b1-chitin and free protein source, a total of five protein sources\u2014beef extract, casein, nutrient broth, yeast extract, and peptone\u2014were combined with \u03b1-chitin used as sole C/N source for fermentation by Serratia marcescens to produce PG. The experimental results in w/w). This designed medium also reached the high PGs yield of 3.21 mg/mL, and as such used for next investigation. To investigate the effect of C/N sources on PG production by 2HPO4 was found to be the most suitable phosphate salt for PG biosynthesis by S. marcescens. Further experiments investigated the optimal added K2HPO4 was 0.05% 2SO4, MgSO4, CaSO4, CuSO4, (NH4)2SO4 as the basal salt solution. CaSO4 demonstrated good effect on PG production with the highest yield of 4.32 mg/mL  in liquid medium with initial pH of 5.65\u20136.15 containing 1% \u03b1-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4, fermentation was kept at 25 \u00b0C for 2 d. To achieve maximum production of prodigiosin, some parameters, including cultivation temperature e, initiaw/w) and used as the sole C/N source at the concentration of 1.5% (w/v) for fermentation by S. marcescens TKU011. PG was primary extracted from the cultured broth by ethyl acetate. The PG from the cell pellet extracted with acetone was mixed with the ethyl acetate layer. After evaporation to dry crude PG, this compound was further purified via silica gel column, and then finally isolated by thin layer chromatography. The procedure is summarized in \u03b1-chitin was mixed with casein with the ratio of 5/3 , A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma). The means of inhibition (%) with the same letter are not significantly different based on Duncan\u2019s multiple range test . CV (%) = 1.979533.50 value. IC50 value is a concentration of sample that may reduce 50% of cancerous cells; therefore, the smallest this value of the sample, the strongest anticancer activity it displayed. As shown in 50 values of 0.04, 0.06, 0.04, and 0.20 against A549, Hep G2, MCF-7, and WiDr, respectively . Four cancerous cell lines\u2014MCF-7 (Human breast adenocarcinoma), A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma)\u2014were purchased from the Bioresources Collection and Research Centre . Mitomycin C and Silicagel  were obtained from Sigma Chemical Co.  and Mitsubishi Chemical Co. , respectively. Reagents, solvents and common chemicals were used at the highest grade available.us study . S. marcus study , S. marcSerratia marcescens TNU01: 16S gene sequence of TGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGC.Serratia marcescens TNU02: 16S gene sequence of CTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTG C.w/w) and used as the sole carbon and nitrogen source with the concentration of 1.5% (w/v) for fermentation. The medium containing 1.5% carbon and nitrogen source, 0.1% K2HPO4 and 0.1% FeSO4(NH4)2SO4 was fermented by S. marcescens TKU011 at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). \u03b1-chitin mixed with casein at the ratio of 5/3 (w/w) reached the greatest PG yield production; as such, \u03b1-chitin/casein was used for comparison in the following experiments evaluating other carbohydrate sources  and proteinous sources. \u03b1-chitin/casein at the ratio of 5/3 (w/w) finally proved best and was used for fermentation in the subsequent investigation, including the effect of added salts and some parameters.\u03b1-chitin and \u03b2-chitin obtained from shrimp shells and squid pens were mixed with casein with 6 ratios of 1/7, 2/6, 4/4, 5/3, 6/2, and 7/1 2SO4, 0.1% phosphate salt, and 1.5% C/N source were fermented at 30 \u00b0C in 1 d, and then at 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). KH2PO4 was found to be the most suitable phosphate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% K2HPO4 and combined with 0.1% FeSO4(NH4)2SO4; the fermentation procedure was conducted at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of phosphate salts and its optimal concentration added to culture medium on PG production: four kinds of phosphate salts\u2014KH4(NH4)2SO4, MgSO4, CaSO4, CuSO4, and (NH4)2SO4 were used as sulfate salts. The medium containing 0.05% K2HPO4 and 0.1% sulfate salt, 1.5% C/N source were fermented at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). CaSO4 was found to be the most suitable sulfate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% CaSO4 and combined with 0.1% K2HPO4; the fermentation was conducted at 30 \u00b0C in 1 d, and then 25 \u00b0C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of sulfate salts and its optimal concentration added to culture medium on PG production: FeSOThe effect of some parameters on PG production: some parameters including temperature programs , initial pH  and period of cultivation time . w/v) hydrated potassium aluminum sulfate was added into this mixture, mixed, and then centrifuged at 1400 g for 5 min. The harvested supernatant was then mixed with a solution of methanol/0.5 N HCl at the ratio of 1/9, v/v. The final solution optical density was measured at 535 nm. PG purified from the culture broth was used as the standard to convert OD535 nm measurement to mass concentration via an appropriate calibration. PG was purified by the method previously described [g for 15 min. The supernatant was collected and mixed with ethyl acetate with the ratio 1/1. The mixture was kept in a funnel for 3 h and immediately shaken every 30 min. The PG dissolved in ethyl acetate layer was collected. The PG from the cell pellet was extracted with acetone, and centrifugated at 10000\u00d7 g for 15 min. The ethyl acetate layer and acetone containing PG were mixed, concentrated by evaporation of the solvent and then dissolved in ethyl acetate for further air oven drying at 55 \u00b0C to get dry crude PG powder. The crude PG was further purified by loading onto a silica open column  for column chromatography, size: 0.040\u20130.063 mm) and eluted with methanol in chloroform with a ratio of 0/10\u20132/8 (v/v). The PG was finally isolated by thin layer chromatography (TLC) with the mobile phase system using methanol in chloroform with a ratio of 2/8 (v/v). After TLC separation, the lane contained PG was cut into small pieces, and methanol was used to dissolve PG. Then PG was concentrated in a rotary evaporator  at 60 \u00b0C under vacuum. Finally, all the residue solvent was removed by keeping the sample in the oil pump in 12 h at 60 \u00b0C. The isolated PG was used to detect UV, MALDI-TOF MS and biological activities. PG concentration was determined according to the method previously described by Wang et al., 2012 . A mixtuescribed  with mod50 values, were analyzed with the use of Statistical Analysis Software (SAS-9.4) provided by the SAS Institute Taiwan Ltd. .Four cancerous cell lines: A549, Hep G2, MCF-7, and WIDR were conducted to evaluate the anticancer activities. The bioassay was done according to the methods described in detail in our previous report . The sig2HPO4, and 0.1% CaSO4 for efficient biosynthesis of bioactive prodigiosin. The fermentation was maintained at 25 \u00b0C for 2 d. The prodigiosin was purified, qualified via UV and Mass. The purified prodigiosin was also evaluated for its anticancer properties. Notably, the purified PG displayed high inhibition on four cancerous cell lines. The results in this study suggest that the purified prodigiosin newly biosynthesized may be a potential candidate for cancer drugs.The current study established the novel designed medium containing 1% \u03b1-chitin, 0.6% casein, 0.05% K"}
+{"text": "We show that CD28 stimulation in the absence of TCR strongly up-regulates IL-22 gene expression and secretion. As recently observed for IL-17A, we also found that CD28-mediated regulation of IL-22 transcription requires the cooperative activities of both IL-6-activated STAT3 and RelA/NF-\u03baB transcription factors. CD28-mediated IL-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. Finally, by using specific inhibitory drugs, we also identified CD28-associated class 1A phosphatidylinositol 3-kinase (PI3K) as a pivotal mediator of CD28-mediated IL-22 expression and IL-22\u2013dependent epithelial cell barrier functions.IL-22 is a member of the IL-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. Dysregulation of IL-22 production has also frequently been observed in acute respiratory distress syndrome (ARDS) and several chronic inflammatory and autoimmune diseases. We have previously described that human CD28, a crucial co-stimulatory receptor necessary for full T cell activation, is also able to act as a TCR independent signaling receptor and to induce the expression of IL-17A and inflammatory cytokines related to Th17 cells, which together with Th22 cells represent the main cellular source of IL-22. Here we characterized the role of CD28 autonomous signaling in regulating IL-22 expression in human CD4 We found that IL-22 gene expression and secretion was strongly up-regulated by CD28 in human CD4+ T cells were enriched from PBMC by negative selection using an EasySepTM isolation kit  and cultured in RPMI 1640 supplemented with 5% human serum , L-glutamine, penicillin and streptomycin. The purity of the sorted population was 95% to 99%, as evidenced by staining with anti-CD3 plus anti-CD4 Abs. Human na\u00efve CD4+CD45RA+ and effector/memory CD4+CD45RO+ T cells were sorted using a high speed cell sorter . Purity of sorted cells was consistently > 98%, and was acquired on a cytometer . PBMCs were derived from buffy coats or anonymous healthy blood (HD) donors provided by the Policlinico Umberto I . Written informed consent was obtained from blood donors and both the informed consent form and procedure was approved by the Ethics Committee of Policlinico Umberto I . CACO-2 epithelial cell line from human colon was provided by ATCC  and cultured in DMEM supplemented with 10% FBS , L-glutamine, penicillin and streptomycin.Human primary CD4\u22121), anti-human CD3 , goat anti-mouse , anti-human CD3-PE (#555333), anti-human CD45RA BV421 (#562885), mouse anti-human STAT3 (#610189), anti-human CD45RO PE (#555493) (BD Biosciences); rabbit anti-human Lck , rabbit anti-human RNA polymerase II , anti-human RelB , anti-human GAPDH (#sc-25778), anti-human mucin 1 (MUC1) (#sc-7313) , rabbit anti-human RelA (#8242S) , mouse anti-human IL-6 , mouse anti-human IL-22  , anti-human CD4 FITC . Human recombinant IL-6 (rIL-6) was from Miltenyi Biotec . The following inhibitory drugs were used: PS1145 , S31-201 , AS605240 .The following antibodies were used: anti-human CD28  in 24 trans-well plates as indicated at 37\u00b0C. At the end of incubation, total cell extracts were obtained by lysing cells for 30\u00a0min on ice in 1% Nonidet P-40 lysis buffer , 1 mM EGTA, 1 mM MgCl2, 50 mM NaF, 10 mM Na4P2O7) in the presence of inhibitors of proteases and phosphatases . Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with the anti-MUC1 or anti-GAPDH (1:400 dilution), extensively washed and after incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit  or HRP-labeled goat anti-mouse  developed with the enhanced chemiluminescence\u2019s detection system . Protein levels were quantified by densitometric analysis using the ImageJ 1.50i program .CACO-2 cells were plated at 2.5 \u00d7 10+ T cells were plated at 2 \u00d7 106 ml\u22121 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells (2.5 \u00d7 105 ml\u22121) and stimulated for the indicated times with control isotype Abs (Ig) or crosslinked anti-CD28 (2 \u03bcg ml\u22121), or anti-CD3 (2 \u03bcg ml\u22121) or anti-CD3 plus anti-CD28 Abs (2 \u03bcg ml\u22121). Secretion of human IL-22, IL-6, and MMP9 was measured from the supernatants of CD4+ T cells cultured alone or with CACO-2 cells and stimulated with control isotype Abs or crosslinked anti-CD28.2 (2 \u03bcg ml\u22121), by using human IL-6 (#DY206), IL-22 (#DY782), and MMP9 (#DY911) ELISA kits (R&D Systems). Data were analyzed on a Bio-Plex . The assays were performed in duplicate. The sensitivity of the assay was 9.4 pg ml\u22121 for IL-6 and 31.2 pg ml\u22121 for IL-22 and MMP9.CD4+ T cells were plated at 2 \u00d7 106 ml\u22121 in 24-well plate or 24-well plate inserts in the experiments of co-culture with CACO-2 cells and stimulated for the indicated times with control isotype matched Abs (Ig) or crosslinked anti-CD28 (2 \u03bcg ml\u22121), or anti-CD3 (2 \u03bcg ml\u22121) or anti-CD3 plus anti-CD28 Abs (2 \u03bcg ml\u22121). Total RNA was extracted by either CD4+ T cells or CACO-2 cells using Trizol according to the manufacturer\u2019s instructions and was reverse-transcribed into cDNA by using Moloney murine leukemia virus reverse transcriptase . TaqMan Universal PCR Master Mix and human IL-6, IL-22, MMP9, MUC1, and GAPDH primer/probe sets were from Thermo Fisher Scientific. The relative quantification was performed using the comparative CT method. The results were expressed as arbitrary units (AU) by using the mean value of cells stimulated with control Ig as CT calibrator or fold induction (F.I.) over control Ig-stimulated cells after normalization to GAPDH.CD4\u2212644 of the human IL-22 promoter, the pIL-22 (-644)-GFP construct was obtained by subcloning the PCR fragment into AseI-Hind III sites within the CMV promoter of pEGFP-N1 vector . The sequence of human IL-22 promoter fragment (\u2212644/+156) was verified by DNA sequencing and was as follow: 5\u2032-ATTAATACAATTTTAAGATATATTTACTTCTGCCTTAATTGTTATGATCACTTAAAAATAGTTCCAAAAAGGGAAGAAAACAATAATTAGATTAGCCAAGACAGTTATTTTTGAAACATAAGTCTGGTTTAGAATTCAGCATGTTTAAAAATGAGATAAAATTATTTTAATAATGGAATGATCTGTTAGCTGTCATTACCATTTACTTTAAAGCAGAGGATATAGGACATGGGTCCTTTTTTTCTGATCACCTCCAATGAGATAAGAATCTATAAAGCTGGTAGGAAAATGAGTCCGTGACCAAAATGCTTACTCGGTCACTATAGGAGATCAAAACATTTTATACTAAATCTGAACTCTACTAAGACAAAACAATTGTGTTCTTTGAAAAATATGTAGGGTTTAGAAAATTTCTGGGATTTGTCTGTAAAATACCCTCCGGGCTCTAATAGTGACGTTTTAGGAAAACACTTGCATCTCAAGGTGGAAAGGATAGAGGTGGTGTTTTGTGGGCTCCTGTGGTGGTTAGGTCGTTCTCAGAAGACAGTACTGGAAATTAGATAATTGCTGATGTCATATTTTTCACAATTAAAAAAAAGTCAGTATCCTGGGGGCTATAAAAGCAGCAGCTTCTACCTTCCCCGTCACAAGCAGAATCTTCAGAACAGGTAGGCGTTTCGGCAAACTTGGTACAATTGGTTAGTTTGACGAAATACTTCTTGACTAATTTTGTTCCTTCACGTTGTCTTCGACCAGGTTCTCCTTCCCCAGTCACCAGTTGCTCAAGTTAGAATTGTCTGCA-3\u2032.The pIL-22 (-644)-GFP construct containing the GFP under the control of the \u2212644 bp region upstream of the transcriptional start site and the +156 region upstream the translational start site was generated as previously described . BrieflyPlasmid vectors expressing HA-tagged human RelA, IKK\u03b1, IKK\u03b2 and NIK were previously described , 38.3 GFP-positive events by gating for SSC and FSC.CD28-positive Jurkat T cells were electroporated  in 0.45\u00a0ml RPMI-1640 supplemented with 10% FBS with 1 \u03bcg of pIL-22(-644)-GFP together with pcDNA3 control vector 10 \u03bcg HA-RelA, or HA-IKK\u03b1 or HA-IKK\u03b2 or 20 \u03bcg HA-NIK. Twenty-four hours after transfection, the cells were analyzed by using a BD FACScan flow cytometer . Mean fluorescence intensity was calculated on a total of 5 \u00d7 107 CD4+ T cells were stimulated as indicated and chromatin immunoprecipitation (ChIP) assays were performed as previously described  was performed for the human IL-22 promoter. Specific enrichment was calculated as previously described ((Ct of control ChIP-Ct of control Input)/2(Ct of specific ChIP - Ct of specific Input). The human IL-22 promoter primers used for each specific ChIP were as follow: RelA I, RelB I and pSTAT3 I, 5\u2032-GCTTACTCAGCCACTATAGGAGATCA-3\u2032 and 5\u2032-CCGGAGGGTATTTTACAGACAAATCC-3\u2032; RelA II, 5\u2032-ACCCTCCGGGCTCTAATAGTGAC-3\u2032 and 5\u2032-AGAACGACCTAACCACCACAGGA-3\u2032; pSTAT3 II and Pol II 5\u2032-CCTGTGGTGGTTAGGTCGTTCT-3\u2032 and 5\u2032-GCTGCTTTTATAGCCCCCAGGAT-3\u2032.10escribed . Brieflyescribed  by using\u22121). CACO-2 cells were plated at 2.5 \u00d7 105 cells ml\u22121 in 24-well plates and treated with 10 \u03bcM AS605240 or DMSO, as vehicle control, for 48\u00a0h. Cytotoxicity was analyzed by a BD Biosciences FACScalibur  by quantifying the percentage of PI positive cells. Results were calculated from at least three independent experiments.The cytotoxicity of AS605240 on CACO-2 cells was evaluated by propidium iodide (PI) staining  was performed to evaluate differences between continuous variables through Prism 5.0  using Student t test. For multiple group comparisons, significant differences were calculated using nonparametric Mann\u2013Whitney + T cells from HD, relapsing-remitting MS (RRMS) and T1D patients (+ T cells from stable RRMS patients (+ T cells from HD with an agonistic anti-CD28 Ab (CD28.2) binding the same epitope recognized by B7 molecules (+ T cells from one representative HD induced a significant increase (p < 0.01) of IL-22 gene expression within 6\u00a0h that further increased 24 to 48\u00a0h and decreased 72\u00a0h after stimulation (+ T cells from a larger sample size (n = 7) (+ T cells from HD. As we previously observed for IL-17A (+CD45RA+ (+CD45RO+ (We have recently found that CD28 stimulation induces the expression of Th17 related cytokines in CD4patients \u201334. Morepatients . In ordeolecules  or anti- (n = 7) . Consist (n = 7) , CD28 str IL-17A  and other IL-17A , the ana+ T cells in a IL-6\u2013dependent manner.These data evidence that CD28 intrinsic signaling regulates IL-22 gene expression and secretion in both na\u00efve and effector/memory CD4+ T cells stimulated with agonistic anti-CD28.2 Abs. To do that, three distinct oligonucleotide probes were used; probe I (\u2212338 to \u2212203) for both STAT3 I and NF-\u03baB I binding sites, probe II (\u2212213 to \u2212110) for NF-\u03baB II binding site and probe III (\u2212131 to \u221220) for both STAT3 II binding site and TATA box (+ T cells from a larger sample size (n = 9) and was associated to CD28-induced transcriptional activation of IL-22 promoter, as evidenced by RNA polymerase II (pol II) promoter occupancy (The human IL-22 gene promoter contains two putative STAT3 responsive elements (STAT3 I and II) and two NF-\u03baB binding sites (NF-\u03baB I and II) upstream of the transcription start site that have been involved in IL-22 promoter trans-activation , 43 Fi, 44 and,ccupancy , 46 and ccupancy  , or anti-CD28, or anti-CD3 or anti-CD3 plus anti-CD28 Abs and co-cultured in trans-well plates with CACO-2 cells for different times. The gene expressions of mucin 1 (MUC1), MMP9 and S100A9 anti-microbial peptide were then analyzed. The kinetic analysis evidenced that both MMP9 . Moreover, the up-regulation and MUC1 gene expression induced by co-culturing CACO-2 cells with CD28-stimulated CD4+ T cells was also associated with a strong increase of MUC1 protein content  required for epithelial barrier functions and protection against extracellular pathogens , 9. We nreported . CD4+ T + T cells   , 58 and  (ROR\u03b3t) , 4. Here (ROR\u03b3t) Figure 1 kinetic  and no s T cells , stimulapression , 60 resupression \u201363.+ T cells. We also found that activated STAT3 was also recruited to the proximal human IL-17A promoter and induced its trans-activation in response to CD28 stimulation . The patients/participants provided their written informed consent to participate in this study.MK performed most of the experiments, analyzed the data, interpreted the results, and helped in writing the manuscript. CA, SF, SC, MS, and MB performed parts of the experiments and data analyses. SA contributed with human samples for the study. MK, CA, SF, MS, and LB were involved in the discussion about the data. LT designed the study, coordinated the work, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by: the Italian Foundation for Multiple\u00a0Sclerosis (FISM 2016/R/29), \u201cProgetto Ateneo\u201d  and Istituto Pasteur Italia-Fondazione Cenci Bolognetti  to LT; the Italian Ministry of Health  and the Italian Foundation for Multiple Sclerosis  to LB; the Italian Ministry of Health (GR-2016-02363725 and GR-2018- 12365529) to MS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Fluorogen-activating proteins (FAPs) are innovative fluorescent probes combining advantages of genetically-encoded proteins such as green fluorescent protein and externally added fluorogens that allow for highly tunable and on demand fluorescent signaling. Previously, a panel of green- and red-emitting FAPs has been created from bacterial lipocalin Blc (named DiBs). Here we present a rational design as well as functional and structural characterization of the first self-assembling FAP split system, DiB-splits. This new system decreases the size of the FAP label to ~8\u201312\u2009kDa while preserving DiBs\u2019 unique properties: strong increase in fluorescence intensity of the chromophore upon binding, binding affinities to the chromophore in nanomolar to low micromolar range, and high photostability of the protein-ligand complex. These properties allow for use of DiB-splits for wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also represent an attractive starting point for further design of a protein-protein interaction detection system as well as novel FAP-based sensors. The central idea is that reconstitution is followed by regain of a specific function which is abolished in the separated parts. Theoretically, many proteins can be divided into such fragments. In practice, the identification of a functional split protein is still nontrivial, although some success in direct evolution-basedin vivo protein-protein interaction detection4. Successful cleavage of the reporter protein, dihydrofolate reductase, fused to the C-terminal fragment of ubiquitin was happening only when both the C-terminal and mutated N-terminal fragments of ubiquitin were expressed as fusions to a leucine zipper homodimerization domain but not when expressed individually.Split proteins were first employed when using ubiquitin for 5, \u00df-lactamase6, thymidine kinase7, or luciferase8. This allows for real time and quantitative analysis of protein interactions in vitro as well as in model organisms. The desire for more user-friendly methods for detecting protein-protein interactions in complex environments and for identification of their precise cellular localization in combination with enormous progress in fluorescent microscopy techniques prompted the creation of fluorescent split proteins. This included split versions of green fluorescent protein (GFP)10, its differently colored derivatives and homologs13, far-red emitting phytochrome-based fluorescent proteins14, or even dual split reporters15.Later, this concept was applied to a number of other proteins. Many of them were enzymes like dihydrofolate reductase12.When used for protein-protein interaction detection, spontaneous self-association of split proteins is highly undesirable. Such self-association events will contribute to the false positive signal and decrease the overall sensitivity of the method. However, spontaneously self-complementing fluorescent split pairs were found to be useful. Their usage allows for substantial decrease of the tag size that is required to be fused to the protein of interest. Therefore, it diminishes potential influence of the tag on the protein of interest behavior16, IFP1.417, iRFP18, and UnaG19 find their ligands  readily available in mammalian cells. Other FAPs like various dye-binding antibodies21, FAST22, DiBs23, and de novo computationally designed mFAPs24 require an exogenous supply of the chromophore. The latter group of FAPs provides multiple benefits. First, available synthetic molecules show a wide range of chemical and photophysical properties allowing for the creation of fluorescent probes with a desired combination of characteristics. Second, external addition of the ligand gives control over the timing and intensity of the fluorescent signal. Third, the noncovalent nature of interaction provides, in some systems, millisecond-scale blinking of the fluorescent signal caused by ligand binding-dissociation events. In these cases, the optimal signal density for high-resolution image reconstruction can be achieved simply by using an appropriate dye concentration. This approach circumvents usage of damaging levels of illumination intensities, which is common for many single-molecule localization microscopy techniques25.Fluorogen-activating proteins (FAPs) are a group of unrelated proteins capable of binding to non-protein ligands (fluorogens) and increase the fluorescence quantum yield and/or change spectral properties of these ligands. Some of these FAPs like miniSOG14 and reversible26 split systems, photoactive yellow protein-based splitFAST27, a label for correlative light and electron microscopy split-miniSOG28, and a bilirubin-binding UnaG-based split reporter uPPI29. Two of these FAP-based splits, IFP PCA26 and splitFAST27, require exogenous supply of the chromophore for imaging in eukaryotic cells. It also has been shown that the full-length UnaG fluorescence recovery under photobleaching conditions needs excess bilirubin added into the solution30. While IFP chromophore, biliverdin, forms a covalent adduct with the protein, only splitFast and uPPI seem to be suitable for use with binding and dissociation events-detecting single-molecule localization microscopy techniques like protein-PAINT23. However, to the best of our knowledge, such an application has not been shown yet for either probe. The reported usage of split-miniSOG for imaging at subdiffraction resolution via electron microscopy is limited to fixed samples.Following the extension of the list of reported FAP systems, FAPs-based splits, also known as bimolecular fluorescence complementation (BiFC) systems, begin to appear. That includes multiple bacteriophytochromes-based irreversiblein vitro screenings we created a panel of FAPs from bacterial lipocalin Blc (named DiBs) capable of recovering the fluorescence of synthetic analogs of green and red fluorescent proteins\u2019 chromophores31. Here we report on DiB-splits, a self-assembling FAP split system which has been inspired by the domain-swapped structure of a full-length DiB protein. This new FAP system reduces the size of the label needed to be conjugated with a protein of interest to ~8\u201312\u2009kDa and is compatible with wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also offer an attractive template for design of a protein-protein interaction detection split system as well as FAP-based sensors.In our previous work, using a combination of computational and 32, previously characterized wild type Blc (wtBlc) protein35, as well as another Blc mutant, DiB1, that has been co-crystallized with the M739 . Our attempts to structurally characterize other DiB proteins23 in apo and bound states resulted in obtaining protein crystals of DiB3 in the apo form at low pH conditions (pH 3.5) which diffracted to 1.6\u2009\u00c5. The asymmetric unit contains only one protein chain. However, it forms a biological assembly (dimer) with a crystallographic symmetry mate. The intertwined dimer is caused by domain swapping: each of the two Blc-like eight-stranded beta-barrel folds is created by the N-terminus of one of two polypeptide chains and the C-terminus of the other , except for the region that connects the exchanging parts of the protein .The lipocalin fold contains a single eight-stranded continuously hydrogen-bonded antiparallel \u03b2-barrel complemented by an \u03b1-helix. This common fold has been observed for other lipocalin protein family membersher Fig.\u00a0. As obsein cellulo protein labelling23, we proposed that the observed domain swapping was driven mainly by the very low pH conditions of the crystallization buffer rather than the private DiB3 mutations (V74F and L141Q). To further evaluate this assumption, we calculated the interaction energies between N- and C-termini fragments of the wtBlc, DiB1, and DiB3 proteins using Rosetta38. Despite the fact that wtBlc protein seems to be slightly more stable, DiB3 is not an outlier . Such property is crucial for the successful creation of a split system. Usually designing a split protein involves laborious screening of multiple protein sites in order to select an appropriate cutting point First, we tested whether N- and C-termini fragments, created by separation of a protein chain in the hinge region, are indeed capable to form the lipocalin-like structure when brought together. We fused each of two fragments of DiBs1\u20133 with one of two leucine zipper peptides as shown on Fig.\u00a035 which locks side chains in the preferable conformation for ligand binding. The opposite effect, weaker binding seen in the case of the red-shifted DiB3-split-Zip:M739 complex, supports the previously suggested hypothesis of the alternative binding mode of the ligand in that complex .Split-Zip proteins showed properties similar to the \u201cparental\u201d full-length variants upon addition of the M739 fluorogen including binding affinities, fluorescence spectra maxima, and extinction coefficients Table\u00a0 and S5. 43. On the other hand, in the case of spontaneous self-assembly, the split form of the labelling system allows for a reduction in the size of the labelling tag that needs to be added to a protein of interest. Hence, it minimizes tags\u2019 influence on that protein44. Therefore, as a next step we checked the ability of the DiB N- and C-termini fragments to self-assemble. For this we deleted the leucine zippers as shown on Fig.\u00a0The behavior of a split system in the absence of additional \u201cattracting force\u201d like leucine zippers or other interacting proteins determines the range of its possible applications. If the assembly of the split protein is conditional (fails to self-assemble spontaneously), it can be used for investigation of protein-protein interactionsSelf-assembling split proteins retain their ability to bind and increase fluorescence of the fluorogen M739 Table\u00a0 and S5, 35 as well as a functional dimer34. However, according to our knowledge, the possibility of a trimer formation was never investigated before. According to size-exclusion chromatography conducted during purification routine of split proteins there were no signs of oligomerization of any kind . The residues of the hinge loop and two adjacent \u03b2-strands are also responsible for the main difference between wtBlc and DiB2-split monomers . We assessed the behavior of these constructs in separate transfections. Cells transfected with the TagBFP-splitC110\u2013177 construct alone showed uniform distribution of the blue fluorescence signal throughout cytoplasm and nuclei of the cells  produced uniformly distributed blue fluorescent signal  TagBFP-splitN1\u2013125\u2009+\u2009TagBFP-splitC126\u2013177 and (2) TagBFP-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177. Upon addition of the M739 chromophore, recovery of the DiB2-specific fluorescent signal in green channel was observed only in the second pair . We speculate that this can be caused by somehow compromised integrity of the new splitN1\u2013125 fragment. For example, because of presence of an alternative conformation of the new C-terminus. Combination of two protein fragments with partial sequence overlap seems to allow for more efficient assembly or/and longer half-time of the functional complex.Next, we tested the ability of the split system to function 1\u2013125 and splitC110\u2013177 fragments in fusion with either histone H2B or vimentin, and conventional fluorescent protein TagBFP. The assembled split would be visible in a distinct localization in green detection channel if self-assembly occurs successfully. The blue detection channel would show the overall distribution of one of the split halves within a cell and allow for detection of aggregation, non-assembled portion, or other undesirable behavior. We first tested two pairs of H2B-fused proteins: (1) splitN1\u2013125 fragment fused with H2B and splitC110\u2013177 fused with TagBFP (H2B-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177), and (2) splitN1\u2013125 fragment fused with TagBFP and splitC110\u2013177 fused with H2B (H2B-splitC110\u2013177\u2009+\u2009TagBFP-splitN1\u2013125). Upon transient transfection of the first pair of fusion proteins (H2B-splitN1\u2013125\u2009+\u2009TagBFP-splitC110\u2013177), the blue fluorescent signal of the TagBFP confirmed absence of aggregation both in the nucleus and in the cytoplasm , DiB2-split can be used as a protein-PAINT tag: bursts of fluorescence from individual protein-ligand interactions are clearly detectable Fig.\u00a0 of labelin vivo and was found to give bright and specific fluorescent signal indistinguishable from the one of the full-length protein.In this study, inspired by the obtained domain-swapped crystal structure of the DiB3 protein in its apo state, we designed and characterized a novel DiB-split FAP system. The two fragments of the split proteins were able to spontaneously reassemble fully restoring fluorogen-activating and spectral properties of the \u201cparental\u201d full-length DiBs. Crystallization of one of these proteins, DiB2-split, further corroborate the preservation of the lipocalin fold by the system. The DiB2-split was tested 23 label of a smaller size for super-resolution imaging in living cells. The decrease of the tag size provided through the split can diminish influence of the tag on the protein of interest. In vitro data suggest near complete assembly of DiB2 from split fragments, making DiB-splits a feasible replacement for full-length DiBs or fluorescent proteins in cases where the size of the molecular tag matters. Moreover, while DiB-split fluorescence does not require post-assembly chromophore maturation unlike self-assembling fluorescent proteins12, it can be used for immediate detection of different biological processes such as protein expression and early trafficking events or as a faster reporter of protein solubility.This DiB-split system presents a proof-of-principle demonstration of the potential of the lipocalin scaffold to create a split system. It is immediately applicable as a protein-PAINT46. Both systems do not require oxygen for their function and might be used in oxygen-deficient systems as it was previously shown with FAST47.DiB-splits as well as the parental full-length DiBs have lower signal-to-noise ratio in comparison with FAST family probes. However, FAST localization density decreased rapidly during data acquisition time in single-molecule localization microscopy regime and successful super-resolution imaging using FAST required protocol modifications and usage of oxygen scavengers1\u2013109) as well as the elongated one (splitN1\u2013125) can be redesigned for better stability. Moreover, mutagenesis of the new N- and C-termini could increase binding affinities for the fluorogen. In addition, there is potential for the design of a variety of other fluorescent tools. For example, redesign of the intramolecular interface of the DiB-split proteins reported here to promote higher independence of the N- and C-termini fragments can result in a new FAP-based tool for protein-protein interactions detection. Such tool would complement the existing mEos3.248 and PAmCherry1-based49 super-resolution imaging compatible BiFC labels providing additional benefit of fast and oxygen level independent measurements. Spatial proximity of the natural N- and C-termini of the protein makes DiB proteins a promising starting point for the design of DiB-based circularly permuted proteins. While the discovered split point is close to the ligand binding site, such circularly permuted proteins represent a promising starting point for DiB-based biosensors design. Successful permutation might also allow for the creation of a new self-assembling split system with smaller parts analogous to self-complementing split fluorescent GFP1110 and sfCherry1112 tags.DiB-splits would benefit from further optimization of the location of the split point. The original N-terminus fragment of the system  and C-fragments (residues 110 to 177) of the DiB mutants23 as well as leucine zipper peptides with adjacent upstream or downstream portions of the vector from the pMRBad-Z-CspGFP and pET11a-Z-NspGFP plasmids, correspondingly, and the upstream portion of the pET11a-Z-NspGFP plasmid including His-tag. Second, we used the overlap PCR to create DNA fragments containing leucine zipper peptides fused with N- or C-fragments of the DiB mutants flanked by upstream and downstream portions of the vector. These fragments were digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors. These vectors were further used to create split fragments without leucine zipper peptides. For this we amplified N-fragments of the DiB mutants with adjacent upstream portion of the vector introducing stop codon and BamHI restriction site instead of leucine zipper peptide coding sequence and C-fragments of the DiB mutants introducing start codon and NcoI restriction site instead of leucine zipper peptide. The PCR products were again digested with BamHI and XbaI or NcoI and BsrGI restriction enzymes and ligated in the original vectors.Plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730) and pET11a-Z-NspGFP (Addgene plasmid #40729)53. The resulted constructs\u2019 amino acid sequences are provided below. The linker sequences are underlined.DiB2-split fusions with H2B, vimentin, and TagBFP were generated by Golden Gate Assembly1\u2013125>H2B-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK110\u2013177>TagBFP-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN110\u2013177>H2B-splitCDPPVATMGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAITKYTSAK1\u2013125>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN1\u2013125>vimentin-splitNDPPVATGMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG110\u2013177-TagBFP>splitCDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG1\u2013109>TagBFP-splitNDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN126\u2013177>TagBFP-splitCDPPVATMRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN>vimentin-DiB2DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWILSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGSG*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEGCorrectness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media supplemented with 100\u2009\u00b5g/mL ampicillin (full-length DiB proteins) or 100\u2009\u00b5g/mL ampicillin and 50\u2009\u00b5g/mL kanamycin (split-Zip and split proteins) at 37\u2009\u00b0C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10\u2009\u03bcM IPTG (split-Zip and split proteins) at 0.8 OD. Cells were harvested after 3\u2009hours of expression at 37\u2009\u00b0C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at \u221280\u2009\u00b0C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75\u2009pg or Superdex 200\u2009pg 10/300 GL column  pre-equilibrated with 50\u2009mM sodium phosphate buffer, pH 6.0.All proteins were expressed in XJb(DE3) Autolysis (Zymo Research) 54 colorimetric assay (Bio-Rad) and bovine serum albumin standard. Single point absorption measurements (595\u2009nm) were performed using FlexStation 3 microplate reader (Molecular Devices). All measurements were performed in triplicate.Protein concentrations were estimated using the Bradford dye-binding method-basedHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.Fluorescence quantum yield was measured relative to a known standard keeping all instrumental conditions identical. Previously characterized DiB:M739 complexes as well as free M739 chromophore were used as standards. Absorbance spectra were detected using double-beam Shimadzu UV-1800 UV/Vis spectrophotometer. Fluorescence spectra were measured using Horiba Jobin Yvon Fluoromax-3 fluorometer.23 using FlexStation 3 microplate reader (Molecular Devices). In brief, constant amount of the chromophore solution was added to protein solutions of different concentrations. The full fluorescence emission spectra were collected using wavelength close to protein-chromophore complex excitation spectrum maximum wavelength. Fluorescence intensity at complex emission spectrum maximum wavelength was extracted and used to determine apparent dissociation constants (Kd).Titrations were performed and analyzed as previously describedDiB3  was crystalized at 21\u2009\u00b0C in 0.8\u2009M sodium citrate, 50\u2009mM sodium borate, 0.1 sodium acetate, pH 3.5 with protein to precipitant volume ratio of 1:1 using hanging drop vapor diffusion technique. Crystals grew within 1\u20133 days and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.DiB2-split  crystals were obtained at 21\u2009\u00b0C in 1.6\u2009M ammonium sulphate, 0.1\u2009M MES, pH 4.5 with protein to precipitant volume ratio of 1:1 or in 1.6\u2009M ammonium sulphate, 0.1\u2009M MES, pH 4.5 supplemented with 0.1\u2009M Iron(III) chloride hexahydrate or 5% w/v n-Dodecyl-b-D-maltoside according to Hampton Research Additive Screen protocol using sitting drop vapor diffusion technique. Crystals grew within 1 week and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.55. The crystal structures were solved by molecular replacement with MOLREP56 using the wtBlc structure (PDB ID 1QWD) as a search model. Models building and iterative refinement were performed with Coot57 and REFMAC58, respectively. The final statistics of the structures are shown in Supplementary Table S1. The models have been deposited into the Protein Data Bank (PDB ID 6UKK and 6UKL). Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using xia2 software suite2. For transient transfections FuGENE 6 reagent (Roche) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer supplemented with 20\u2009mM Hepes).HEK293 and HeLa Kyoto cells were grown in Dulbecco\u2019s modification of Eagle\u2019s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50\u2009\u00b5g/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco) and 10% fetal bovine serum  at 37\u2009\u00b0C and 5% CO\u22122 of 488\u2009nm laser light intensity. Typical acquisitions were 10 000 frames taken at a frequency of 30\u2009Hz.Widefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope, Zyla 5.5 sCMOS camera (Andor), CoolLED pE-300 light source, GFP and BFP filter sets. Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S (ONI). Imaging in HILO mode was performed with \u20091.1\u00a0kW\u2009cm59.Localizations during acquisition were detected using NimOS 1.6.1.9898 . Super-resolution image reconstruction was performed using default values of photon, precision and sigma filters in NimOS. Data analysis was performed using a custom-made Python script. Image resolution was determined by decorrelation analysis pluginTo prepare DiB3 structure for analysis we reconstructed the intertwined dimer using crystallographic symmetry and saved the N-terminus portion of one protein chain (residues 24 to 109) and the C-terminus portion of the other protein chain (residues 114 to 175) as a single pdb file. Both wtBlc (PDB ID 1QWD) and DiB1 crystal structures contain two protein chains in the asymmetric unit. We separated the chains into different pdb files and used both for analysis. Each \u201coriginal\u201d chain was further separated into two chains mimicking DiB3-split and only residues 24 to 109, and 114 to 175 were left to make the length of all structures equal. Each of the 5 prepared structures was then relaxed and the interface energy between two chains was calculated using the following protocol:<ROSETTASCRIPTS\u2009>\u2009<SCOREFXNS/\u2009>\u2009<TASKOPERATIONS/\u2009>\u2009<MOVERS\u2009>\u2009<FastRelax name\u2009=\u2009\u201cfastrelax\u201d relaxscript\u2009=\u2009\u201cdefault\u201d/><ClearConstraintsMover name\u2009=\u2009\u201cclear\u201d/><InterfaceAnalyzerMover ligandchain\u2009=\u2009\u201cB\u201d name\u2009=\u2009\u201ciface_analyzer\u201d pack_input\u2009=\u2009\u201c0\u201d pack_separated\u2009=\u2009\u201c1\u201d packstat\u2009=\u2009\u201c0\u201d scorefxn\u2009=\u2009\u201cREF2015\u201d tracer\u2009=\u2009\u201c0\u201d/></MOVERS\u2009>\u2009<FILTERS/\u2009>\u2009<APPLY_TO_POSE/\u2009>\u2009<PROTOCOLS\u2009>\u2009<Add mover\u2009=\u2009\u201cfastrelax\u201d/><Add mover\u2009=\u2009\u201cclear\u201d/><Add mover\u2009=\u2009\u201ciface_analyzer\u201d/></PROTOCOLS\u2009>\u2009<OUTPUT scorefxn\u2009=\u2009\u201cREF2015\u201d/\u2009>\u2009</ROSETTASCRIPTS\u2009>\u2009The protocol was repeated independently 100 times for each of the starting structures. The data obtained for two separate chains in the asymmetric unit from wtBlc and DiB1 crystal structures were combined. The distribution of the obtained interface energies between N- and C-terminus fragments is shown on Supplementary Fig. S2.Supplementary information.Video1Video2"}
+{"text": "N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells\u2014in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type  The occurrence of lectins, formerly designated as hemagglutinins due to their capacity to agglutinate red blood cells from humans and various animals, in marine seaweeds has been recognized for a long time, following the pioneering works of Boyd et al.  and BlunGriffithsia sp., exhibited inhibiting properties towards HIV-1 [Kappaphycus alvarezii (KAA-2) [Eucheuma serra (ESA-2) [Halimeda renschii (HRL-40) [N-glycans (HM-glycans) occurring at the surface of the influenza virus [Boodlea coacta (BCA) [Oscillatoria agardhii [Nostoc ellipsosporum [Microcystis aeruginosa [Scytonema varium [N-glycans associated to different glycoprotein receptors, e.g., hemagglutinin from influenza virus [Griffithsin (GRFT) from the red alga ds HIV-1 . High-ma (KAA-2) ,13, Euch (ESA-2) , and Hal(HRL-40)  also recza virus ,11,12,14za virus ,11,13. Tagardhii , cyanovisosporum ,19,20,21ruginosa ,23, and a varium . Antivira varium , influena varium ,19, hepaN-glycans decorating the spikes arrayed on the surface of the SARS-CoV-2 envelope, essentially comprised of complex N-glycans and high-mannose N-glycans [Taking into account the antiviral properties and the well known diversity of -glycans ,26, one SARS-CoV-2 spikes consist of homotrimers of S-glycoproteins and play a key role in both the recognition and the subsequent membrane fusion events, resulting in the infection of the host cells ,28. SpikN-glycans of the S-glycoproteins forming the SARS-CoV-2 spikes [Due to the high number of high-mannose 2 spikes ,26,35, hAccording to their carbohydrate-binding specificity towards simple sugars, (seaweed) lectins can been classified in five main groups of Man-specific lectins, GlcNAc-specific lectins, Gal/GalNAc-specific lectins, Fuc-specific lectins, and Sia-specific lectins. Until now, the research on seaweed lectins has focused especially on the Man-specific lectins, and these lectins have been characterized in more detail.To date, a large number of seaweed lectins have been screened, but only a few lectins have been studied in detail or have been characterized at the molecular/structural level. In spite of these limitations, the amino acid sequences and some structural information have become available for some Man-specific seaweed lectins from the red algae (Rhodophyta), the yellow-green algae (Ochrophyta), and the green algae (Chlorophyta) .-Galanthus nivalis agglutinin)-related family of lectins consists of protomers organized in a \u03b2-prism II or \u03b2-trefoil. The red alga Grateloupia chiangii lectin (GCL) and the green alga Boodlea coacta lectin (BCA) present this type of structural organization.The GNA , Nannochloropsis , Ostreococcus , and Porphyra .The legume lectin-related family is made of protomers organized in a \u03b2-sandwich or jelly roll fold (two \u03b2-sheets). This structural scaffold occurs in a few lectins from the genera Man-specific seaweed lectins belong to a few well defined protein families which have been previously identified and characterized for the molecular organization of their protomers, especially in plants :-The GNAN-acetyllactosaminic type glycans and high-mannose type glycans.Man-specific seaweed lectins readily accommodate Man, oligomannosides, and high-mannose type glycan chains. In addition, most of them recognize the tri-mannosyl core Man\u03b11,3-Man\u03b11,6-Man occurring in both Grateloupia chiangii lectin (GCL) offers a nice example of a Man-specific red alga lectin with a \u03b2-prism II structure. The \u03b2-prism II scaffold consists of three bundles of four antiparallel \u03b2-strands arranged into a flattened trefoil-shaped structure around a central pseudoaxis. The GCL lectin dimer consists of two covalently linked swapped protomers organized in a \u03b2-trefoil in such a way that both protomers become oriented almost orthogonally  with a \u03b2-prism II structure [The ogonally A. Each pogonally B,C. The tructure .Porphyra umbilicalis lectin (PUL) illustrates the \u03b2-sandwich organization of the lectin protomer, resulting from the covalent superposition of two strands of \u03b2-sheets connected by more or less extended loops, forming the front and back faces of the \u03b2-sandwich, respectively (Nannochloropsis gaditana (BU14), and in the green alga Ostreococcus tauri (OtL) [The ectively A,B. Thisectively B, form tectively C. The CBectively D. Two otri (OtL)  , and two additional stacking interactions with aromatic residues Y28 and Y110 C. The CBThe CBS of griffithsin also accommodates dimannosides, e.g., Man\u03b11,6Man, via a similar network of 8 hydrogen bonds and stacking interactions with the Y28 and Y110 residues, but the second Man unit located at the reducing end of the disaccharide does not participate in the interaction A,B. A fr8) to the lectin  towards high-mannose glycans, has been studied in detail by Sato et al. [The carbohydrate-binding specificity of another Man-specific lectin with a \u03b2-prism I structure from the red alga o et al. . The hig5) to the modeled KAA-2 lectin, showed the existence of four CBS located at both ends of the \u03b2-barrel forming each protomer of the KAA-2 dimer , e.g., cyanovirin-N (CV-N) from the cyanobacterium sosporum , microviruginosa ,23, scyta varium , and theinin OAA . All of inin OAA .N-glycans of the N-acetyllactosaminic type  and three O-glycosylation sites T323, S325 and T678 are actually glycosylated [The spike S-glycoprotein consists of a single polypeptide chain of 1273 amino acids (140 kDa), containing 22 potential osylated ,35.N-glycosylation sites NXT/NXS are highlighted in dark blue and the O-glycosylation sites are shown in bold letters highlighted in yellow:As shown below the complete amino acid sequence of the S glycoprotein of SARS-CoV-2 is made of two S1 and S2 subunits. The RBD of subunit S1 is highlighted in green and the S1/S2 cleavage site for cathepsin and serine protease TMPRSS2 is highlighted in red. All the VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFEKN- and O-glycans attached to the potential N- and O-glycosylation sites decorating the amino acid sequence of the S-glycoprotein of SARS-CoV-2 , are almost exclusively occupied by often sialylated, bi-, tri- and tetra-antennary N-glycans of the complex type , and GalNAcGalNeuAc  A large diversity was observed in the types of N-glycans and high-mannose N-glycans. Moreover, high-mannose glycans predominantly occur at the top of the S-glycoprotein whereas complex N-glycans are localized at the bottom of the glycoprotein, close to the viral envelope surface.Bi- and tri-antennary glycans are predominantly represented, across all categories of the complex N-glycosylation sites, 331NIT and 343NAT, predominantly occupied by high-mannose N-glycans that should be readily accessible to Man-specific seaweed and cyanobacterial lectins.An interesting note is that the RBD , only contains two O-glycosylation sites T323 and S325, are rather buried at the top of the S-glycoprotein, in such a way that the O-glycans are not identified as key targets for the binding of GalNAc/T-Tn-specific lectins to the S-glycoprotein trimer. However, O-glycosylation site T678 is pretty well exposed at the bottom of the S-glycoprotein and therefore, should be accessible to the GalNAc/T-Tn-specific lectins. Accordingly, seaweed lectins with the corresponding specificity should not be relevant glycan probes for SARS-CoV-2, except for the single exposed O-glycosylation site T678  and two rather buried sites for O-glycosylation (T323 and S325)  . In addist cells ,35.The spikes S-glycoprotein trimers covering the SARS-CoV-2 virions, mediate the binding to the ACE2 receptor through their RBD (S1 subunit), and the subsequent fusion of the viral membrane with the cell membrane (S2 subunit). The spike S-glycoprotein exhibits some flexibility and conformational motions of the S-glycoprotein are pH-dependent . HoweverBoodlea coacta, were shown to possess antiviral properties against various enveloped virus including influenza, herpes, and hepatitis C viruses, and HIV-1  or gp120 (HIV-1) glycoproteins (n = 0) to 100% (n = 3) (A detailed study of the binding-activity towards pyridylaminated (PA-)-oligosaccharides measured for Man-specific lectins of red algae , green a coacta ) and theagardhii ), showedproteins . The bin (n = 3) .An interesting note is that some of the high-mannose glycans of both the hemagglutinin of the influenza virus and the gp120 of HIV-1 recognized by the lectins, also decorate the S-glycoprotein forming the spikes occurring at the surface of the SARS-CoV-2 envelope. Accordingly, the algal Man-specific lectins should similarly interact with the SARS-CoV-2 through the recognition of their spike S-glycoproteins. In this respect, griffithsin (GRFT) was shown to inhibit both the replication and cytopathy of the coronavirus SARS-CoV .Kappaphycus alvarezii), HRL-40 , BCA (Boodlea coacta) and OAA (Oscillatoria agadhii), shows they are the best exposed at the surface of the S-glycoprotein monomer ), N122, N234, N331, N343, N616 and N717 occupied by the high-mannose glycans recognized by the Man-specific lectins KAA-2  and seaweed lectins specific for terminal Neu5Ac residues, could interact with the S-glycoprotein of SARS-CoV-2  RS-CoV-2 . Looking at T678   [http://www.rcsb.org/pdb/) [Burkholderia oklahomensis (PDB code 4GU8 and 4GK9) [Oscillatoria agardhii (PDB code 3OBL) [The atomic coordinates of griffithsin GRFT from de 2GTY) , and lecde 2GTY) , 6\u03b1-mannde 2GTY)  and highde 2GTY) , were targ/pdb/) . Similarnd 4GK9)  and the de 3OBL)  were alsGrateloupia chiangii [Kappaphycus alvarezii [Porphyra umbilicalis [Homology modeling of other lectins including the \u03b2-prism II folded GCL from chiangii , the \u03b2-blvarezii , and theilicalis , was perilicalis  using vailicalis , ANOLEA ilicalis , and theilicalis ,97, wereDocking of simple sugars and oligosaccharides was performed with YASARA and SwissDock . HydrophGriffithsia sp., readily recognized the high mannose N-glycans located on the very similarly glycosylated SARS-CoV S-glyco- protein [The S-glycoprotein on the surface of the SARS-CoV-2 virus is a highly glycosylated protein. Due to the exposed localization of high-mannose glycans at the top of the S-glycoprotein trimers many of these glycans are readily accessible to carbohydrate-binding proteins. Seaweed lectins represent well adapted glycan probes for the specific recognition of this type of viruses. In this respect, the Man-specific lectin griffithsin (GRFT) of the red alga  protein ,63. More protein , GRFT waMoreover, the binding of seaweed lectins to SARS-CoV-2 virus could be applied in biomedical research, e.g., using Man-specific seaweed lectins (1) for detection purposes of the virus on various contaminated surfaces such as doorknobs or furniture elements, (2) as an efficient barrier to avoid the shedding into the environment of contaminating virions and, (3) as control reagents for the occurrence of viral particles in biotic/abiotic samples. Depending on the case, whether properly labelled, e.g., fluorochrome-labelled, Man-specific seaweed lectins could be used directly as glycan probes or unlabelled lectins could be further detected using properly labelled, e.g., fluorochrome-labelled, specific anti-lectin antibodies.Galanthus nivalis (snowdrop), NPA from Narcissus pseudonarcissus (daffodil) and APA from Allium porrum (leek). In addition, two targets for these Man-specific lectins in the replication cycle of SARS-CoV have been identified, one in the early phase of the replication cycle during viral attachment, and a second target at the end of the infection cycle [Lablab purpureus), which specifically recognizes N-glycans of the complex type occurring on the surface of coronavirus envelope, was demonstrated to neutralize SARS-CoV-2 and prevent both viral protein production and cytopathic effects in host (mice) cells [The antiviral properties of Man-specific seaweed lectins and the application of these lectins as blocking agents for the replication of enveloped viruses still requires more investigation. So far, the antiviral properties of Man-specific seaweed lectins, have been demonstrated essentially in in vitro conditions . Indeed,on cycle . More ree) cells .Hippeastrum hybridum, at the end of the SARS-CoV infection cycle [At the molecular level, the mechanism of action for Man-specific lectins is primarily referred to a masking effect of the molecular surface of S-glycoprotein RBDs due to their interaction with the Man-containing glycans, thus hampering the proper attachment of the virions to the host cell receptors and preventing the viral replication. However, the identification of a second target for HHA, the Man-specific lectin from on cycle , suggestAlthough lectins remain attractive anti-coronavirus candidates, at present it remains difficult to correctly assess the actual role of these natural compounds in the therapeutic armamentarium, to fight against SARS-CoV-2, the coronavirus responsible for the highly transmissible infectious COVID-19 ,105,106."}
+{"text": "Correction to: IMA Fungus (2019) 10:15https://doi.org/10.1186/s43008-019-0015-5Incorrect sequence:The published article (Zhang and Zhang \">NAD4L_NC_001715Correct sequence:ATGCTTTTAGAAATAATAACAGCTTATAAAATAGGAACAATCTTATTTTTAATTGGAATTTTAGGTTTCATTATCAATAGACAAAATATTCTTTTACTTATTATCTCTATTGAAATGACTTTATTAGCTATTAGTTTTATTATTATTTGTTCTGCTCTTTTCCTTGATGATTCTGCAGCAGCTTGTTTTTCACTTTATATTTTAGCTCTTGCTGGTTCAGAAGCTGCAATTGGTCTTTCACTTTTAGTTTTATTCCATAGATTTAGAGGATCAGTATTAATTTCAGCTTCTCGACAATAG\"\">nad4L_NC_036382ATGAGTTTAACTTTAGTACTTTTTTTAATAGGAATCTTAGGATTCGTATTTAATAGAAAAAATATAATATTAATGCTTATTTCTATAGAAATAATGCTATTATCTATAACATTTTTAATATTGGTAAGTTCTATTAATCTTGACGATATAATAGGACAAACATATGCTATATACATTATAGTAGTTGCTGGTGCAGAATCTGCTATCGGTTTAGCTATTTTAGTAGCTTTTTATAGACTAAGAGGAAGTATCGCAATAGAATATAAATAA\"To be in concordance with this change, \u201cP263\u201d in the nad4L column should be changed to \u201cP239\u201d in Table 3.The authors would like to apologise for any inconvenience caused."}
+{"text": "Bacillus species, B. CMC1) and a regulator molecule  by a vapor-phased encapsulation method with simple steps of water sublimation and poly-p-xylylene deposition in chemical vapor deposition (CVD) process. Mechanically, the capsule construct exhibited a controllable shape and dimensions, and was composed of highly biocompatible poly-p-xylylene as the matrix with homogeneously distributed bacteria and CMC molecules. Versatility of the encapsulation of the molecules at the desired concentrations was achieved in the vapor-phased sublimation and deposition fabrication process. The discovery of the fabricated capsule revealed that viable living B. CMC1 inhabited the capsule, and the capsule enhanced bacterial growth due to the materials and process used. Biologically, the encapsulated B. CMC1 demonstrated viable and functional enzyme activity for cellulase activation, and such activity was regulatable and proportional to the concentration of the decorated CMC molecules in the same capsule construct. Impressively, 13% of cellulase activity increase was realized by encapsulation of B. CMC1 by poly-p-xylylene, and a further 34% of cellulase activity increase was achieved by encapsulation of additional 2.5% CMC. Accordingly, this synergistic effectiveness of the capsule constructs was established by combining enzymatic B. CMC1 bacteria and its regulatory CMC by poly-p-xylylene encapsulation process. This reported encapsulation process exhibited other advantages, including the use of simple steps and a dry and clean process free of harmful chemicals; most importantly, the process is scalable for mass production. The present study represents a novel method to fabricate bacteria-encapsulated capsule for cellulose degradation in bioremediation that can be used in various applications, such as wastewater treatment and transforming of cellulose into glucose for biofuel production. Moreover, the concept of this vapor-phased encapsulation technology can be correspondingly used to encapsulate multiple bacteria and regulators to enhance the specific enzyme functions for degradation of various organic matters.A regulatable bioremediation capsule material was synthesized with isolated single-strain bacteria ( Bioremediation provides promising solutions for the removal of environmental pollutants, toxic elements, and poisoning management for clinical purposes ,2,3. Varp-xylylenes of United States Pharmacopeia (USP) class VI with high biocompatibility and chemical resistance to strong acids, bases, and solvents was used for encapsulation during the fabrication process. The fabrication is performed in one step with a dry and clean vapor phase, which is desirable for sensitive biological substances such as cells, enzymes, growth factors, and other functional peptides and proteins [p-xylylene polymer on a template substrate that is eventually sublimated, and by manipulating the mass transport during the processing conditions, diverse species with distinct thermodynamic properties were subjected to sublimation and deposition within the confined space of the templates. Finally, transformation of the template resulted in the construction of composite materials with defined physical properties in terms of porosity, bulk size and geometry, and chemical functionality by the compartmentalized functional entities and the devised interfacial chemistries [Bacillus species CMC1 (hereafter referred to as B. CMC1) with specific enzymatic function and the same precision to localize and distribute B. CMC1 due to the encapsulation technique, enabling well-controlled bacterial growth activities and functions, and (ii) a regulator molecule, carboxymethyl cellulose (CMC), with a customizable encapsulation concentration to provide the same customizable stimulation dosages to regulate the enzymatic functions of the neighboring B. CMC1 bacteria. The fabricated and encapsulated capsules were composed of inhabitant bacteria from (i) and the surrounding conditioning regulator molecules from (ii), and the synergistic activities exhibited by (i) and (ii) were able to deliver a combination of controlled enzymatic factors for bioremediation in the devised microenvironments , 0.2 g CaCl2 , 0.2 g KNO2 , 2 g NH4NO3 , 0.3 g Na2HPO4 , 5 g glucose , and 2.5 mL tween 80  were added and then pH with NaOH to 7.0. The genomic DNA of the isolated bacterial strain was extracted using a commercial genomic DNA extraction kit . Then, the sequence of its 16S rRNA was confirmed by polymerase chain reaction by using the primers F8 (5\u2032GAGAGTTTGATCCTGGCTCAG3\u2032) and R1492 (5\u2032GGTTACCTTGTTACGACTT3\u2032). The obtained 16S rRNA sequence from the isolated bacteria wasWastewater and activated sludge were collected from the sewage of a pig farm in Taoyuan, Taiwan. All tools and reservoirs were sterilized before sampling. Activated sludge was stored immediately at 4 \u00b0C after collection. The wastewater and activated sludge were used for further culturing of environmental bacteria in liquid nutrient medium. In one liter of culture medium, 3 g peptone , 1 g yeast extract , 5 g NaCl , 0.2 g MgSOTGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGC.https://www.ezbiocloud.net/. The isolated bacteria were the Bacillus species with the top ranking of B. subtilis and was named Bacillus species CMC1 (B. CMC1) in the study.The sequence was further confirmed according to the 16S rRNA database at B. CMC1 was cultured for 30 h at 30 \u00b0C and then washed twice with diH2O by centrifugation and resuspended into final solutions , 1.5% CMC, or 2.5% CMC) to prepare samples with the same concentration of bacteria (~1 \u00d7 108 bacteria/mL) on the same day of capsule fabrication. The prepared bacterial solution with/without CMC was solidified by liquid nitrogen for later encapsulation. A home-built vapor deposition system was used for the encapsulation and fabrication process in this study , 0.2 g KCl , and 1 g MgSO4\u00b77H2O  were added, and then the pH was adjusted to 6.3 with NaOH. Then, 9 g agar  and 10 mg trypan blue  were added. A single bacterial capsule (100 \u00b5L in volume) was seeded in the center of one 9 cm plate and left at 30 \u00b0C for up to 140 h for subsequent enzyme functioning zone quantification. For the quantification of DNA and RNA, a single bacterial capsule (100 \u00b5L in volume) was seeded in a 50 mL centrifuge tube with 30 mL of growth culture medium inside, and the culture was left at 30 \u00b0C for 50 h on an orbital shaker with a 100 rpm shaking speed. The same volume of the culture (5 mL) from encapsulated bacteria was centrifuged for genomic DNA extraction using a commercial extraction kit  for the following DNA quantification. The same number of bacterial cells (2 \u00d7 108 cells) was used for total RNA extraction by using a commercial extraction kit  for total RNA quantification. The concentrations of DNA and RNA were measured by a NanoDrop Spectrophotometer . For this, 1 \u00b5L of purified DNA and RNA were used in the analysis. Note that only the DNA and RNA with the 260/280 ratio between 1.8 and 2.0 were used for concentration measurements, ensuring the sufficient purity of DNA and RNA for further quantification analysis.Cellulase activity was examined on 9 cm agar plates by quantifying the size of the enzyme functioning zones. To one liter of cellulase visualizing agar, 1 g yeast extract , 1 g NHB. CMC1 in the porous parylene structure. A VK-9510 3D profile microscope  was used to analyze the external architecture of the bacterial capsules and the existence of the bacteria inside the structure. SEM images were recorded using a NovaTM NanoSEM  operated at a primary energy of 10 keV and a pressure of 5 \u00d7 10\u22126 Torr to detailed the internal structure of the bacterial capsule. EDS elemental point analyses were captured for the quantification of the studied elements. 3D analysis of the interior structure was performed by using Bruker Skyscan 2211  at 2.0 \u03bcm/pixel resolution. The setting of the voltage was 40 kVp, whereas the current was 700 \u03bcA at 8 Watt output with microfocus mode. Image reconstruction, ring artifacts, and beam-hardening correction were performed using reconstruction software, Instarecon . FT-IR spectra of the fabricated capsules were recorded by a Spectrum 100 spectrometer equipped with an ATR detector . The recorded spectra ranged from 600 to 4500 cm\u22121 with 4 scan times at 4 cm\u22121 resolution.A Nikon ECLIPSE 80i fluorescence microscope  with a visible light source was used to visualize the crystal violet-stained p-xylylene in one continuous step [p-xylylene molecules to replace the resulting space when the sublimated ice/water molecules evaporated from the ice templates. The resultant construct consisted of a three-dimensional porous poly-p-xylylene matrix with encapsulated B. CMC1 bacteria in the matrix , and confocal microscopy to verify the localization and distribution of the bacteria . The imaructures a. The anB. CMC1, we hypothesized that the bacteria were resistant to low temperatures during iced template preparation and low-pressure conditions (approximately 10\u22123 Torr) during the sublimation and deposition process. Cultured samples of encapsulated B. CMC1 were compared to samples of B. CMC1 without encapsulation (positive control), and their cell densities were monitored by OD600 measurement. The results showed comparable bacterial growth patterns and activities in liquid medium for both types of bacteria during the culture time frame of 10 h  various concentrations of CMC . Subsequently, the same sublimation and deposition process was performed to resolve the capsule construct that comprised a poly-p-xylylene matrix with simultaneous encapsulation (i) and (ii) the same matrix structure. In addition to the already confirmed encapsulation of B. CMC1, the use of FT-IR analysis indicated that the intensity of the characteristic \u2013C=O and \u2013CH\u2013O\u2013CH2 peaks from CMC observed at 1558 and 1050 cm\u22121, respectively, gradually increased from a low concentration to higher concentrations of CMC content in the fabricated capsules  from poly-p-xylylene, additionally showed consistency by stoichiometry of the encapsulated CMC concentrations  exhibited approximately 10 h of bacterial growth in the lag phase and showed consistency with the samples with only B. CMC1 encapsulation  were studied in parallel to bare samples with unregulated B. CMC1 (0% CMC), and their enzymatic cellulase activities were first examined on agar plate results for up to 140 h  to a 0.25% regulation was found, and an anticipated enhancement of the expression with increasing CMC regulation compositions was accordingly discovered with a 13.1% increase for 1.5% CMC and 27.4% for 2.5% CMC of the studied capsule samples. On the other hand, based on the same bacteria number to extract their total RNA, the results in B. CMC1 was regulated for the synergistic capsules, and the synergy was found to be consistent with the same regulatable and enhancement of the bacterial population in the aforementioned study. Collectively, the results unambiguously verified the hypothesis that synergic effectiveness was achieved by vapor encapsulation of (i) B. CMC1 and (ii) the CMC regulator in the same fabricated capsule constructs, and the regulation was achievable through the versatile use of the encapsulated CMC compositions.More evidence showing the enhancement of the enzyme activities was further verified through the analysis of the genomic information, including the amount of genomic DNA and total RNA from the cultured samples of the capsules at a time point of 50 h, which was in the early stage of stationary phase in the bacterial growth curve. Calculation and quantification of the genomic DNA was performed based on the same volume of bacteria obtained from the capsule samples, and as shown in B. CMC1 was achieved in the current study. Because of the versatility of various types of remedial bacteria and regulator molecules, unlimited applications are expected beyond those shown in the report. In addition, the use of water/ice templates and a dry and clean vapor-phased process preserved the sensitive biomolecules and their biological functions, and the final fabricated capsule construct was composed of a USP Class VI compatible poly-p-xylylene matrix. Due to this specific production process, this novel fabrication process can not only encapsulate functional bacteria, regulating molecules, but also other potential absorbing material to concentrate the organic matters for degradation. It was reported that the encapsulation of phenol metabolic bacteria in microfiltration membrane capsules alone can create a confined environment to enhance the bioremediation of efficiency of encapsulated bacteria [p-xylylene fabrication technology can constitute a defined inner porous structure to capture different size of organic particles to further concentrate organic matters and a controllable size and shape of the products to fit to different situations. Until now, most of the present studies still focused on identifying the bacteria with specific enzyme functions to use in the bioremediation process [Synergistic and regulatable bioremediation of cellulose by encapsulated bacteria . The pol process ,25,26. W"}
+{"text": "Scientific Reports 10.1038/s41598-020-68172-2, published online 09 July 2020Correction to: The Supplementary Information file that accompanies this Article contains errors in Supplemental Table 1.her9 F, \u201cCCTGACGGAGAACTGAACACAAGACACACA\u201dThe primer sequence reported for should read:\u201cCGCCACACACACGCTCGTGT\u201dher9 R, \u201cTTTCTCAATGGTACGGCGGGTGCTCTGGGC\u201dThe sequence reported for should read:\u201cCGGCTTGTGGAACGCCCGAA\u201dher9 CR F, \u201cAAGCTTCCTGACGGAGAACTGAACACAAGACACACA\u201dThe sequence reported for should read:\u201cAAGCTTTGGCTTCTACCGGACTCAACTTTGGTGTTT\u201dher9 CR R, \u201cGAATTCTTTCTCAATGGTACGGCGGGTGCTCTGGGC\u201dFinally, the sequence reported for should read:\u201cGAATTCCCATGAAAACTTTATAAGTTCATATGAGATGCGC\u201d"}
+{"text": "Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.  CARs are synthetic chimeric receptors consisting of an antibody based extracellular part to recognize specific antigens expressed on the surface of tumor cells and an intracellular part containing (co)stimulatory signals derived from CD3\u03b6, CD28 and/or 4_1BB ,10,11. CH) and variable light (VL) regions and the generation of a single-chain variable fragment (scFv) for target recognition [H and VL of the different scFvs [CARs that obtained market authorization and those that are clinically tested rely on the generation of a monoclonal antibody, the sequencing of the variable heavy  found in Camelidae [H gene family III and their small size [Nanobodies can potentially be an alternative antigen-binding moiety. Nanobodies, first described by Hamers-Castermans, are isolated from the Vamelidae . The strall size ,29. No oall size ,31,32,33all size . Finallyall size . Therefoall size . Both siall size ,40,41,42all size .We previously described the use of a dual specific CAR based on nanobodies . To expa1 CH2CH3 (Fc) spacer, the CD28 transmembrane domain and the CD28 and CD3\u03b6 intracellular signaling domains. The 4_1BB:\u03b6 construct contains the CD8\u03b1 hinge and transmembrane region followed by the 4_1BB and CD3\u03b6 intracellular signaling moieties. Both CAR backbone constructs were cloned in a retroviral plasmid that contains an IRES-eGFP sequence to allow easy detection of CAR-expressing cells. At the 5\u2032 position we incorporated a BamHI restriction site . The nanobody specific sequences were amplified by PCR using primers with 15\u201320 nucleotide overhangs complementary to the 5\u2032 retroviral plasmid (forward primer) or the 3\u2032 CAR sequence (reverse primer). The forward primer also encoded a leader sequence derived from the L-kappa murine leader sequence. The PCR products were purified, and Gibson Assembly was used to introduce the amplified sequence into the CAR vector . The nanobody sequences specific for human CD33 were derived from a llama nanobody phage library. This library was constructed from peripheral blood lymphocytes obtained after immunization of a llama with the extracellular domain of human CD33 protein. Three different nanobody clones were randomly selected and amplified by PCR, purified and cloned into the CAR backbone by Gibson Assembly, resulting in three CD33-specific nanoCAR constructs. After sequence confirmation, we produced retroviral particles and subsequently transduced peripheral blood mononuclear cells (PBMC). NanoCAR expression was confirmed by flow cytometry using an antibody specific for the nanobody protein . Transdu\u2212 ovarian cancer cell line SKOV3 was used as negative control. Most acute myeloid leukemia cells express CD33. A panel of AML cell lines was analyzed for CD33 expression A. Flow c+ cell lines. The cytotoxicity was driven by CAR binding to cognate antigens, since CD33+ cell lines were neglected by non-transduced T cells and CD33\u2212 SKOV3 cells did not elicit target cell lysis. Next, we evaluated cytokine production of our CD33-specific nanoCAR T cells 5 h after co-incubation with our target cell panel by intracellular staining for interferon-\u03b3 (IFN-\u03b3) and interleukin-2 (IL-2). The nanoCAR-expressing cells were able to produce both IFN-\u03b3 and IL-2 after incubation with CD33+ MOLM13 cells. Incubation with the CD33\u2212 cell line SKOV3 resulted in no significant expression of IL-2 or IFN-\u03b3  mice were intravenously injected with Thp1 cells expressing firefly luciferase. The cells were allowed to engraft and expand. After checking engraftment 7 days after injection, a single tail vein injection of nanoCAR T cells was administered day 0 in A. Follow+ hematopoietic precursor cells (HPC) were isolated from different cord blood donors and analyzed for CD33 expression. Only CD34dimCD38dim HPC expressed CD33 although at a lower level compared with leukemic cell lines  through their CH2 domain. Earlier studies of the IgG1 Fc spacer showed off-target CAR T cell activation by FcR\u03b3+ myeloid and lymphoid cells and speculated that potential activation-induced cell death (AICD) could occur [We also noticed no survival benefit nor disease control in NSG mice injected with CD33-1-CD28:\u03b6 nanoCAR T cells. Furthermore, we were not able to detect any eGFPld occur . Other gld occur ,55,56,57In summary, we have shown that our technique of using nanobodies, PCR and Gibson Assembly is a rapid and efficient way to generate nanoCAR T cells with a 100% success rate for the six randomly selected nanobody clones. We chose two completely different antigens: CD20, a tetraspanner and CD33, a single pass receptor, to test our technique. We strongly believe that the use of nanobodies is advantageous over the use of scFvs, since nanobodies are monomeric structures that (i) will probably not aggregate on the T cell surface and therefore not induce premature T cell activation and exhaustion ; (ii) wiProcedures for immunization of llamas, preparation of mRNA, construction of the library, and panning were performed as previously described .l-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin  . SKOV3 was cultured in DMEM  supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin . Thp1 was cultured in RPMI  supplemented with 0.05 mM \u03b2-mercaptoethanol, 10% fetal calf serum , 2mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin .All the cell lines were cultured as per American Type Culture Collection  recommendations. RL, Raji, MOLM13, U937, were cultured in RPMI , supplemented with 10% fetal calf serum , 2 mM L-glutamine , 100 IU/mL penicillin  and 100 IU/mL streptomycin . Jurkat and HL60 were cultured in IMDM  supplemented with 10 % fetal calf serum , 2 mM Escherichia coli (High Efficiency), NEB) and plated on agar. After overnight incubation, colonies were selected and grown in liquid lysogenic broth  overnight. Plasmids were isolated and sequenced. Colonies containing the correct plasmid were further cultured and midipreps (Qiagen) were performed. The different constructs, as shown in 5 cells per 6 cm dish  and placed overnight in a 7% CO2 incubator at 37 \u00b0C. Next day, the plasmids encoding the nanoCAR constructs were transfected using calcium phosphate as follows: per 6 cm dish, 10 \u00b5g plasmid DNA was diluted in 36 \u00b5L 2M CaCl2 (homemade) and subsequently nuclease free water  was added to a total volume of 300 \u00b5L. The DNA-CaCl2 solution was pipetted dropwise into a 15 mL polystyrene tube containing 300 \u00b5L 2\u00d7 HEPES-buffered saline (HBS) solution (homemade) while blowing air bubbles in the 2\u00d7 HBS buffer. The mixture was incubated for 15 min at room temperature and then added dropwise to the cells. Ten minutes before the addition of the DNA mixture, 2 mL medium was added to the cells containing 1 \u00b5L of a 200 mM chloroquine  solution. Cells were placed in a 7% CO2 incubator at 37 \u00b0C overnight. Medium was refreshed at day one. At day two, cells were analyzed for transfection efficiency and transgene expression and reseeded in a T75 culture flask  in selection medium containing 2 \u00b5g/mL puromycin (Sigma). After an additional two days, selection medium was replaced by medium without puromycin. At day six and day 10, the selection cycle was repeated. Finally, at day fourteen, retroviral supernatant was collected and was frozen at \u221280 \u00b0C until use. Phoenix A cells were analyzed for nanoCAR and eGFP expression at days of reseeding and at day fourteen. Viral particles were produced using the Phoenix A packaging cell line. Phoenix A cells were seeded at 7.5 \u00d7 10+ cells was determined by flow cytometry and T cells were stimulated with Immunocult Human CD3/CD28/CD2 T cell activator  per fabricator instructions in cIMDM, in the presence of 10 ng/mL IL-12 . Cells were harvested 72 h after stimulation and resuspended in retroviral supernatant and centrifuged for 90 min at 1000\u00d7 g at 32 \u00b0C on retronectin  coated plates. Buffy coats from healthy donors were obtained from the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital, after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated by Lymphrop  gradient centrifugation. The percentage of CD3Transduced cells were detected by eGFP expression and after staining with a nanbody specific antibody. Transduced cells were sorted and expanded on irradiated allogenic feeder cells, consisting of a mixture of 40 Gy irradiated peripheral blood mononuclear cells and 50 Gy irradiated JY cells. Cells were cultured in cIMDM, supplemented with 1 \u03bcg/mL phytohemagglutinin  (Roche) was added on day five and day ten. Cells were restimulated every seven\u2013fourteen days.Staining of surface markers was performed as described earlier . The fol+, Raji, RL and SKOV3. Cytotoxicity assay was performed as previously described . The folDetection of cytokine producing cells was performed as previously described , with th4 Thp1 cells) in cIMDM. Cells were stained with CD3, CD4 and CD8\u03b1 at the start of the co-culture and at day three, seven, ten and fourteen. At day seven of co-culture, 2 \u00d7 104 Thp1 cells were added to the remaining wells. Cell numbers were determined by flow cytometry. T cells expressing nanoCARs were incubated with Thp1 cells at an effector/target ratio of 0.025:1  was performed after intraperitoneal injection of 150 mg/kg bodyweight d-luciferin (Perkin Elmer) and 5 \u00d7 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by IVIS imaging. Mice were checked for overall health status and scarified when humane endpoints were reached. NSG mice were injected intravenously with 2 \u00d7 106 RL cells were subcutaneously injected. The cells were allowed to form a solid mass and at day eighteen, 5 \u00d7 106 nanoCAR T cells were intravenously injected. Tumor progression was followed up by caliper. Mice were checked for overall health status and scarified when human endpoints were reached. For the RL model, 2 \u00d7 10+ HPC were purified using CD34 MicroBead Kit (MACS Miltenyi Biotech) per manufactures instructions. T cells expressing the nanoCARs were cultured with CD34+ HPC in cIMDM supplemented with stem cell factor , thrombopoietin  and FMS-like tyrosine kinase 3 ligand , all three at 100 ng/mL with PBMC at a 1:1 ratio. At the start and at 24 h, 48 h and at 72 h of the co-culture, cells were harvest and stained for CD3, CD34, CD38 and CD33. Subsequently, cells were analyzed by flow cytometry and absolute cell counts were determined.Cord blood was acquired through the Belgian Red Cross and used following the guidelines of the Medical Ethical Committee of Ghent University Hospital (EC/UZG 2015/0768), after informed consent had been obtained, in accordance with the Declaration of Helsinki. PBMC were isolated using density gradient centrifugation. CD34Underlined sequences indicate overlap with the LZRS vector, cursive sequences indicate BamHI site, sequences in bold indicate leader sequence, sequences in upper case indicate overlap with nanobody sequences and sequences in upper case and bold indicate overlap with CAR backbone.Nanobody specific sequence amplification.Fw primer:ggatccgggtggaccatcctctagactgccgccatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaCAGGTGCAGCTGCAGGAG3\u20325\u2032Rev primer 4_1BB:\u03b6 backbone:ggatccACTGAGGAGACGGTGACCTG3\u20325\u2032GGTCGCGGCGCTGGCGTCGTGGTCRev primer CD28:\u03b6 backbone:ggatccACTGAGGAGACGGTGACCTG3\u20325\u2032AGGAGATTTGGGCTCGGCGGGCDNA sequence of CD28:\u03b6 CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCTTCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGATAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTDNA sequence of 4_1BB:\u03b6 CAR backbone construct:gggtggaccatcctctagactgccggatccgccggatccGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAatggattttcaggtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtctagaGTData were analyzed with GraphPad Prism Software . Statistical differences between groups or conditions were determined by two-way ANOVA, followed by Bonferroni post hoc test. Survival curves were compared using the log-rank Mantel\u2013Cox test."}
+{"text": "Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-\u03b2 in macrophages, the latter being a key component for augmented T cell\u2013mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.New strategies are needed to enhance the efficacy of anti\u2013programmed cell death protein antibody (anti\u2013PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti\u2013PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not   Inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in murine melanoma models. While there have been extensive efforts to combine other T cell\u2013stimulating factors with anti\u2013programmed cell death protein antibody anti\u2013PD-1 Ab), there is an increasing interest in identifying T cell\u2013independent strategies that will augment the efficacy of anti\u2013PD-1 Ab. Tumor cell autophagy has been identified as a major resistance mechanism to targeted therapy and chemotherapy. Chloroquine and hydroxychloroquine (CQ and HCQ) are the only drugs that have been tested as autophagy inhibitors in clinical trials in patients with cancer. However, there are conflicting reports about whether as a single agent CQ derivatives augment, impair, or have no effects on antitumor immunity  Ab, ther.Ppt1 (siPpt1) were all able to convert M2 to M1 tumor-associated macrophages (TAMs). Conditioned media from macrophages treated with Ppt1 inhibitors were able to enhance T cell\u2013mediated cancer cell killing. Interestingly, the combination of HCQ and anti\u2013PD-1 Ab resulted in a change in TAM polarization and a significant reduction in myeloid-derived suppressor cell (MDSC) infiltration in vivo while each single agent did not. The mechanism by which PPT1 inhibitors produced macrophage phenotype switching was dependent on mitochondrial calcium release and p38 activation. PPT1 inhibition also induced the cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 (cGAS/STING/TBK1) pathway to induce IFN-\u03b2 release from macrophages. PPt1 inhibition\u2013induced IFN-\u03b2 release was critical for the augmentation of antigen-primed T cell killing of melanoma cells. These data suggest that this combination, which can be immediately tested in the clinic, could provide an alternative rational combination approach for melanoma immunotherapy.Recently, we identified the major molecular target of CQ derivatives as the lysosomal protein palmitoyl-protein thioesterase 1 (PPT1) , 4. Meanhttps://doi.org/10.1172/jci.insight.133225DS1). Tumors harvested at the end of the experiment were significantly smaller with combination compared with monotherapy arms , and Atg7. KD of these 3 genes in B16 cells produced reduced autophagic flux as evidenced by increase in p62. While Lc3b-II levels did not change much with siUlk1 or siPik3c3 as is often observed  to an antitumorigenic M1) phenotype or mouse macrophages in vitro and in vivo  phenotyp. Morpholhenotype . KD of crization . Ppt1 KD+ T cells play a major role in the elimination of tumor cells and regression of tumors by their cytotoxic activity  activates Cre recombinase, and melanocytes express mutant Braf and lose Pten. Spontaneous melanoma tumors arise on the skin of these mice with 100% penetrance. In concordance with the B16 tumor model, we found that HCQ significantly enhanced the antitumor response of anti\u2013PD-1 Ab  (+CD11b+Ly6C+Ly6G\u2013CX3CR1+ monocytes and spleen eosinophils (CD45+CD11b+Siglec F+) and no significant change in DCs (CD45+CD11b+CD11c+) and PMN-MDSCs (CD45+CD11b+Ly6CloLy6Ghi)  T cells compartment in the spleen and tumor  . There woLy6Ghi) . There wnd tumor . Taken tPpt1 also resulted in increased levels of p-p38 compared with siNon-target control  . HCQ, DC control . PPT1 in control . Inhibit control .+/Ca2+ exchanger, completely abrogated the increased p38 phosphorylation induced by lysosomal inhibitors , while a comparison of DC661 versus control showed 47 significantly changed proteins out of a total of 3166 proteins identified with high confidence . MCM was subjected to proteome analysis. Comparison of HCQ versus control showed 64 significantly changed proteins  CAGCATCTTCTTGGCAGACATAAATCAAGAGAGGTGTGTCAATGAGTCCTACAAGAAGAACCTGATGGCCCTCAAGAAGTTTGTGATGGTGAAATTCTTTAATGATTCCATTGTGGACCCTGTCGACTCTGAGTGGTTTGGATTTTACAGAAGTGGCCAAGCTAAGGAAACCATTCCCCTCCAGGAGAGCACTCTATACACAGAGGACCGCCTGGGGCTAAAGAAAATGGACAAAGCAGGAAAGCTAGTGTTTCTGGCTAAGGAAGGGGACCATCTTCAAATATCTAAAGAATGGTTTACTGCCCACATCATACCTTTTCTTAAGTGATGCCCTGGCACTTTATAGCAGAGTTCATGAAACCACAGCTCTTCCAAGCCATGTACATAGTTCATGCTCAGCCTGAACTCTAATCTAGCCTGCAACCAGCCCTTCTCTCCTCTTATCATCTAACATACCCTACTTGGAAAGATCTAAGATCTCAATCTTATCCTTTGCCGCCT. PpT1 siRNA pool 2 (Santa Cruz Biotechnology sc-142398) AGGCGGCAAAGGATAAGATTGAGATCTTAGATCTTTCCAAGTAGGGTATGTTAGATGATAAGAGGAGAGAAGGGCTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTTCTTGAGGGCCATCAGGTTCTTCTTGTAGGACTCATTGACACACCTCTCTTGATTTATGTCTGCCAAGAAGATGCTG. Ppt1 siRNA single duplex  CTGGTTGCAGGCTAGATTAGAGTTCAGGCTGAGCATGAACTATGTACATGGCTTGGAAGAGCTGTGGTTTCATGAACTCTGCTATAAAGTGCCAGGGCATCACTTAAGAAAAGGTATGATGTGGGCAGTAAACCATTCTTTAGATATTTGAAGATGGTCCCCTTCCTTAGCCAGAAACACTAGCTTTCCTGCTTTGTCCATTTTCTTTAGCCCCAGGCGGTCCTCTGTGTATAGAGTGCTCTCCTGGAGGGGAATGGTTTCCTTAGCTTGGCCACTTCTGTAAAATCCAAACCACTCAGAGTCGACAGGGTCCACAATGGAATCATTAAAGAATTTCACCATCACAAACTT. Fluo-4, AM, was used to stain the cells for calcium as per the manufacturer\u2019s instructions .DC661 was provided in-house. Purity of the sample was determined by NMR spectroscopy and liquid chromatography-mass spectrometry. Mouse melanoma B16-F10 (CRL-6475) and RAW 264.7 (TIB-71) were purchased from ATCC. Cell lines were tested for mycoplasma biannually and authenticated using short-tandem repeat fingerprinting. Cells were cultured in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose, sodium pyruvate, phenol red, and 10% fetal calf serum . BMDMs were isolated as described previously .Total RNA was isolated by RNA isolation kit  according to the manufacturer\u2019s protocol. Complementary DNA (cDNA) was synthesized using iScript Reverse Transcriptase kit with 500 ng of purified RNA as per manufacturer\u2019s protocol . The qPCR reaction was set up using SYBR Green PCR Master Mix  containing 1 \u03bcL of cDNA. All measurements were carried out in duplicate, and Hsp90 was used as internal standard for \u0394CT calculations. Gene expression analysis was done using the following primers: iNos forward (AGGAGGAGAGAGATCCGATTTAG), iNos reverse (TCAGACTTCCCTGTCTCAGTAG); Retnla/Fizz1 forward (TGCCAATCCAGCTAACTATCC), Retnla/Fizz1 reverse (GCAAAGCCACAAGCACAC); Hsp90 forward (GGGAGCTCATCTCCAATTCATC), Hsp90 reverse (GTCCTGTTTGCTGGGAATGA); Arg1 forward (TACCTGCTGGGAAGGAAGAA), and Arg1 reverse (CTGTAAGATAGGCCTCCCAGA).Atg7 gRNA AACTCCAACGTCAAGCGGGT sequences for targeting mouse cells were used as described previously (XhoI and NheI sites of pLX-sgRNA followed by restriction digest and ligation. Sanger sequencing was used to confirm that the Atg7 gRNAs and nontargeting control RNA were correctly subcloned in the pLX-sgRNA vector. B16 cells were first transfected with the pCW-Cas9 vector and selected with puromycin 4 \u03bcg/mL for 6 days and then transfected with the pLX-sgRNA containing the Atg7 gRNA and nontargeting gRNA using Lipofectamine 3000 (Thermo Fisher Scientific) based on the manufacturer\u2019s instructions. After 48 hours\u2019 incubation, growth medium was changed to selection medium containing blasticidin  6 \u03bcg/mL. After blasticidin selection for 12 days, the cells were treated with doxycycline 0.5\u20131 \u03bcg/mL to express Cas9 and induce Atg7 deletion. Doxycycline was replenished every 2 days for 2 weeks, after which the cells were harvested and analyzed by immunoblotting for Atg7 deletion (The nontargeting guide RNA (gRNA) TAGCGAACGTGTCCGGCGT and eviously . The 2 seviously . Subclondeletion .Whole-cell lysates and lysosomal extracts were immunoblotted as previously described , 6.5) with an equal volume of Matrigel  in the right flank of C57BL6/J mice. Daily injection of HCQ (60 mg/kg) and every-other-day injection of anti\u2013PD-1 Ab (200 \u03bcg) commenced at the tumor size of 50 mm3. CA PtenloxP Tyr::CreERT2BRaf mice (The Jackson Laboratory) were treated topically on the back with 4-HT to elicit V600EBRaf and to silence Pten expression. Tumors were measured using electric calipers . Tumor volume was calculated as L \u00d7 W2 \u00d7 0.5. In all animal experiments 2-tailed t test or 2-tailed t test for unequal variance was used to test the hypothesis that the addition of HCQ to anti\u2013PD-1 Ab is significantly different compared with anti\u2013PD-1 Ab + Veh.Tumor generation, measurement, and harvesting were performed as previously described , 9. BrieTumor was excised and digested with a tumor digestion kit  according to the manufacturer\u2019s protocol.6) from the digested tumor were stained with antibodies (please see Cells (1 \u00d7 105) were cultured and irradiated with 25 Gy x-rays. Splenocytes were then cultured with (primed) or without (unprimed) irradiated B16 cells in the presence of IL-2 (5 IU/mL) and cocultured for 48 hours. Splenocytes cultured with concanavalin A (10 \u03bcg/mL) were used as a nonspecific T cell priming control. Priming was confirmed by IFN-\u03b3 ELISA of the supernatant. Primed splenocytes were then cocultured with freshly cultured B16 cells with various target (B16) to effector (splenocytes) ratios. The B16 cell death\u2013associated LDH release and then percentage cytotoxicity were measured according to the manufacturer\u2019s protocol .B16 tumor cells  to remove cellular debris. The supernatant was passed through a 0.2 \u03bcm filter, concentrated 50-fold using an Amicon Ultra 3K membrane, electrophoresed for 0.5 cm into an SDS-PAGE gel, and stained with colloidal Coomassie . The entire stained region was excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with a Nano-ACQUITY UPLC system using a 245-minute gradient and acquisition parameters as previously described  were computed for each mouse using linear regression of the natural logarithm of tumor volume on time; the estimated slopes for the HCQ and anti\u2013PD-1 Ab or anti\u2013PD-1 Ab + Veh groups were analyzed using a 2-sample t test. All t tests were 2 tailed. A P value less than 0.05 was considered significant. P values presented in the figures are for the test of the hypothesis that the expected mean when HCQ is added to anti\u2013PD-1 Ab is significantly different from the expected mean for anti\u2013PD-1 Ab + Veh. Adjusted P values are indicated as * when the adjusted P < 0.05 and as ^ when the adjusted P < 0.10. All analyses were done using SAS/STAT software, version 9.4 of the SAS system for Windows.For continuous variables, a 2-sample All animal experiments were performed in accordance with the protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee.GS, RO, ENO, EJW, DG, XX, DWS, PAG, and RKA conceived experiments. GS, RO, ENO, JM, JA, VWR, SL, SP, JLL, SH, AR, VJ, MCN, JDW designed the experiments, conducted the experiments, and collected the data. GS, XX, ENO, EJW, DG, JDW, DWS, PAG, and RKA supervised experiments and analyzed the data. GS, DWS, DG, PAG, and RKA wrote the manuscript with assistance from all the other authors."}
+{"text": "A large number of GEPP with varied 1O2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511C71G), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation  of GEPPPM-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR. Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen ( Cholecystokinin 1 receptor (CCK1R) is expressed prominently in highly restricted brain regions, such as the basal ganglia, hippocampus, thalamus, hypothalamus, medulla oblongata ,6,7,8,9,1O2 or 1O2), usually generated in type II photodynamic action with chemical photosensitizer sulphonated aluminum phthalocyanine (SALPC) after a brief cellular incubation [CCK1R is unique among A class G protein-coupled receptors (GPCR) in that it is permanently activated ligand-independently by the lowest lying excited state molecular oxygen, the delta singlet oxygen . Energy transfer to ground state molecular oxygen results in the production of 1O2 (type II) [1O2 quantum yield (\u03d51O2) in type II photodynamic action. In contrast to chemical photosensitizers such as porphyrins and phthalocyanines, genetic manipulations will ensure that GEPP could be targeted to specified cell types or subcellular organelles with high precision. A typical photodynamic action involves three elements: light, a light-absorbing molecule (photosensitizer), and molecular oxygen. After absorption of a photon of a certain wavelength by a photosensitizer, the excited state photosensitizer molecule eventually undergoes either electron transfer or the energy transfer process. Electron transfer leads to the production of oxygen radicals (type I), such as superoxide anion (Otype II) ,30,31. T2\u2212.) [1O2 also [1O2 quantum yield (\u03d5\u03941O2 0.008) of more than eight-fold higher than O2\u2212. [KillerRed is the first major GEPP to emerge, initially thought to generate solely superoxide anion (O2\u2212.) ,34,35 bu1O2 also ,36, and han O2\u2212. . The minhan O2\u2212. ,39 but hhan O2\u2212. ,41, moduhan O2\u2212. , and forhan O2\u2212. . 1O2 quantum yields  [Q103V) [1O2 quantum yield [1O2-generating flavin-binding fluorescent protein (FbFP) photosensitizers originally from other source organisms, such as Pp2FbFP (from Pseudomonas putida), DsFbFp (from Dinoroseobacter shibae), EcFbFP (from Bacillus subtilis), CreiLOV (from Chlamydomonas reinhardtii), Mr4511C71G (from Methylobacterium radiotolerans), and AsLOV2 (from Aveva sativa), have also appeared, all with desirably sufficient 1O2 quantum yields [Different variants of KillerRed and miniSOG have also appeared, either to monomerize the KillerRed dimer (SuperNova and GreenSuperNova) ,45, to br SOPP3) ,50,51,52 [Q103V) . A red fum yield . 1O2-genm yields ,57,58,59In view of the above developments, it has become pertinent to examine whether the newly emerged GEPP could also be used for photodynamic CCK1R activation. Further, numerous works have reported that G protein-coupled receptors (GPCR) function not only from PM but, also, from intracellular membranes ,61. The C71G, and DsFbFP in AR4-2J cells after light irradiation all photodynamically activated endogenous CCK1R in the rat pancreatic acinar tumor cell AR4-2J, triggering persistent calcium oscillations, with DsFbFP being the least effective. Interestingly, light irradiation of AR4-2J cells with miniSOG expressed at the plasma membrane (PM), mitochondria (MT), or lysosomes (LS) was found to trigger similarly persistent calcium oscillations, which were inhibited by CCK1R antagonist devazepide 2 nM with a graded sensitivity of PM > MT > LS. Therefore, GEPP expressed either at PM or intracellularly are both effective to photodynamically activate CCK1R, suggesting that photodynamic action may be particularly suited for the study of intracellular GPCR without the need for extracellularly added agonists to overcome multiple diffusion barriers. In the present work, it was found that plasma membrane (PM)-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr4511PM vector was bought from Evrogen . Ampicillin and kanamycin were from CWBio . Endotoxin-free plasmid extraction kit and DH5\u00e0 competent cells were from TianGen Biochemicals . MitoTracker\u2122 Red FM was from Invitrogen . LysoTracker Red was from Beyotime . Sulfated cholecystokinin octapeptide (CCK) and CCK1R antagonist devazepide were from Tocris Cookson . Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)/F12 medium was bought from Invitrogen . Fura-2 AM was from AAT Bioquest . JetPRIME transfection reagent was from PolyPlus-transfection SA . Fetal bovine serum (FBS) was from Thermo Scientific . pKillerRed2 incubator under humidified atmosphere (5% CO2/95% air) at 37 \u00b0C, as reported previously [AR4-2J was bought from The American Type Culture Collection  and cultured in DMEM/F12 supplemented with 20% fetal bovine serum in a COeviously ,62,63,64E. coli medium LB/kana and LB/amp were sterilized and culture plates made. Liquid E. coli medium LB/kana and LB/amp had the same composition but without agar. Solid PMpKillerRed was bought from Evrogen , proliferated in and harvested from competent E. coli. A mammalian codon-optimized miniSOG gene (GenBank accession number JX999997) was synthesized de novo from nucleotides at Genscript  with the following full sequence: ATGGAAAAGAGCTTTGTGATTACCGATCCGCGCCTGCCAGACAACCCGATCATTTTCGCGAGCGATGGCTTTCTGGAGTTAACCGAATATTCTCGTGAGGAAATTCTGGGTCGCAATGGCCGTTTCTTGCAGGGTCCGGAAACGGATCAAGCCACCGTGCAGAAAATCCGCGATGCGATTCGTGACCAACGCGAAATCACCGTTCAGCTGATTAACTATACGAAAAGCGGCAAGAAATTTTGGAACTTACTGCATCTGCAACCGATGCGCGATCAGAAAGGCGAATTGCAATATTTCATTGGTGTGCAGCTGGATGGCTAG. This synthesized full miniSOG gene sequence was inserted into plasmid PMpKillerRed  to replace the KillerRed sequence. Competent E. coli were infected with the recombinant plasmid, cultured on solid LB/kana. Bacteria colonies were picked and further cultured in liquid LB/kana with shaking overnight. Proliferated plasmid was extracted with sequence verification. The plasmid so obtained was named PMpminiSOG due to the presence of the PM-localization sequence in the original Evrogen plasmid. After transfection with plasmid PMpKillerRed or PMpminiSOG, positive expressing AR4-2J cells were named KillerRedPM- or miniSOGPM-AR4-2J cells, as reported before [Plasmid d before ,32. PMpminiSOG2, the miniSOG2 [miniSOG sequence in plasmid PMpminiSOG was replaced with the synthesized miniSOG2 sequence to obtain plasmid PMpminiSOG2 . The miniSOG2 sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCCGCCTGCCTGACAACCCAATCATCTTCGCCAGCGACTCCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACCCACGGTTCCTGAGAGGACCTGAGACCGACCAGGCAACCGTGCAGAAGATCCACGACGCCATCCGCGACCAGAGGGAGATCACCGTGCAGCTGATCAACTACACCAAGAGCGGCAAGAAGTTCTGGAACCTGTTCCGGCTGCAGCCAATCAGAGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCAGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpminiSOG2 were named miniSOG2PM-AR4-2J cells. For the construction of plasmid miniSOG2  gene wasPMpSOPP, the SOPP amino acid sequence [SOPP gene was synthesized de novo after rat codon optimization. The miniSOG sequence in plasmid PMpminiSOG was replaced to obtain plasmid PMpSOPP , where the SOPP gene sequence was: ATGGAGAAGAGCTTCGTGATCACCGACCCCAGGCTGCCTGACAACCCAATCATCTTCGCCAGCGACGGCTTCCTGGAGCTGACCGAGTACTCCAGGGAGGAGATCCTGGGAAGGAACGGCCGGTTCCTGCAGGGACCCGAGACCGACCAGGCCACCGTGCAGAAGATCAGAGACGCCATCAGAGACCAGCGCGAGATCACCGTGCAGCTGATCAACTACACCAAGTCCGGCAAGAAGTTCTGGAACCTGCTGCACCTGCAGCCCATGCGGGACCAGAAGGGCGAGCTGCAGTACTTCATCGGCGTGCTGCTGGACGGCTAA. AR4-2J cells transfected with plasmid PMpSOPP as verified were named SOPPPM-AR4-2J cells.For the construction of plasmid sequence  was usedC71GPMpMr5411, the Mr5411C71G protein sequence from [C71GMr5411 gene was synthesized de novo with rat codon optimization, which was used to replace the miniSOG sequence in PMpminiSOG . The C71GMr5411 sequence was ATGGAGACCGGAGGAACCGCCACCAGCCACGTGCCAGACGAGCTGAAGGCAGAGTCCCACAGAGGCGACCCTTTCGCCGCAGCCGTGAGGGCAACCAGGATGCCCATGATCATCACCGACCCTGCCCAGCACGACAACCCAATCGTGTTCGTGAACGACGCCTTCCTGAAGCTGACCGGCTACACCAGGATGGAGGTGGTGGGAAGAAACGGCCGCTTCCTGCAGGGACCAGACACCGAGGCAGCAGCAGTGGACAGACTGAGGGCAGCCATCAGGCGGGAGGAGGACATCAGAGTGGACCTGCTGAACTACCGCAAGGACGGCAGCACCTTCCAGAACGCCCTGTACGTGGGACCCGTGAGGGACGAGGCAGGACGGGTGGTGTACTTCTTCGCCAGCCAGCTGGACGTGTCCGAGCACTACGCCCTGACCGCAGAGATCGAGAGGCTGAAGGCCGCCCTGGCCGAGGCCGAGGCCAAGCTGGCCGCCCGGTAG. AR4-2J cells transfected with plasmid C71GPMpMr5411 were named C71GPMMr5411-AR4-2J cells.For the construction of plasmid nce from  was usedDsFbFP gene [miniSOG from plasmid PMpminiSOG . The DsFbFP gene sequence was ATGAGGCGGCACTACCGCGACCTGATCAGGAACACCCCCATGCCTGACACCCCACAGGACATCGCAGACCTGCGCGCCCTGCTGGACGAGGACGAGGCCGAGATGAGCGTGGTGTTCAGCGACCCATCCCAGCCCGACAACCCTATGATCTACGTGTCCGACGCCTTCCTGGTGCAGACCGGATACACCCTGGAGGAGGTGCTGGGAAGGAACGCAAGATTCCTGCAGGGACCAGACACCAACCCACACGCAGTGGAGGCAATCAGGCAGGGCCTGAAGGCAGAGACCAGATTCACCATCGACATCCTGAACTACAGGAAGGACGGCAGCGCCTTCGTGAACAGACTGCGCATCAGGCCTATCTACGACCCAGAGGGCAACCTGATGTTCTTCGCCGGCGCCCAGAACCCCGTGCTGGAGTAG. Positive AR4-2J cells after transfection with plasmid PMpDsFbFP were named DsFbFPPM-AR4-2J cells.The bFP gene  was syntMTpminiSOG was prepared by replacing the PM-localizing sequence ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC in PMpminiSOG with the mitochondrial (MT)-targeting sequence (MTS: ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGATCCATTCGTTGGGGGATCCACCGGTCGCCACC) . LSpminiSOG was prepared by replacing the PM-localizing ATGCTGTGCTGTATGAGAAGAACCAAACAGGTTGAAAAGAATGATGAGGACCAAAAGATC sequence in PMpminiSOG with the lysosomal (LS) sequence (LTS: ATGAAGGGACAGGGAAGCATGGACGAGGGAACCGCCGACGAGAGGGCCCCCCTGATCCGGACC). Competent E. coli were infected with the plasmid, further cultured on solid LB/kana. Bacteria colonies were picked and cultured in liquid LB/kana with shaking overnight. Propagated plasmids were extracted for sequence verification. The plasmid constructs were designated MTpminiSOG and LSpminiSOG; transformed AR4-2J cells were named miniSOGMT-AR4-2J and miniSOGLS-AR4-2J cells, accordingly.The plasmid ex 543 nm for KillerRedPM, and \u03bbex 488 nm for miniSOGPM, miniSOG2PM, Mr4511C71GPM, and DsFbFPPM. Mitochondrial or lysosomal expressions of miniSOG (\u03bbex 488 nm) were verified by colocalization with MitoTracker Red or LysoTracker Red (\u03bbex 543 nm) in confocal imaging (Zeiss LSM510 META), objective \u00d7 60 oil. AR4-2J cells were cultured in 6-well plates with one round glass cover-slip in each well, and to be transfected, cells were allowed to grow to 50\u201370% confluence. Plasmid (2 \u03bcg/well) and JetPRIME transfection reagent (4 \u03bcL/well) in JetPRIME buffer (200 \u03bcL) were added; then, AR4-2J cells were cultured for a further 24 h. Positive cellular GEPP expression was verified by confocal imaging of GEPP fluorescence: \u03bbPM-AR4-2J cells. AR4-2J cells cultured in a Petri dish (35 mm) were transfected; twenty-four hours later, cells were washed in PBS before the extraction of RNA. RNA concentration was determined with a Nanodrop2000 nanospectrometer . mRNA was reverse-transcribed with a GoScript Reverse Transcription Kit A5001  to obtain cDNA. To a PCR tube was added Oligo (dT) 1 \u03bcL, RNA 1 \u03bcg, 70 \u00b0C denaturation for 5 min, cooled on ice for 5 min, before the addition of the reaction buffer (\u00d75) 5 \u03bcL, RNAase inhibitor 1 \u03bcL, M-MLV reverse transcriptase 1 \u03bcL, dNTP (10 mM) 1.25 \u03bcL, topped up with diethyl pyrocarbonate (DEPC)-treated water to 25 \u03bcL. Reverse transcription conditions: 40 \u00b0C, 60 min and 70 \u00b0C, 15 min to obtain cDNA. PCR reaction: 2-\u03bcL cDNA template, 15-\u03bcL 2\u00d7Taq Master Mix , primers 1 \u03bcL each, topped up to 30 \u03bcL. Initial de-naturation 95 \u00b0C, 5 min; PCR cycles: 94 \u00b0C, 30 s, 60 \u00b0C, 30 s, 72 \u00b0C, 1.5 min, 30 cycles, and final prolongation 72 \u00b0C, 5 min. The RT-PCR product was run on 1% agarose gel with 0.01% GoodView  added, 120 V, 40 min before imaging. PCR primers for DsFbFP were: forward 5\u2032-GGCACTACCGCGACCTGATC-3\u2032 and reverse 5\u2032-CTACTCCAGCACGGGGTTCT-3\u2032. Primers for internal reference GAPDH were: forward 5\u2032-GTGGAGTCTACTGGCGTCTT-3\u2032 and reverse 5\u2032-CCAGGATGCCCTTTAGTG-3\u2032.RT-PCR (reverse transcription-polymerase chain reaction): HiPure Total RNA Plus Mini Kit  was used as instructed in the manufacturer\u2019s manual for RNA extraction from AR4-2J and DsFbFPPM-AR4-2J cells were irradiated with white light  from a halogen cold light source  equipped with a condenser (HLL201). AR4-2J cells expressing PM-localizing miniSOG, miniSOG2, SOPP/miniSOGQ103L, Mr4511C71G, DsFbFP, MT- or LS-localizing miniSOG were irradiated with blue LED  . Power density was measured at the level of attached cells in the Sykes-Moore perfusion chamber with a power meter . Light-responding transfected AR4-2J cells were identified as GEPP-positive cells. KillerRed340/F380 and plotted against time with SigmaPlot , as reported before [N  identical experiments.Parental control or transfected AR4-2J cells grown on glass cover-slips in 6-well plates were loaded with Fura-2 AM  for 1 h after assembly in the Sykes-Moore perfusion chamber. Cytosolic calcium was measured in an inverted fluorescent microscope (Nikon TE-2000U)  coupled to a Photon Technology International  calcium measurement system with alternating excitations at 340 nm/380 nm (DeltaRam X); emitted Fura-2 fluorescence  was detected with a charge-coupled device (CCD) camera . Calcium concentration was expressed as Fura-2 fluorescence ratios Fd before ,63,64,66N (as indicated) independent experiments were presented in bar graphs as mean \u00b1 SEM, unless specifically stated otherwise. Student\u2019s t-test was used for statistical analysis against controls, and p < 0.05 was taken as significant and indicated with an asterisk (*).All calcium tracings and other graphs were plotted with SigmaPlot. For calculation and comparison of the strength of induced calcium responses, the calcium peak area above the baseline was integrated . Statistical data from PMpKillerRed, PMpminiSOG, PMpminiSOG2, PMpSOPP, C71GPMpMr4511, and pDsFbFP , DsFbFP demonstrated markedly dimmer fluorescence, suggesting poorer protein expression, as shown in the confocal images  in parental AR4-2J cells . In parallel experiments, white light irradiation  from the halogen light source was found to induce sustained calcium oscillations in KillerRedPM-AR4-2J cells  elicited persistent calcium oscillations in miniSOGPM-  like previously reported ,32, we niniSOGLS a, with aoTracker b,c. \u22122, 1.5 min) was found to have no effect on the baseline calcium level in parental AR4-2J cells, but in these cells, CCK 20 pM induced marked calcium responses  induced long-lasting calcium oscillations in miniSOGMT-AR4-2J cells  similarly induced long-lasting calcium oscillations in miniSOGLS-AR4-2J cells  identical experiments was calculated and plotted as bar graphs (i). Note the sharp difference in the calcium response after LED light irradiation in parental AR4-2J cells, and dark or light responses in MT or LS miniSOG-transfected AR4-2J cells (i). Quantitative analysis of calcium responses in original tracings, as represented in PM-AR4-2J cells  were inhibited nearly completely by the CCK1R antagonist devazepide 2 nM  but no effect on miniSOGLS photodynamic action  . For the representative experiment shown in MT photodynamic action was inhibited without significance by devazepide 2 nM (from 100% to 65%) but significantly by devazepide 10 nM (from 100% to 29%)  d. miniSO to 29%) d. C71G, and DsFbFP after light irradiation all photodynamically activated CCK1R to induce persistent cytosolic calcium oscillations in AR4-2J cells, but the photodynamic effect of DsFbFP was much reduced in comparison, likely due to poor protein expression. Permanent photodynamic CCK1R activation was achieved in AR4-2J cells by miniSOG expression not only at the plasma membrane (PM) but, also, in mitochondria (MT) and lysosomes (LS). Calcium oscillations induced by miniSOG photodynamic action at intracellular sites showed reduced sensitivity to inhibition by CCK1R antagonist devazepide 2 nM with the order of PM > MT > LS. In the present work, it was found that PM-expressed KillerRed, miniSOG, miniSOG2, SOPP, Mr45111O2 generated in type II photodynamic action with SALPC, KillerRed, or miniSOG as the photosensitizer [\u22122, 4 min and miniSOG with blue LED 450 nm, 85 mW\u2027cm\u22122, 1.5 min) triggered long-lasting calcium oscillations in both KillerRedPM-AR4-2J and miniSOGPM-AR4-2J cells (Q103L), Mr4511C71G, and DsFbFP expressed at the plasma membrane  [bl50% miniSOG: 2.85 min and DsFbFP: 0.35 min) [PM-AR4-2J cells after blue LED irradiation (Dinoroseobacter shibae) has been known to be expressed in CHO-K1 cells as a dimer, which might also affect its fluorescence, unlike the monomeric miniSOG, miniSOG2, or SOPP, all sourced from Arabidopsis thaliana [Although the DsFbFP protein level was low, as shown by the dim DsFbFP fluorescence, DsFbFP mRNA were expressed at sufficient levels in AR4-2J cells, as verified by RT-PCR experiments g. Althou = 0.35)  is the h.35 min) . This waadiation g. DsFbFPthaliana ,70. The thaliana  might fa1O2 after light irradiation in a type II photodynamic action [1O2 of 0.03 (as reviewed in [2\u2212.) [1O2 [1O2 than O2\u2212., with an \u03d51O2 of 0.008, whilst monomeric KillerRed SuperNova has an \u03d51O2 of 0.02 [SOPP or miniSOGQ103L) has a \u03d51O2 of 0.25 [C71G sourced from Methylobacterium radiotolerans has a \u03d51O2 of 0.19 [1O2 among the lot at 0.33 [1O2 from 0.008 to 0.33 . Killerin [2\u2212.) , but mor\u2212.) [1O2 ,36,43. P of 0.02 . miniSOG of 0.02 . The sin of 0.25 . Mr4511C of 0.19 . DsFbFP  at 0.33 . Althoug0.33 see , no signefficacy . This in\u22122, 1 min) induced phototoxicity in a small percentage of cells two hours after light irradiation of miniSOG-HEK293, miniSOG2-HEK293, and SOPP-HEK293 cells, but no marked difference was found in HEK293 cells expressing SOPP (9%) with a \u03d51O2 of 0.25 or miniSOG (11%) with a \u03d51O2 of 0.03 [1O2 of only 0.03 to permanently activated photodynamically CCK1R in miniSOGPM-AR4-2J cells in the present work might be related to the progressive photochemical transformation of the fluorophore flavin mononucleotide (FMN) to lumichrome and the photo-oxidization of internal residues in miniSOG to significantly increase its \u03d51O2 up to 10-fold [1O2 is not determined; see Avena sativa phototropin 1, light irradiation was found to induce progressive photochemical dissociation or the release of FMN from the AsLOV2 protein moiety, leading to significantly increased \u03d51O2 [It has been found by others that blue LED irradiation  of the visible spectrum (full-spectrum white light was used in the present work), whereas all others by blue light (450 nm)   (for the(450 nm) . Althoug(450 nm)  might waC71G, and DsFbFP. It would be ideal if KillerRed could be subjected to further annotations, to shift its maximal excitation peak toward even longer wavelengths (a red shift), possibly by genetic code expansion [C71G, DsFbFP -induced calcium oscillations were inhibited by CCK1R antagonist devazepide 2 nM significantly in miniSOGPM-CHO-K1 cells, slightly in miniSOGMT-AR4-2J cells, but not at all in miniSOGLS-AR4-2J cells  or lysosomal (LS) miniSOG were found to photodynamically trigger persistent calcium oscillations similarly . LED irr2J cells , althoug2J cells . The cal2J cells . We beli present ,76,77. S present  and are  present ; therefonometers  could eaLS photodynamic CCK1R activation and the little inhibition afforded by CCK1R antagonist devazepide in miniSOGLS-AR4-2J cells. The insensitivity of lysosomal CCK1R to the antagonist devazepide 2 nM might also be due to limited accessibility of lysosomal CCK1R to extracellularly added devazepide 2 nM. Such a reduced sensitivity of intracellular GPCR to ligands was noted before for the nuclear membrane GPCR in cardiomyocytes, for example [The lysosomal accumulation of endocytosized CCK1R and partial CCK1R degradation ,79,80,81 example . 1O2 generation, due to its limited lifetime of 1 \u00b5s [1O2 in the cellular milieu has been suggested to be in the tens of nanometers or more (20\u2013150 nm) [1O2 could diffuse from the plasma membrane (PM), mitochondrial (MT), or lysosomal (LS) membranes as the origin to within a circle with a radius of 20\u2013150 nm. Although the emphasis in the present work was the subcellular localization of miniSOG expression and therefore of subcellular  of 1 \u00b5s ,84,85 th\u2013150 nm) ,86,87,881O2 could well help to further investigate GPCR functions at these intracellular sites. Photodynamic GPCR activation/modulation might offer distinct advantages over conventional receptor pharmacology in that no ligand is needed for photodynamic activation after GEPP  expression at defined intracellular sites. Only light irradiation is required to permanently activate the intracellular GPCR. The limited diffusion distance of photodynamically generated 1O2 (20\u2013150 nm) may ensure spatial precision and specificity. It may be noted that there is abundant evidence for GPCR expression, localization, and function at nuclear , mitocho1O2 activation of CCK1R. In the present work, all GEPP examined were found to elicit persistent calcium oscillations  photodynamically, either from the PM, MT, or LS. We have previously found that the SALPC photodynamic activation of CCK1R in rat pancreatic acini involved the near quantitative transformation of the CCK1R protein dimer to the monomer . In addiC71G, and DsFbFP) reported in the literature were found to photodynamically activate the endogenous CCK1R in AR4-2J cells after plasma membrane expression. The miniSOG expression at intracellular sites was also found to induce persistent calcium oscillations or CCK1R activation (In conclusion, representative GEPP (KillerRed, miniSOG, miniSOG2, SOPP, Mr4511tivation . The pre"}
+{"text": "Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore\u2013microtubule attachments in yeast and mouse meiosis. In  Formation of a diploid embryo requires that sperm and egg contribute exactly one copy of each chromosome. The cell division in charge of reducing ploidy of the genome is meiosis, which involves two chromosome segregation steps after a single round of DNA replication . Female Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase composed of a catalytic subunit C (PPP2C), a scaffolding subunit A (PPP2R1), and a regulatory subunit B (PPP2R2\u2013PPP2R5) . While tPP2A:B56 can be targeted to distinct sites by different proteins, and two mechanisms have been characterised at the structural level. While an N-terminal coiled coil domain in Shugoshin/MEI-S332 binds PP2A:B56 , other sCaenorhabditis elegans, the meiotic role of PP2A remains unexplored. The core components of the PP2A holoenzyme in C. elegans are LET-92  and PAA-1 (scaffolding A subunit), and the regulatory B subunits are B55SUR-6  and paa-1(RNAi),\u00a0oocytes microtubules do not organise into a bipolar structure (let-92(RNAi) oocytes, displaying a cluster of small foci  activity (i.e. anaphase progression). Cytoplasmic securinIFY-1 degradation proceeded at similar rates in wild-type and LET-92-depleted oocytes  in vivo, we attempted to tag endogenous LET-92 with GFP but were unsuccessful, presumably due to disruption of its native structure, in agreement with a recent report . We geneeiosis I . The fuseiosis I , in agreeiosis I . During eiosis I .C. elegans B56 orthologues, PPTR-1 and PPTR-2, and the single B55 orthologue, SUR-6  during metaphase of meiosis I  with RNAi-mediated depletion of B56\u03b1PPTR-1 (\u2018pptr-1(RNAi)\u2019), which we will refer to hereafter as \u2018PPTR-1/2 depletion\u2019. While there was no significant change in GFP::PAA-1 localisation upon depletion of B56\u03b1PPTR-1 or deletion of B56\u03b3PPTR-2, no PAA-1 signal is detected associated with chromosomes upon double PPTR-1/2 depletion  does not inhibit B56 localisation  involved in targeting PP2A:B56 to meiotic chromosomes. Two of the most studied proteins involved in B56 targeting are Shugoshin and BubR1. The BubR1 orthologue, Mad3 spindle  and Mad3lisation . We thery CRISPR  and obse1 levels  and a sl2 levels . ParticuC.\u00a0elegans B56 subunits display a dynamic localisation pattern similar to that of the kinase BUB-1 throughout meiosis I   . Sequenc 282\u2013287 . When co 282\u2013287 . In addi elegans . The put elegans , with seL282A,V285A was indistinguishable from that of wild-type BUB-1 during metaphase I , but segregation was achieved in 100% of the cases with no PBE\u00a0defects were detected. Interestingly, the newly identified LxxIxE motif specifically recruits PP2A:B56 to metaphase chromosomes and anaphase central spindle, but not anaphase chromosomes.Using a fluorescence polarisation-based assay, we confirmed that the LxxIxE motif of BUB-1 binds to purified recombinant PPTR-2, and this binding is abolished by the L282A,V285A mutations . While laphase I , localisA mutant . B56\u03b3PPTxE motif . In cont spindle  without  spindle . Some GFteractor , is not teractor . Mutatioteractor , indicatPPTR-2 with higher affinity than non-phosphorylated LxxIxE peptide  had a similar effect to that of the BUB-1L282A,V285A mutant: it significantly reduced B56\u03b1PPTR-1 localisation  destabilises the kinase domain and prevents its interaction with Mad1MDF-1  the two pools of B56\u03b3PPTR-2.These results show that the BUB-1 kinase domain is important for recruitment of B56\u03b3C. elegans. PP2A is essential for meiosis I: depletion of the catalytic or scaffold subunits leads to severe spindle assembly defects, lack of chromosome segregation, and failure to achieve PBE. These effects are likely brought about by a combination of different PP2A subcomplexes with varying regulatory B subunits. PP2A:B56 regulates chromosome dynamics prior to segregation since depletion of the two B56 orthologues, PPTR-1 and PPTR-2, leads to alignment defects. We have uncovered a new phospho-regulated B56 LxxIxE motif in C. elegans BUB-1 that recruits PP2A:B56 and is important for chromosome alignment during meiosis I.In the present article, we have uncovered new roles for PP2A during oocyte meiosis in Depletion of the sole catalytic or scaffold PP2A subunits leads to massive meiotic failure and embryonic lethality. The earliest effect we could see in our experimental set\u00a0up is a failure to assemble a bipolar spindle. This will of course have a direct impact on any process that should occur after spindle assembly, including chromosome alignment and segregation, followed by PBE. However, BUB-1 depletion leads to severe spindle assembly and alignment defects, yet in the majority of cases, chromosomes segregate (with visible errors \u2013 lagging chromosomes) and polar bodies do extrude. Therefore, lack of a proper bipolar metaphase spindle is not sufficient to result in lack of segregation or PBE. PP2A complexes harbouring the B56 subunits PPTR-1 and PPTR-2 are required to target the phosphatase to chromosomes to regulate chromosome alignment in metaphase I. Furthermore, this chromosomal B56 pool is recruited by BUB-1 through its LxxIxE SLiM motif. Mutation of this motif that prevents binding to B56 subunits leads to alignment defects. B56 subunits however are not involved in spindle pole targeting of PP2A, suggesting that this spindle pole pool is the one relevant for spindle assembly. Our attempts to address a possible role for other B subunits were focused on RSA-1 and SUR-6 given their reported centrosomal roles during mitosis . HoweverC. elegans oocytes is highly dynamic, and differences have been observed between metaphase and anaphase. For example, while kinetochore localisation of the CLASP orthologue CLS-2 during metaphase depends on BUB-1, a pool of CLS-2 is able to\u00a0localise in the anaphase central spindle in a BUB-1-independent manner  resemble those reported in the absence of kinetochore proteins . InteresC. elegans, the Aurora B orthologue, AIR-2, concentrates in the interface between homologous chromosomes  closely resembling the consensus for PP2A:B56 reported in During meiosis in bivalent . Consistbivalent . During tochores  and it iC. elegans BUB-1 .Strains used in this study were maintained at 20 degrees unless indicated otherwise. For a complete list of strains, please refer to For RNAi experiments, we cloned the different sequences in the L4440 RNAi feeding vector .600 of 0.6\u20130.8. Isopropyl-\u03b2-d-thiogalactopyranoside (IPTG)\u00a0was added to a final concentration of 1 mM, and cultures were incubated overnight at 20\u00b0C. Bacteria were then seeded onto NGM plates made with agarose and allowed to dry. L4 worms were then plated on RNAi plates and grown to adulthood at 20\u00b0C for 24 hr in the case of let-92(RNAi), bub-1(RNAi), and paa-1(RNAi) and 48 hr in all other cases.All sequences were inserted into L4440 using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and transformed into DH5a bacteria. The purified plasmids were then transformed into HT115(DE3) bacteria . RNAi clFor the generation of in situ-tagged GFP::SUR-6, we used the self-excising cassette method . In brieK718R,D847N, BUB-1L282A,V285A, BUB-1S283A,\u00a0and BUB-1L282A,V285A, K718R,D847N were generated by Sunybiotech.The strains AID::GFP::GSP-2, GFP::PAA-1, BUB-1.CTCGACAATATCATCTCCAGATTATTGGAAGATGcctaaagatccagccaaacctccggccaaggcacaagttgtgggatggccaccggtgagatcataccggaagaacgtgatggtttcctgccaaaaatcaagcggtggcccggaggcggcggcgttcgtgaagAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGACGTAGAAAAGCTTAAT..GCTACTGACGACGCGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGGAGGTGGATCCGGTGGTGGATCCTCGGTTGTCGAAGAA.The wild-type sequence:AAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGwas mutated to:CGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAgtatgggaaaacatttgttaattttagacgttatcttttttcagGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTGTAACCGATGATCAAAGGACAGTAGCTGTGThe introduced changes are shown in red, and synonymous mutations are shown in cyan.The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGAACGCCAATCTAAATCCTAGAAGACGTCATThe wild-type sequence:CATCTTTCACCAGTCAGTGAGAAAACGGTT GATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTwas mutated to:CACCTTGCGCCAGTCAGTGAGAAAACGGTTGATGATGAGTTCAACGCCAATCTAAATCCTAGAAGACGTThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See The wild-type sequence:CTTTCACCAGTCAGTGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGGACAGTAGCTGTGAAGTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCGACTGGGGACGTGCCATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATwas mutated to:GCATCACCAGCTAGCGAGAAAACGGTTGATGATGAGGAGGAAAAGCGAAGCCGGATTTATTCGCCGCTGGTTGCAACGAAGGATGCTCACAGACCTGCACTTCGGAGCAAAATTGAGAATCCTCCAGCGACAGTGACACTTTCGTCGGATACAAAGTCTGCTTCGGAGAAAGATGTTAGTGATTCCGATGATGCAGATGATGATGAAAGACTCAAGATTATGACTGCCGGCAGAAAAGATGGTAACCCTCCAGACCGTTCCACAAGCATATCTTCCAACTATTCAACTGCTTCTGCAAGAACATCAAAGAGTGGAGCTGGATTGGATTTGATGGCGGAAAATAAGTGTTTGGAGGCACATGCTATGTTTTCCGACACTGTACATCTTGCTAGCGAAAAGACAATGGTCCTTGGCGATGATTCTGTCTTCGTTCCAGAAAGATCTTTAGCTACTACGCAGATAGTTACTGACTTTTCCGTGCTCTGTGATCCTGATCCGACAATGACCATTACACAGGAGCGTCCGAAAAAAGTGTCGAATGGGTTGAATGTTGTTTACGATGAGGCAGCCGAACCGGAAGAATCTCAGAAAGTTGAGGAATCTGAAGTACAACCCGAAATTGTCCTAGTTTCTCCAGTGACGCAAACCTCACCAGCTACAATGTTTAATGATAGTGGGTTTATCGAAAAATATAACAACTTATGTTTTAATTTTTAGTTTATGACGATGAAATCGAGTTTGGCTTTTTCAAACCGTCTCGTGGTAATTTCGTCACATCGACCCCCGCACAAGGAGTTCATTTGGTCAACATTGATGAATATTTCGGAAATAAAGAGGAGGAAAGCACTCACGAACAGGAAGCTCCAGTATTTGTTGCTCCAACCAGCAGTACTTTCAGTAAATTAGTAAGTGCCAGACAAATTTTCGACATACTATTCAAACTTTTTCAGACACGTCGAAAGTCACTAGCAGCAAATCAAGCCGTTCAGCCCTCAGTCACAGAGTCATCAAAGCCTGAACGATCAGATCCTAAAGATTCATCTATCGATTGTTTGACAGCTAATCTAGGAAGACGTCTTTCAATTGGTGCTGATGAAATTCCAAATCTCACTGAAAACAACGAATCTGAAATCACTGGTTGCAAGATTCGTCGGCGCAGTGAAATTATCAAGCAAGGAGACATCAATCCATGGGACGAAACTCTTCGAAAAAAATTGATGTGTCTTGTGCGTCCTCCCCAGAATATGCACGAGTTCCAAGAACGAGCACCGAAGATTCAAGCTCTGAGAGACTGCGAGGTTAGCGGAGAAAAGCTCCACATTCAAACTCTTATTGGTCAAGGTGGATACGCTAAAGTATACCGGGCTGTAACCGATGATCAAAGAACAGTAGCTGTGCGCTACGAGGTGCCATCATGTTCGTGGGAAGTGTACATTTGCGACCAAATGCGGAATCGCCTGAAAGATCGAGGTTTGGAGCTGATGGCCAAATGTTGCATTATGGAAGTGATGGATGCTTATGTTTATTCAACTGCTTCGCTTCTTGTTAATCAGTACCACGAATATGGAACGCTGCTTGAATATGCGAATAACATGAAGGATCCGAATTGGCACATAACCTGCTTCTTGATTACCCAAATGGCCCGAGTTGTGAAGGAAGTCCATGCCTCTAAAATTATTCATGGAGATATCAAACCGGATAATTTTATGATCACCAGAAAGTATGGGAAAACATTTGTTAATTTTAGACGTTATCTTTTTTCAGGATCGATGATAAATGGGGCAAAGATGCTCTGATGAGTAACGACAGCTTTGTCATCAAGATTATCAATTGGGGACGTGCGATTGACATGATGCCACTGAAGAACCAGCGTAACGCCAATCTAAATCCTAGAAGACGTCATThe introduced changes are shown in red, and synonymous mutations are shown in cyan.See C. elegans oocytes was used with minor modifications PVSEKTC. Serum was adsorbed with a non-phosphorylated peptide (RRRHLSPVSEKTC) followed by affinity purification with the antigenic, phosphorylated peptide. Different fractions were tested in immunofluorescence by incubating different dilutions  with 1 \u00b5M and 10 \u00b5M of either non-phosphorylated or phosphorylated peptide. Only the phosphorylated peptide compited out the signal. Additionally, we used the BUB-1d-lysine -coated slide, and a 24\u00a0\u00d7\u00a024 cm coverslip was gently laid on top. Once the worms extruded the embryos, slides were placed on a metal block on dry ice for\u00a0>10 min. The coverslip was then flicked off with a scalpel blade, and the samples were fixed in methanol at 20\u00b0C for 30 min. After blocking in phosphate-buffered saline\u00a0(PBS) buffer plus 3% bovine serum albumin\u00a0and 0.1% Triton X-100 (AbDil), samples were incubated overnight at 4\u00b0C with anti-BUB-1 . We used the strain HY604, which is a temperature-sensitive allele of the the APC component MAT-1, that arrests in meiosis I prior to spindle rotation when moved to the restrictive temperature.For GFP immunoprecipitations, we followed a published protocol  with minIP samples were run on 4\u201312% Bis\u2013Tris sodium dodecyl sulfate gels with MOPS running buffer, and the gel was stained using Quick Coomassie Stain (Generon). Bands of interest were cut and washed with water:acetonitrile (50:50), followed by a wash with 100 mM ammonium bicarbonate. The gel pieces were then washed with 100 mM ammonium bicarbonate:acetonitrile (50:50), followed by a final wash with acetonitrile. Gel pieces were dried using a SpeedVac.Samples were reduced with 10 mM DTT in 20 mM ammonium bicarbonate and alkilated\u00a0with 50 mM IAA (iodoacetamide)\u00a0in 20 mM ammonium bicarbonate. Samples were then washed sequentially with 100 mM ammonium bicarbonate, 100 mM ammonium bicarbonate:acetonitrile (50:50), and acetonitrile. Gel pieces were dried using a SpeedVac.Trypsin solution (12.5 \u00b5g/ml stock in 20 mM ammonium bicarbonate) was added to cover the gel pieces and incubated for 30 min on a shaking platform and incubated sample overnight at 30\u00b0C on a shaker. Peptides were extracted by standard procedures and reconstituted in 10 \u00b5l of 5% formic acid/10% acetonitrile. After vortexing for 1 min, water was added to 50 \u00b5l.Samples were run on an Ultimate 3000 RSLCnano system (ThermoFisher Scientific) coupled with\u00a0a Q-Exactive Plus Mass Spectrometer (Thermo Fisher Scientific). Peptides initially trapped on an Acclaim PepMap 100 (Thermo Fisher Scientific) and then separated on an Easy-Spray PepMap RSLC C18 column (Thermo Fisher Scientific). Sample was transferred to mass spectrometer via an Easy-Spray source with temperature set at 50\u00b0C and a source voltage of 2.1 kV. The mass spectrometer was operated on in data-dependent acquisition mode (top 15 method). MS resolution was 70,000 with a mass range of 350\u20131600. MS/MS resolution was 17,500.C. elegans proteome and BUB-1 specifically, using the Mascot Search Engine (Mascot Daemon Version 2.3.2). Type of search used was MS/MS Ion Search using Trypsin/P. Carbamidomethyl (C) was set as a fixed modification, and variable modifications were as follows: acetyl (N-term), dioxidation (M), Gln to pyro-Glu (N-term Q), oxidation (M), deamidation (NQ), and phosphorylation (STY).RAW data files were extracted and converted to mascot generic files (.mgf) using MSC Convert. Extracted data then searched against Peptide mass tolerance was\u00a0\u00b110 ppm (# 13C\u00a0=\u00a02) with a fragment mass tolerance of\u00a0\u00b10.6 Da. Maximum number of missed cleavages was 2.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner C. elegans PPTR-1 (UniProtKB O18178) and PPTR-2 (UniProtKB A9UJN4-1). A guide tree was calculated from the distance matrix generated from sequence pairwise scores.All sequence alignments were performed using Clustal Omega , versionC. elegans PPTR-1 (S420-V435) and PPTR-2 (S449-A464) were used for the alignment. The canonical sequences of each B56 isoform were retrieved from UniProt , \u03b4 (S454-A469), \u03b2 (S409-V424), \u03b1 (S403-V418), \u03b5 (S395-V410) and  UniProt .C. elegans BUB-1 , human BubR1 , and RepoMan  were aligned with Clustal Omega  or H1. At t\u00a0=\u00a00, 15 ng/\u00b5l Cdk1/Cyclin B was added, before incubation at 30\u00b0C for 30 min. Aliquots were taken immediately after Cdk1/CyclinB addition at t\u00a0=\u00a00 and then again after the 30 min incubation. All samples were then incubated at 70\u00b0C for 15 min in a final concentration of 1\u00d7 LDS buffer. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis\u00a0(SDS-PAGE) was then conducted on a NuPage 4\u201312% Bis\u2013Tris gel (Thermo) with MES buffer before being stained with ProQ Diamond (Thermo) and imaged using Bio-Rad ChemiDoc. Once fluorescence was recorded, Coomassie staining was performed. For the western blot, SDS\u2013PAGE was conducted as above with 67 ng of substrate protein per well before the western was conducted using a nitrocellulose membrane  and 1\u00d7 NuPage transfer buffer (Thermo). The membrane was blocked using Intercept PBS blocking buffer (LI-COR), the primary antibodies used were anti-GST at 1:1000 (made in sheep), and anti-phospho\u00a0Ser 283 1:20,000 (made in rabbit). Secondary antibodies were anti-sheep IRDye 680RD anti-rabbit 800CW (LI-COR),\u00a0both at 1:50,000. The membrane was then imaged using LI-COR Odyssey CLx.Forty microlitre\u00a0reactions were set up containing 40 mM Tris\u2013HCl pH 7.5, 100 \u00b5M ATP\u00a0(Adenosine triphosphate), 10 mM MgClEscherichia coli strain BL21 (DE3) and the bacterial culture was incubated overnight at 37\u00b0C with shaking at 220 rpm. Bacteria were grown in TB medium at 37\u00b0C with shaking at 220 rpm until OD600 reached\u00a0~0.6\u20130.8 and induced with 150 \u03bcM IPTG. Induction was performed at 18\u00b0C with shaking at 220 rpm for\u00a0~16 hr. Cells were pelleted and lysed by sonication in 50 mM NaP, 300 mM NaCl, 10 mM imidazole, 10%\u00a0glycerol, 0.5 mM TCEP,\u00a0(tris(2-carboxyethyl)phosphine) and protease inhibitors. After binding to a Ni-NTA column, protein was washed with 20 mM imidazole and then eluted with 350 mM imidazole. The tag was cleaved with 6xHis-tagged TEV (Tobacco Etch Virus Protease)\u00a0protease overnight at 4\u00b0C, and the tag and protease were removed from the sample by binding to Ni-NTA. PPTR-2 was concentrated and further purified using a HiLoad 16/600 Superdex 200 pg size exclusion column.PPTR-2 was expressed from pHISTEV30a vector as a 6xHis- and Strep-tagged protein (plasmid fgp_445) in The following peptides were synthesised by peptides and elephants GmbH: BUB-1 FITC-Ahx-NPRRRHLSPVSEKTVDDEEE, pBUB-1 FITC-Ahx-NPRRRHLphSPVSEKTVDDEEE, and LAVA FITC-Ahx-NPRRRHASPASEKTVDDEEE. Reactions (35 \u00b5l) were set up in FP buffer  containing peptide concentrations 0.5\u20131 \u00b5M. These were then used to create a 1:2 serial dilution series using FP buffer containing the same peptide concentrations as above as well as the indicated concentration of PPTR-2. Reactions were left for 30 min before triplicates of each dilution were aliquoted into a black 384-well plate, centrifuged , and analysed using the PheraStar FS.For time-dependent analysis, metaphase I was taken as time\u00a0=\u00a00 s. This frame was chosen as the previous frame where the first indication of chromosome separation was visible. All image analysis was done in Fiji . For totCentral spindle to chromosome ratio for PPTR-2::GFP  was obtaFor the alignment/congression analysis, we selected videos in which the spindles were contained in a single Z-plane at \u221280 s. There, we established the spindle axes with a line extending from pole to pole and the \u2018metaphase plate\u2019 with a perpendicular line in the middle of the spindle see . A line Contingency tables were analysed using the Fisher\u2019s exact test (two\u00a0tailed), and the p\u00a0values are presented in the figures and/or in the text.The 4D TIFF files were converted to video (.avi) files using a custom-made macro that uses the StackReg Fiji plugin for image registration. Videos were assembled using maximum-intensity projections; hence, the videos might not match a specific panel within the main figures, which are single slices or partial projections in order to highlight specific characteristics. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Using a combination of biochemistry, genetics and live imaging, Borja et al. show that the kinase BUB-1 recruits, through two regulatory subunits, the phosphatase PP2A to meiotic spindle chromosomes during oocyte meiosis I to promote proper congression of the chromosomes prior to anaphase. This recruitment occurs independently of the conserved protein Shugoshin, which has been shown by others to promote PP2A recruitment to chromosomes during mouse oocyte meiosis I. Moreover, Borja et al. show that phosphorylation of a peptide motif in BUB-1 promotes this recruitment, and that this BUB-1motif is likely targeted by CDK-1 for phosphorylation to provide proper temporal regulation of these events.Decision letter after peer review:[Editors\u2019 note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]C. elegans Oocytes\" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jakob Nilsson (Reviewer #2); Patrick Meraldi (Reviewer #3).Thank you for submitting your work entitled \"PP2A:B56 Regulates Meiotic Chromosome Segregation in eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in Reviewer #1:C. elegans oocyte meiotic cell division. They show that PP2A and two conserved B56-type regulatory subunits are localized to spindle poles, chromosomes and the central spindle (with some differences for the B56 subunits), and that all required for spindle assembly, chromosome congression to the metaphase plate, and for chromosome segregation (with some redundancy for the two B56 subunits). The further show that a conserved LxxIxE motif in BUB-1 recruits most of one and some of the other B56 subunit, and that the kinase domain of BUB-1 is required to recruit the other (presumably through an intermediary). Thus in C. elegans, a novel form of PP2A recruitment functions, with the other known recruiters, Shugoshin and Mad3 , appearing not be required. The authors use mutational analysis to nicely document the requirements for both parts of BUB-1 in PP2A recruitment, and also identify a phosphorylated residue in BUB-1 that may be involved in CDK-1 regulation of the recruitment. The authors propose that BUB-1 recruitment of PP2A to the central spindle is important for chromosome segregation during anaphase in C. elegans oocyte meiosis I.Borja et al. describe their analysis of the requirements for the phosphatase PP2A during C. elegans oocyte meiosis, with results that will be of substantial interest to investigators studyng oocyte meiotic cell division, the advances are in my opinion to incremental, and also lacking in conclusiveness as to the actual mechanism involved. Therefore as written the manuscript is not suitable for publication in eLife, as summarized in the major comments below.While the authors provide an extensive analysis of the requirements for PP2A during 1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out. Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other 3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in eLife.Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of 1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 \u2013 IP or purified components3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on 3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim. [Editors\u2019 note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors\u2019 response to the first round of review.]You will see from the reviews that all the reviewers agree that your study will be of interest to the meiosis community in identifying that BUB1 rather than Sgo recruits PP2A, but that also all agree that more experiments are required to provide firm evidence for a number of your conclusions that would be necessary for publication in eLife; in particular whether BUB1 directly binds to PP2A, through the LxxIxE motif, and whether Cdk phosphorylation of this is important. After discussion, they concluded that the amount of time and effort that will be required for these experiments is such that the fairest thing to do is to return the manuscript to you.The new version of the manuscript has numerous new experiments to address reviewers\u2019 comments/concerns. Relating to the particular two points highlighted by the reviewing editor, we provide data showing that the BUB-1 LxxIxE motif binds the B56 orthologue PPTR-2 in vitro and Ser 283 phosphorylation increases affinity. Furthermore, mutating Ser 283 to Ala drastically inhibits B56 recruitment in vivo resulting in a chromosome alignment defect similar to the L282A,V285A mutant.We also performed in vitro kinase assays and showed that Cdk1 can phosphorylate Ser 283 in BUB-1 and developed a phospho-specific antibody recognising phosphor-Ser 283 and show that phosphorylated BUB-1 is detected \u2026 in vivo.Thanks to the new quantitative phenotypic analysis we performed, it became clear that B56 subunits play a role during chromosome alignment/congression prior to anaphase. Furthermore, this relies on B56 recruitment by the newly identified BUB-1 LxxIxE motif.Reviewer #1:[\u2026]1) The authors provide clear evidence that PP2A is required for spindle assembly, congression of chromosomes to the metaphase plate, and chromosome segregation (with some caveats as to chromosome segregation noted below). While the authors favor and propose that the chromosome segregation defects are due to central spindle defects , they in fact provide no evidence to support this conclusion. It is entirely possible that earlier spindle assembly defects are responsible for the subsequent defects in both congression and segregation. The authors fail to address this possibility and provide no evidence to rule it out.We agree we might have oversimplified the phenotypes and the connection between them. Our biggest mistake was to base the alignment analysis on subjective observations its classification as either \u201caligned\u201d or \u201cmisaligned\u201d.Firstly, we performed a new analysis related to the chromosome alignment phenotype. This is now detailed in the Materials and methods section and it provides more rigorous and less subjective quantifications which lead us to a better characterisation of this phenotype. This had a tremendous impact on the manuscript, because it is precisely this process of chromosome alignment where BUB-1 targeted PP2A:B56 plays a role. To avoid any potential amplification of the phenotypes due to partial loss of function in PP2A subunits, we decided to measure the phenotypes on the GFP::tubulin expressing strain and not on the strains with tagged PP2A subunits.PP2A is essential for the assembly of a bipolar spindle  and chromosome alignment and segregation. While we agree with the reviewer that in this case it is difficult to tease out whether the alignment and segregation are only a direct consequence of this, depletion of the B56 subunits PPTR-1 and PPTR-2 does not affect spindle assembly but perturbs chromosome alignment/congression . Since this phenotype is measurable and independent from any noticeable spindle defect, we focused on this \u201cclean\u201d phenotype and sought to understand the molecular mechanisms driving it.Without some idea as to which targets PP2A acts through, the advance is incremental relative to what is known in other systems, only showing a variation on how LxxIxE motifs can recruit PP2A to spindle structures through BUB-1 instead of other factors. The authors discuss the AuroraB kinase AIR-2 as a possible target but provide no analysis of such a role. Without more mechanistic insight, the manuscript is of substantial interest but seems more appropriate for a more specialized journal such as Molecular Biology of the Cell.C. elegans, it was not clear what role(s) BUB-1 play. Therefore, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we think this will require an in depth analysis that will be the focus of another paper.While we respect the reviewer\u2019s view on the advance provided by our manuscript, we do believe we provide mechanistic insight into the targeting of PP2A:B56 in a context in which the previously known/characterised regulators (BubR1 and Shugoshin) do not play a role. On the other hand, while BUB-1 is known to be important during female meiosis in C. elegans mutants that chromosomes segregation and polar extrusion are not tightly correlated. The authors should establish a more direct approach to quantifying the defects in chromosome segregation; as written only representative examples are shown.2) In figures throughout the manuscript, the authors use polar body extrusion as a proxy for chromosome segregation defects. However, it is known from work on other We apologise for the misunderstanding. We did not intend to use PB extrusion as a proxy for chromosome segregation defects, but rather analysed it as an independent process. We are now making this clear in the manuscript. We analyse chromosome alignment defects (see below), lagging chromosomes, and polar body extrusion. For these three phenomena, we provide the quantitative assessment for each condition and show a representative image (or sets of images).3) The authors show that there are severe spindle assembly defects after knockdown of PP2A but only show microtubules and chromosomes to document the defects. Given that the spindle assembly defects might be primarily responsible for subsequent defects in chromosome alignment and segregation, the authors should better characterize the spindle assembly defects using pole marker(s). Do mutant oocytes even establish a bipolar spindle? From the images, it seems likely they do not.As requested by the reviewer, we have performed new experiments using GFP-ASPM-1 as a pole marker. As expected, LET-92-depleted oocytes fail to assemble a bipolar spindle . As mentioned above, in order to differentiate subsequent phenotypes from the spindle defect, we focused on the alignment/congression defects which are observed even in the presence of a seemingly normal bipolar spindle.4) The authors generate point mutations in both the LxxIxE motif and the kinase domain of BUB-1 to nicely document requirements for recruiting PP2A. However, the authors do not include any mention of whether these mutations are essential for oocyte meiotic cell division, or if the mutations are associated with any embryonic lethality . In fact, from the supplemental tables it appears that both mutant strains are homozygous viable, although this is never mentioned in the text. If these motifs are indeed responsible for recruiting PP2A and its essential functions to the spindle, then the mutants should result in defects identical to the PP2A knockdowns. Thus to verify the importance of these mutations, the authors would need to construct a balanced strain in which both motifs are mutated and document defects much like those observed in the PP2A knockdowns. More generally, the authors should provide a genetic analysis of the viability of the single mutants .Full analysis of embryo viability and brood size analysis is now presented for all the mutants used in the study .Reviewer #2:C. elegans. Although the shugoshin proteins have been shown to be important regulators of cohesin during meiosis by recruiting PP2A-B56 in other systems the authors show this is not the case in C. elegans. Instead they show that the Bub1 protein recruits PP2A-B56 through a LxxIxE motif that resembles the one found in human BubR1. Mutation of this motif prevents the recruitment of the PPTR1 (one of the B56 isoforms) to the midbivalent and the central spindle and reduces the recruitment of the PPTR2 to the central spindle. The Bub1 variant with a mutated LxxIxE motif displays misalignments suggesting an imbalance in phosphorylations. The phenotype of the Bub1 mutant is not as severe as Bub1 RNAi but this could be because the kinase domain of Bub1 helps in recruiting PPTR2.This manuscript explores female meiosis and the role of Bub1 in targeting PP2A-B56 to chromosomes and central spindle for proper meiosis in Overall the paper is easy to read and the data are consistent. However, the paper is fairly descriptive and lacks to some degree mechanistic insight. I think that some additional experiments would make the work more interesting for the readers of eLife.1) Characterization of the double Bub1 mutant with a mutated LxxIxE motif and mutations in the kinase domain. This would clarify if there is some redundancy in recruitment mechanisms.We have performed the suggested experiment. The results indicate that LxxIxE motif-mediated recruitment of B56 subunits operates mostly in the midbivalent and central spindle, whereas the kinase domain mediates mostly chromosome chromosomal recruitment of the B56 subunits .2) Direct evidence that the LxxIxE motif of Bub1 facilitates binding to PPTR1/2 \u2013 IP or purified components.We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and B56. We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. We could observe that PPTR-2 bound the wild - type sequence and that L282A,V285A mutations abolished binding . On the contrary, binding was enhanced when the peptide was phosphorylated at Serine 283 .3) Characterization of the LAPI mutation of the LxxIxE motif to determine if Cdk1 phosphorylation of this site is important. Reading the manuscript as it is now this is purely speculative despite they go through the effort of showing phosphorylation.We have now expanded on the LxxIxE motif phosphorylation and its role in vivo.1) in vitro assays demonstrate that Cdk1 phosphorylates Serine 283 in vitro. This is now part of the New Figure 5C-E.S283A, achieving a similar effect to that of BUB-1L282A,V285A. Previous data showed that substituting I for V in the LxxIxE motif decreases affinity and we believe in this case this renders the motif more dependent on phosphorylation. These results are included in the New Figure 6.2) To analyse the role of Serine 283 phosphorylation in vivo, we mutated it to alanine in endogenous BUB-1. PPTR-1 and PPTR-2 localisation to the midbivalent and central spindle was significantly affected in BUB-13) In addition to the previously reported mass spec data, we now show the localisation of the L(pS)PVSE motif by immunofluorescence with a newly generated phospho-specific antibody .Reviewer #3:C. elegans anaphase. It finds that PP2A plays a crucial role in chromosome alignment and segregation. This role depends on the B56 regulatory subunits. Moreover, the author show that the recruitment of the B56 subunits to the spindle apparatus and kinetochores in anaphase depends on the Bub-1 kinase.The study by Bel Borja and colleagues studies to which extent the PP2A phosphatase contributes to chromosome segregation in C. elegans anaphase, and is generally of good technical quality (see below), yet at the same time feels a bit thin in terms of results. In particular several major claims of the discussion are not supported by the data. Moreover, it is not clear to which extent the reported experiments are reproducible.The study explores a novel role of PP2A in 1) The authors do not indicate the number of independent experiments or the number of embryos on which the conclusions are based on. This information is essential to evaluate the reproducibility of the results. The authors use good statistical tests, but the reader must know whether the experiments are based on 5, 10 or 20 embryos.We have now included all the information in the graphs. For intensity measurements, \u201cN\u201d is the number of experiments, and \u201cn\u201d is the number of oocytes. For the congression and alignment analysis, \u201cN\u201d is the number of spindles (=number of oocytes) and \u201cn\u201d is the number of bivalents analysed. In the latter analysis, information on the number of experiments used to obtain the data is stated in the figure legend.C. elegans anaphase by RECRUITING PP2A:B56 to the spindle apparatus. The presented experiments only show that the localization of the B56 depends on Bub-1, which is not the same thing. The authors could test by IP and in vitro pull-down that the claimed interaction is direct (in vitro), or that the proteins are part of the same complex (in vivo). This should be feasible since the authors already have performed IP experiments.2) The authors claim the Bub-1 acts on We thank the reviewer for raising this concern and we have now changed the wording when referring to this. We have performed in vitro binding experiments and present evidence for the direct interaction between the LxxIxE motif of BUB-1 and PPTR-2 . We expressed recombinant, full-length PPTR-2 in bacteria and performed fluorescence polarisation experiments using fluorescently labelled LxxIxE motif peptides. PPTR-2 bound the wild type sequence and that binding was abolished by the L282A,V285A mutations and enhanced when the peptide was phosphorylated at Serine 283.3) The authors claim that PP2A:B56 counteracts Aurora-B during anaphase, but have no evidence for this claim. Does a Aurora-B hypomorph mutant attenuate the B56 loss phenotype? While a negative result would not mean that the model is wrong, a positive result would certainly help to bolster that claim.As stated above, we think we are providing a significant advance on the mechanisms in place to regulate meiosis I by specific recruitment of PP2A/B56. While we are actively working on identifying PP2A substrates and its potential role in antagonising Aurora B, we believe this will require an in-depth analysis that will be the focus of another paper. We have now focused our discussion on our actual data and left this as an interesting, yet unsupported, hypothesis."}
+{"text": "To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live\u2010cell knockout strategy that is applicable to numerous proteins. This modular protein\u2010tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag\u2010mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence\u2010Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11\u2010amino\u2010acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG\u201013, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. \u00a9 2020 The Authors.Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT\u2010HaloTagBasic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions Targeted protein degradation using proteolysis targeting chimeras (PROTACs) is a rapidly growing research area and an exciting new modality of therapeutic treatment  donor vector. After CRISPR pools are generated, further details are provided for enrichment of HaloTag positive cells using the HaloTag fluorophore, Janelia Fluor 646 (JF646) HaloTag ligand  be placed immediately following the endogenous target start codons or any type of signal sequence that could be cleaved. If introducing HaloTag or HaloTag\u2010HiBiT at the C terminus, place the tag(s) immediately upstream of the native stop codon. Generation of genomic maps that include the CRISPR insertions will aid in ensuring the tags are placed in the proper reading frame. As CRISPR insertion success can be highly dependent upon the choice of guide RNA, it is advisable to test a minimum of two different guide RNA sequences. Initial studies appending the tag(s) on the N\u2010 or C\u2010terminal end of the protein using expression vectors can be used to determine the tag(s) effects on the protein's expression, binding interactions, and folding abilities. However, overexpression is not recommended for degradation studies with HaloPROTAC3, as little degradation will be detected due to the high expression levels of the fusion protein.Alt\u2010R CRISPR\u2010Cas9 tracrRNA Alt\u2010R CRISPR\u2010Cas9 CRISPR RNA Nuclease Free Duplex Buffer Alt\u2010R S.p. Cas9 Nuclease Desired cell line for CRISPR modification that is amenable to nucleofectionDPBS 0.05% trypsin/EDTA  or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceMirus Ingenio Solution HaloTag or HiBiT\u2010HaloTag double\u2010stranded (ds) DNA donor plasmid Janelia Fluor 646 (JF646) HaloTag Ligand 100\u00d7 Antibiotic/Antimycotic solution FACS buffer see Optional, for use of HiBiT\u2010HaloTag or HaloTag\u2010HiBiT CRISPR insertions: Nano\u2010GloHiBiT Lytic Detection System Heat block 50\u2010ml conical tubes Mirus Ingenio Kit Bio\u2010Rad Gene Pulser Xcell Electroporation SystemAppropriate incubator for cell lineClear, tissue\u2010culture\u2010grade 96\u2010well plates 5\u2010ml round\u2010bottom tube with cell strainer cap BD FACS Melody or similarWhite 96\u2010well plates Optional, for use of HiBiT\u2010HaloTag or HaloTag\u2010HiBiT CRISPR insertions: Luminometer such as the GloMax Discover Microplate Reader  or CLARIOstar Plus (BMG Labtech)Additional reagents and equipment for basic cell culture techniques, including cell counting  complex by combining 75 pmol Cas9 and 120 pmol tracrRNA:crRNA duplex:Add the Cas9 very slowly to avoid precipitation, and swirl with pipet tip.5Incubate RNP complex 10\u201020 min at room temperature.66 cells/reaction), remove cells from plasticware using appropriate splitting techniques, centrifuge cells at 200 \u00d7 g for 5 min, rinse cells with DPBS, and centrifuge again. For example, if using HEK293 cells, wash with DPBS, detach cells with trypsin, inactivate trypsin with DMEM + 10% FBS, and transfer cells to a conical tube for centrifugation.Determine total number of cells needed based on number of CRISPR reactions  for the cell type, and split cells if they become confluent. If performing HiBiT\u2010HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be performed to determine insertion efficiency  in triplicate to a white 96\u2010well assay plate.29Dilute the LgBiT Protein 1:100 and the Nano\u2010Glo HiBiT Lytic Substrate 1:50 into an appropriate volume of room\u2010temperature Nano\u2010Glo HiBiT Lytic Buffer in a new tube. Mix by inversion.30Add 100 \u00b5l Nano\u2010Glo HiBiT Lytic Reagent to each well.31Mix the samples by placing the plate on an orbital shaker (300\u2010600 rpm) for at least 10 min.32Read luminescence with GloMax Discover Microplate Reader using a 0.5\u2010s integration time Fig. .Basic Protocol 2ent\u2010HaloPROTAC3  is an alternate procedure for the live\u2010cell kinetic degradation analysis of HiBiT\u2010HaloTag insertions to better understand degradation rate, optimal time of treatments, and kinetic dose\u2010response curves. The use of HiBiT for protein level detection is highly quantitative, directly correlative to the endogenous target protein level, and does not require the use of antibodies 0.05% trypsin/EDTA  or 0.25% trypsin/EDTA , depending on cell lineComplete growth medium for cell type of choiceHaloPROTAC3 ligand ent\u2010HaloPROTAC3 ligand Dimethyl sulfoxide Optional materials for live\u2010cell kinetic degradation of HiBiT\u2010HaloTag fusions:LgBiT Expression Vector Nano\u2010Glo Endurazine Live Cell Substrate 2\u2010independent medium COFBS Six\u2010well platesAppropriate incubator for cell lineDilution reservoirs White 96\u2010well plates Optional material for live\u2010cell kinetic degradation of HiBiT\u2010HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader  or CLARIOstar Plus (BMG Labtech)1Culture CRISPR/Cas9\u2010edited cells carrying HaloTag, HiBiT\u2010HaloTag , or HaloTag\u2010HiBiT  insertions appropriately in preparation for the assay.If performing live\u2010cell degradation assays with HiBiT\u2010HaloTag CRISPR insertions proceed to optional live\u2010cell HiBiT\u2010HaloTag degradation assay at the end of Basic Protocol 2For adherent cells, wash cells with DPBS and trypsinize.3ent\u2010HaloPRTOAC3, and one for DMSO control.Count cells and plate 800,000 cells per well in a six\u2010well plate. Plate one well for HaloPROTAC3, one for 4For adherent cells, incubate cells overnight (18\u201024 hr) in an appropriate incubator. For suspension cells, proceed to step 5.5ent\u2010HaloPROTAC3, and (c) a volume of DMSO equivalent to that added in the PROTAC treatments.Add ligands to growth medium in well to obtain final concentrations of (a) 300 nM HaloPROTAC3, (b) 300 nM For example, if there is 2 ml of growth medium in well, add 500 \u00b5l 1.5 \u00b5M HaloPROTAC3 stock to make a solution of 300 nM final concentration.6Incubate cells in an appropriate incubator overnight.These recommended HaloPROTAC3 concentrations and treatment times can be increased or decreased depending upon target and desired level of degradation.7Detect degradation of endogenous HaloTag target fusion. If using HiBiT\u2010HaloTag CRISPR insertions, HiBiT lytic luminescence assays can be carried out to quantitate degradation following Basic Protocol 8Transfect the LgBiT vector into HaloTag\u2010 or HiBiT\u2010HaloTag\u2010edited cells using standard transient transfection and following manufacturer's recommendations.9Incubate plates in an appropriate incubator overnight (18\u201024 hr).102\u2010independent medium with the appropriate percentage of FBS for the cell type, or proper assay medium for cells, by diluting the stock reagent of the substrate 1:100. If the luminometer provides CO2 injection or cells do not require CO2, regular assay medium can be used.Prepare a 1\u00d7 solution of Nano\u2010Glo Endurazine Live Cell Substrate in CO11Aspirate cell culture medium from plate, and add 90 \u00b5l of the Endurazine solution to each well.122 level (if possible or needed) of cell line in use.Incubate plate for at least 2.5 hr at in an incubator set to the appropriate growing conditions for the cells to equilibrate the luminescence. During this incubation period, it is advisable to pre\u2010equilibrate the luminometer to the growth temperature and CO13ent\u2010HaloPROTAC3 either in CO2\u2010independent medium containing the appropriate percentage of FBS for the cell type or in proper assay medium for the cells.Prepare 10 \u00b5M solutions of HaloPROTAC3 and 14ent\u2010HaloPROTAC3 in dilution reservoirs using assay medium that contains DMSO at the same concentration as the 10 \u00b5M stock. Reserve the last well of the dilution series to contain DMSO alone, as a negative control. This results in a 10\u00d7 dilution series, which will be further diluted upon addition to cells for a final treatment concentration of 1 \u00b5M at the highest point.Perform threefold dilutions of 10 \u00b5M HaloPROTAC3 or 15ent\u2010HaloPROTAC3 ligand to each well of the 96\u2010well plate.Add 10 \u00b5l of the diluted HaloPROTAC3 or 16Immediately place the plate in a luminometer plate reader pre\u2010equilibrated to the growth temperature of the cell line in use.17Read luminescence at desired intervals. A recommended starting point is every 15 min over a 24\u2010hr total time frame.18ent\u2010HaloPROTAC3\u2010treated wells by those from the DMSO control . Examples of kinetic degradation profiles of HiBiT\u2010HaloTag CRISPR insertions are shown in Figures Calculate the fractional RLUs at each time point by dividing the RLU value from HaloPROTAC3\u2010treated or 1\u00d7 Hank's Balanced Salt Solution 10 mM HEPES 0.2% BSA 1\u00d7 Antibiotic/Antimycotic Solution N\u2010terminal HaloTag sequence:500\u2010bp upstream homology arm\u2010GCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC\u2010500\u2010bp downstream homology armC\u2010terminal HaloTag sequence:500\u2010bp upstream homology arm\u2010GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGT\u2010500\u2010bp downstream homology armN\u2010terminal HiBiT\u2010HaloTag sequence:500\u2010bp upstream homology arm\u2010GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTCGAGATTTCCGGCGAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAAC\u2010500\u2010bp downstream homology armC\u2010terminal HaloTag\u2010VS\u2010HiBiT sequence:500\u2010bp upstream homology arm\u2010 GAGCCAACCACTGAGGATCTGTACTTTCAGAGCGATAACGATGGATCCGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCTACTCTGGAGATTTCCGGTGTCTCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC\u2010500\u2010bp downstream homology armKey: blue, HiBiT; red, HaloTag; green, linker.Targeted protein degradation has resulted in an explosion of new avenues of research, from therapeutic drug discovery and clinical trials to the expansion of E3 ligase studies and phenotypic studies  as compared to smaller insertions, for which single\u2010stranded oligodeoxynucleotide (ssODN) synthesis is possible and homology arms (30\u201050bp each) are shorter. The use of dsDNA donor vectors in general results in low insertion efficiency, and expected pool percentage of edited cells with this approach could range between 0.1% and 15%. For insertion of HaloTag or HiBiT\u2010HaloTag, the use of donor vector without a promoter is important to promote specific, on\u2010target insertion and minimize random integration of vector, which could then result in expression of the tag alone. To identify CRISPR HaloTag target\u2010edited cells from random integration, it is important to assess that the proper\u2010sized fusion is made by amplifying the genomic region by PCR or visualizing protein size on a protein gel with the HaloTag TMR ligand  on untagged alleles, resulting in a knockout of the untagged protein copies. As a result, the entire target protein pool in these clones is expressed as a fusion to HaloTag; therefore, these clones are sufficient for phenotypic experiments with HaloPROTAC3.HaloPROTAC3 will degrade HaloTag target fusion proteins that are recruited to the VHL E3 ligase component, incorporated into active E2/E3 ligase complexes for ubiquitination, and then trafficked to the proteasome. VHL is expressed throughout the cytoplasm and nucleus, as well as in numerous cell types (Buckley et\u00a0al., As endogenous target proteins have highly variable expression levels that will not be significantly altered by HaloTag insertion, varying concentrations of HaloPROTAC3 may be necessary for successful and maximal degradation. Because HaloPROTAC3 binds irreversibly to HaloTag (Buckley et\u00a0al., ent\u2010HaloPROTAC3 (Buckley et\u00a0al., ent\u2010HaloPROTAC3 has significantly reduced affinity for VHL engagement (Buckley et\u00a0al., With any PROTAC degradation study, it is important to be certain that observed target protein loss is due to the specific PROTAC mechanism. For endogenous HaloTag proteins, this can be achieved using For the alternate procedures utilizing HiBiT\u2010HaloTag fusion proteins, protein levels are easily measured with luminescence (Daniels et\u00a0al., In the schematic shown in Figure ent\u2010HaloPROTAC3 at either 3 or 24 hr (Fig. As an example of HaloPROTAC3 degradation and phenotype studies of a target with no available degraders or specific PROTACs, a homozygous CRISPR clone of \u03b2\u2010catenin\u2010HaloTag\u2010HiBiT was generated in HEK293 cells stably expressing LgBiT. In Figure  hr Fig. , demonst hr Fig. . These r hr Fig.  and degr hr Fig. . Because hr Fig. , an orth hr Fig.  and E. U hr Fig. , includi hr Fig.  and E. T hr Fig.  and loss hr Fig. .ent\u2010HaloPROTAC3 (Fig. ent\u2010HaloPROTAC3 shows that protein loss of each of these targets is specific to HaloPROTAC3. Each of the HaloPROTAC3 degradation profiles was then used to calculate the degradation rate (Fig. 50, the concentration of HaloPROTAC3 that gave half the degradation maximum (Fig. 50 values for each of these targets indicates that it is primarily HaloTag which is driving the degradation, with minimal influence by the different targets. This is desirable for a fusion tag PROTAC system as it needs to be broadly applicable to numerous targets. The kinetic analysis also shows how the optimal dose of HaloPROTAC3 and time of treatment to achieve degradation can be clearly understood from the profiles, saving significant time and yielding more detailed information as compared to western blot analysis.To demonstrate the ability to degrade endogenous HaloTag proteins at a variety of cellular locations, as well as to understand the quantitative parameters of HaloPROTAC3 degradation, the HiBiT kinetic studies outlined in the optional live\u2010cell luminescence degradation detection of HiBiT\u2010HaloTag CRISPR insertion protocol steps were performed Fig. . For theAC3 Fig. \u2010F to proAC3 Fig. \u2010F. All tAC3 Fig. , includiAC3 Fig. . This isAC3 Fig. , suggestate Fig. , as wellmum Fig. , for allmum Fig. . The simF36V\u2010HiBiT at the C terminus in HEK293 cells (Fig. F36V has low efficiency of insertion and edited cells cannot easily be identified or fluorescently enriched from the CRISPR pool.To quantitatively compare HaloPROTAC3 degradation with the dTAG PROTAC system, CRISPR clones of the EPOP target protein were generated by insertion of either HaloTag\u2010HiBiT or FKBP12lls Fig. . These clls Fig.  or dTAG\u2010lls Fig.  were obtlls Fig. , and dTAlls Fig. , showed lls Fig. , which wlls Fig. . A hook lls Fig. . This rells Fig. . The Hallls Fig. . TogetheBasic Protocol Basic Protocol Optional HiBiT Lytic and kinetic protocols: The HiBiT lytic assay to determine insertion efficiency within CRISPR pools or measure degradation of HiBiT\u2010HaloTag target proteins takes 30\u201060 min. The kinetic degradation assay takes 3 days to perform including plating, Endurazine equilibration, HaloPROTAC3 degradation, and data analysis.Elizabeth A. Caine: Data curation; formal analysis; investigation; writing\u2010original draft; writing\u2010review & editing. Sarah D. Mahan: Data curation; methodology. Rebecca L. Johnson: Methodology. Amanda N. Nieman: Methodology. Ngan Lam: Methodology; supervision. Curtis R. Warren: Conceptualization; supervision. Kristin M. Riching: Conceptualization; formal analysis; investigation. Marjeta Urh: Conceptualization; supervision; writing\u2010original draft; writing\u2010review & editing. Danette L. Daniels: Conceptualization; data curation; formal analysis; investigation; project administration."}
+{"text": "ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.The mechanisms underlying  Sufficient protein dosage of ETV4 can directly transform prostate epithelial cells, which recapitulates early human disease. ETS family transcription factors including ERG and three highly conserved polyomavirus enhancer activator 3 (PEA3) subfamily members\u2014ETV1, ETV4, and ETV5\u2014are prevalent and mutually exclusive in prostate cancer; they occur early in the natural history of the disease  models that overexpress ERG or ETV1 showed either minimal or no phenotype early time points  and stabilized ETV4 , harboring mutations at the COP1-binding sites and therefore resistant to COP1-mediated degradation. We demonstrate that the stabilized ETV4AAA significantly increased the protein dosage of ETV4. ETV4AAA expression alone initiates widespread mPIN throughout the prostate epithelium within 2 weeks of activation. The mPIN fails to further progress to invasive cancer due to ETV4-mediated simultaneous activation of the p53-dependent senescence program, and ETV4AAA cooperates with Trp53 loss to promote the development of focally invasive prostate cancer. The neoplastic cells retain expression of prostate lineage differentiation markers such as Nkx3.1 similar to most cases of localized human prostate cancer. In contrast, Pten loss\u2013driven murine prostate cancers generally lose Nkx3.1 and are de novo castration resistant and distinct in human primary adenocarcinoma . We expressed enhanced green fluorescent protein (EGFP) vector control, ETV4WT, and ETV4AAA in A375 melanoma cells and found that, at baseline, protein level of ETV4AAA was significantly higher than that of ETV4WT. Treatment with MG132 that inhibits proteosomal degradation increased ETV4WT protein levels and endogenous ETV4 in vector-infected control cells but only minimally affected ETV4AAA protein level, indicating that ETV4AAA is constitutively stable (fig. S1A). To evaluate the possibility that, beyond protein stability, the mutations introduced in ETV4AAA may alter its transcriptional function, we expressed EGFP, ETV4WT, and ETV4AAA in two prostate cancer cell lines, 22Rv1 and PC3, and performed RNA sequencing (RNA-seq) analysis. In 22Rv1 cells, the protein level of ETV4AAA was significantly higher than that of ETV4WT due to degradation by COP1. PC3 cells harbor a deletion in COP1 and have increased baseline protein levels of ETV4 (AAA and ETV4WT exhibited similar protein levels (fig. S1B). Principal components analysis (PCA) of RNA-seq showed that the first principal component (PC) was separated by cell line and that the second PC was separated by ETV4 expression. In 22RV1 cells, ETV4AAA caused a much greater expression change as ETV4WT in the same direction. In PC3 cells that have baseline higher levels of ETV4, the transcriptome perturbation was less in general, and ETV4WT caused a greater change in gene expression (fig. S1C). We compared the global transcriptome change induced by ETV4AAA and ETV4WT. In 22Rv1 cells, there was a high correlation [Pearson correlation coefficient (r) =\u00a00.72], and ETV4AAA induced a greater change (fig. S1D). In PC3 cells, the global transcriptome change was also strongly correlated (Pearson r\u00a0=\u00a00.76), and ETV4WT induced slightly higher gene expression changes. These data suggest that ETV4AAA regulates a similar transcriptome to ETV4WT.Previous studies showed that transgenic and conditional WTETV4 and AAAETV4 followed by IRES-EGFP driven by the CAG promoter knocked into the Rosa26 locus  mice  with enlarged nuclei, prominent nucleoli, and cribriform growth in the anterior and dorsal prostate (WTETV4-expressing mice were indistinguishable from those expressing EYFP. While GFP immunohistochemistry (IHC) showed prevalent EGFP- or EYFP-positive cells in murine prostates of all three genotypes, ETV4 IHC detected weak nuclear ETV4 protein in ETV4WT prostate but strong nuclear ETV4 expression in the AAAETV4-expressing prostate cells . This highlights a lobe-specific sensitivity to specific oncogenic transformation as observed in other genetic engineered models of prostate cancer .To determine whether high ETV4 protein expression alone is sufficient to drive progression to invasive cancer, we administered TAM in 6- to 8-week-old mice and aged them for 6, 9, and 12 months for phenotypic assessment. Unexpectedly, the mPIN of the anterior and dorsal prostate of  ETV4AAA . The proAAA in the mouse anterior prostate is comparable to that in human prostate cancer, we performed ETV4 IHC using the same conditions on a patient-derived xenograft (PDX) model with TMPRSS2-ETV4 fusion (MDA PCa 175-6) and a control PDX (MDA PCa 173-2) from MD Anderson (MDA PCa program) deposited in biobank , the most commonly used Cre driver in the prostate epithelium, that mediates continued recombination in prostate epithelial cells from puberty . The areas of mPIN were marked by positive EGFP staining. We reasoned that the continuous induction of ETV4AAA though a nonconditional Cre driver results in relatively constant phenotype over time. Despite the continuous induction that maintained mPIN throughout the time studied, there was no evidence of disease progression over time. These data suggest that the expression of AAAETV4 can directly induce mPIN with little latency and that persistent mPIN phenotype requires maintenance of the AAAETV4 expression in prostate epithelial cells; however, AAAETV4 expression alone is insufficient to mediate progression to invasive cancer.We chose ETS translocation alters the chromatin enhancer landscape and the AR cistrome in prostate cancer  and RNA-seq, respectively. We chose the 2-week time point to enrich for direct targets. To specifically analyze prostate epithelial cells with the recombined allele, we sorted EPCAM-positive and EYFP- or EGFP-positive prostate epithelial cells by fluorescence-activated cell sorting (FACS) from T2; EYFP, WTT2; ETV4, and AAAT2; ETV4 murine prostates (fig. S4A).We next examined the molecular impact of stabilized ETV4 in the unique phenotypes associated with prostate tumorigenesis. Recent data have shown that WTETV4- and EYFP-expressing cells clustered closely, while AAAETV4-expressing cells exhibited marked changes in the accessible chromatin landscape  > 2], with approximately equal number of peaks with significantly increased and decreased ATAC signals . The FOX and AP1 motifs were significantly enriched at both ETV4AAA\u2013up-regulated and ETV4AAA\u2013down-regulated peaks, and the nuclear receptor motif was enriched at the down-regulated peaks . Unsupervised clustering of ATAC signal intensity showed that andscape . We foun signals . Among tagnitude , indicated peaks . Consistpression  .eq sites . ATAC-seAAA-induced transcriptome changes. Compared to EYFP-expressing control, AAAETV4 expression induced a greater number of statistically up-regulated genes (537) than down-regulated genes (250)   . Hierarc control . Integra control . These dAAA in prostate epithelial cells, we performed gene set enrichment analysis (GSEA) using ~3000 curated genes sets and custom prostate cancer gene sets. There were many more gene sets enriched among genes significantly up-regulated by ETV4AAA expression than among genes down-regulated by ETV4AAA expression, also suggesting that ETV4AAA is primarily a transcriptional activator (fig. S4D). Among enriched gene sets are those genes associated with cell cycle and proliferation (CHANG_CYCLING_GENES), prostate cancer tumorigenesis in human prostate cancer (TCGA_PrCa_UP), and ERG target genes in prostate cancer (VCAP_siERG_DN and VCAP_Lenti_ERG_UP)  . We furtWT- and ETV4AAA-expressing prostate cells; canonical ETS transcriptional targets, e.g., Dusp6 and Plat, were mildly induced by ETV4WT and much more significantly induced by ETV4AAA (Trp53), its downstream target p21 (Cdkn1a), and senescence-associated genes Cdkn2b, Cxcl1, Spp1, and Tgfb1 were all significantly up-regulated in ETV4AAA-expressing prostate cells compared to that in EYFP and ETV4WT controls. ETV4AAA prostates stained positive for p53 by IHC and for senescence-associated \u03b2-galactosidase \u2013EGFP was similar between ETV4 ETV4AAA . p53  is significantly higher in ETS fusion\u2013positive samples (42.42%) than that in ETS fusion\u2013negative samples (15.83%), consistent with analysis of the Memorial Sloan Kettering Cancer Center cohort . These data indicate that aberrant high level of ETS expression activates p53 and downstream senescence programs and that p53 loss might be a cooperative event that overcomes the barrier for human prostate tumorigenesis and progression.We analyzed the TCGA dataset of primary prostate cancer to correlate r cohort  (20). Wee cancer . To assee cancer : (i) TCGAAA-mediated senescence and tumor progression, we crossed in the LoxP/LoxPTrp53 mice into the AAAT2; ETV4 mice to generate LoxP/LoxP; ETV4AAAT2; Trp53 mice. Two weeks after TAM injection, Western blot of prostate lysates shows decreased p53 protein , which include 19,854 genes, with a median of 1973 genes per cell and a median of 2039 cells per mouse. To visualize single cells of the global atlas, we used uniform manifold approximation and projection (UMAP) for dimension reduction. We then performed Leiden clustering  on FACS-sorted live cells from prostates of T2; EYFP wild-type mice, there was expansion of epithelial cells  over time from 2 weeks to 4 months  , L2, and L3 that had been found in previous studies (fig. S5A and table S6)  that highly expresses lateral prostate marker genes Msmb and Trpv6 (Tacstd2 (Trop2), Ly6a (Sca1), Psca, and Krt4; and exhibit enhanced progenitor characteristics (Foxi1 (fig. S5A and table S6).We next analyzed the identities of luminal cell clusters by the marker gene expression. We then annotated the luminal clusters based on the highly expressed marker genes and quantified the cell numbers in each group (fig. S5A). We first focused on T2; EYFP wild-type prostates, WTT2;ETV4 prostates did not comprise any new luminal clusters, suggesting that ETV4WT cannot transform luminal cells. However, WTT2;ETV4 prostates exhibited expansion of Luminal_1 clusters compared with T2;EYFP that is more pronounced from 2 weeks to 4 months . Furthermore, within the WTT2;ETV4 prostates, the expansion of Luminal_1 clusters over time was mostly due to WTETV4-expressing cells (dashed green and dashed cyan). These data suggest that, while expression of WTETV4 did not cause detectable histologic phenotype or new gene expression cluster, its expression was able to cause luminal expansion over time at the single-cell level.Compared to AAA in AAAT2; ETV4 and AAA;Trp53LoxP/LoxPT2; ETV4 prostates generated two new luminal clusters that express AAAETV4  with distinct transcriptome features from previously described luminal clusters  cluster  using GSEA using GSEAPY package . Pathway analysis showed that extracellular matrix (ECM) organization and multiple growth factor pathways are up-regulated (fig. S6B). To verify the scRNA-seq results, we analyzed the gene expression of L2 marker gene Tacstd2/Trop2 using immunofluorescence staining (fig. S6C). While EGFP (indicating ETV4)\u2013positive cells are Tacstd2 negative, there is a marked expansion of Tacstd2-positive cells in AAAETV4 mice and LoxP/LoxP; ETV4AAATrp53 mice. These Tacstd2-positive cells were found in regions of EGFP-positive, ETV4AAA-expressing cells, suggesting that ETV4AAA-positive cells, through paracrine interactions, expand the normal L2 population.In addition to ETV4 ETV4AAA . This suT2; EYFP and WTT2; ETV4 mice, Basal_2 cells were found exclusively in ETV4AAA-expressing mice, and Basal_3 cells had an intermediate gene expression and were found in all groups of mice (fig. S7A). Basal_2 cells highly express genes Col17a1, Areg, Ly6d, and Cd44 (fig. S7B). We performed immunofluorescence staining of Col17a1 and found a high expression in Ck5-positive basal cells adjacent to EGFP-positive (ETV4AAA-expressing) luminal cells in ETV4AAA mice (fig. S6D), suggesting that ETV4AAA expression in the luminal cells induced paracrine gene expression changes in basal cells.We next identified and quantified subclusters in basal, stroma, and hemopoietic populations. We identified four basal expression clusters. Basal_1 cells were found predominantly in Lama2, Zeb1, Wnt2, Wnt6, Wnt10a, and Rorb; Mesenchymal_2 is characterized by expression of Sult1e1, Fgf10, and Rspo1; Myofibroblast is characterized by expression of Acta2, Myh11, and Notch3. These clusters represent previously described Mesenchymal_1, Mesenchymal_2, and myofibroblast subsets (table S6) . Mesenchymal_3 is characterized by expression of Sod3 and Osr1, which are mesenchymal stem cell markers .We identified five stroma clusters: Mesenchymal_1 to Mesenchymal_4 and myofibroblast (fig. S7C). Mesenchymal_1 is characterized by expression of The myeloid cells and lymphocytes are characterized using the expression of the marker genes (fig. S7F). Marker genes of M1 macrophages, M2 macrophages, and myeloid-derived suppressor cells (MDSCs) are adopted from other publications  level and a high dosage (ETV4AAA) level of ETV4 protein through increased ETV4 protein stability by mutating the conserved ExxVPD motifs responsible for COP1 degradation  is significantly higher in ETS fusion\u2013positive samples than that in ETS fusion\u2013negative samples . MecAAA-driven murine prostate cancer model is that it maintains the characteristics of early-stage human prostate cancers, including prostate differentiation markers, such as Nkx3.1 expression. Comparably, prostates of Pten deletion, the most well-studied GEM model of prostate cancer, exhibit increased basal marker expression, lose Nkx3.1 expression, and are castration resistant de novo  and mutated ETV4AAA cDNA into the plasmid in the middle of the two cassettes. The targeting plasmid was electrophoresed into albino C57BL/6J embryonic stem cells, and G418-resistant clones were isolated by the standard procedures. The clones were screened by Southern blotting. Positive clones were injected into C57BL/6J blastocysts by the MSKCC Mouse Genetics Core Facility, and chimeras were mated with albino C57BL/6J females. Germline transmission was confirmed in albino offspring using Southern blotting. For subsequent generations, genotyping was performed by quantitative polymerase chain reaction (PCR) using the following primers: hETV4_E3_F: GCCGCCCCTCGACTCTGAA; hETV4_E4_R: GAGCCACGTCTCCTGGAAGTGACT.Rosa26 targeting was as described by Srinivas GTACCAGAC(GCAGCAGCC)AGTGATGAGCAGTTTGTTCCTGAT(GCTGCTGCT)TTCCATTCAGAAAACCTAGCTTTCCACAGCCCCACCACCAGGATCAAGAAGGAGCCCCAGAGTCCCCGCACAGACCCGGCCCTGTCCTGCAGCAGGAAGCCGCCACTCCCCTACCACCATGGCGAGCAGTGCCTTTACTCCAGTGCCTATGACCCCCCCAGACAAATCGCCATCAAGTCCCCTGCCCCTGGTGCCCTTGGACAGTCGCCCCTACAGCCCTTTCCCCGGGCAGAGCAACGGAATTTCCTGAGATCCTCTGGCACCTCCCAGCCCCACCCTGGCCATGGGTACCTCGGGGAACATAGCTCCGTCTTCCAGCAGCCCCTGGACATTTGCCACTCCTTCACATCTCAGGGAGGGGGCCGGGAACCCCTCCCAGCCCCCTACCAACACCAGCTGTCGGAGCCCTGCCCACCCTATCCCCAGCAGAGCTTTAAGCAAGAATACCATGATCCCCTGTATGAACAGGCGGGCCAGCCAGCCGTGGACCAGGGTGGGGTCAATGGGCACAGGTACCCAGGGGCGGGGGTGGTGATCAAACAGGAACAGACGGACTTCGCCTACGACTCAGATGTCACCGGGTGCGCATCAATGTACCTCCACACAGAGGGCTTCTCTGGGCCCTCTCCAGGTGACGGGGCCATGGGCTATGGCTATGAGAAACCTCTGCGACCATTCCCAGATGATGTCTGCGTTGTCCCTGAGAAATTTGAAGGAGACATCAAGCAGGAAGGGGTCGGTGCATTTCGAGAGGGGCCGCCCTACCAGCGCCGGGGTGCCCTGCAGCTGTGGCAATTTCTGGTGGCCTTGCTGGATGACCCAACAAATGCCCATTTCATTGCCTGGACGGGCCGGGGAATGGAGTTCAAGCTCATTGAGCCTGAGGAGGTCGCCAGGCTCTGGGGCATCCAGAAGAACCGGCCAGCCATGAATTACGACAAGCTGAGCCGCTCGCTCCGATACTATTATGAGAAAGGCATCATGCAGAAGGTGGCTGGTGAGCGTTACGTGTACAAGTTTGTGTGTGAGCCCGAGGCCCTCTTCTCTTTGGCCTTCCCGGACAATCAGCGTCCAGCTCTCAAGGCTGAGTTTGACCGGCCTGTCAGTGAGGAGGACACAGTCCCTTTGTCCCACTTGGATGAGAGCCCCGCCTACCTCCCAGAGCTGGCTGGCCCCGCCCAGCCATTTGGCCCCAAGGGTGGCTACTCTTACTAG.ATGGAGCGGAGGATGAAAGCCGGATACTTGGACCAGCAAGTGCCCTACACCTTCAGCAGCAAATCGCCCGGAAATGGGAGCTTGCGCGAAGCGCTGATCGGCCCGCTGGGGAAGCTCATGGACCCGGGCTCCCTGCCGCCCCTCGACTCTGAAGATCTCTTCCAGGATCTAAGTCACTTCCAGGAGACGTGGCTCGCTGAAGCTCAGVPD(AAA)SDEQFVPD(AAA)FHSENLAFHSPTTRIKKEPQSPRTDPALSCSRKPPLPYHHGEQCLYSSAYDPPRQIAIKSPAPGALGQSPLQPFPRAEQRNFLRSSGTSQPHPGHGYLGEHSSVFQQPLDICHSFTSQGGGREPLPAPYQHQLSEPCPPYPQQSFKQEYHDPLYEQAGQPAVDQGGVNGHRYPGAGVVIKQEQTDFAYDSDVTGCASMYLHTEGFSGPSPGDGAMGYGYEKPLRPFPDDVCVVPEKFEGDIKQEGVGAFREGPPYQRRGALQLWQFLVALLDDPTNAHFIAWTGRGMEFKLIEPEEVARLWGIQKNRPAMNYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCEPEALFSLAFPDNQRPALKAEFDRPVSEEDTVPLSHLDESPAYLPELAGPAQPFGPKGGYSY.MERRMKAGYLDQQVPYTFSSKSPGNGSLREALIGPLGKLMDPGSLPPLDSEDLFQDLSHFQETWLAEAQPb-Cre4 Hze/JB6.Cg-Gt(ROSA)26Sor) mice of conditional CAG-driven EYFP expression were purchased from the Jackson Laboratory  in which exons 4 and 5 are flanked by LoxP sites were used as previously described . For mouse prostates, all tissues were fixed at 4\u00b0C overnight in 4% paraformaldehyde. Tissue processing, embedding, sectioning, and IHC staining was performed. Antibodies for IHC and Western blotting are as follows: chicken anti-GFP , rabbit anti-AR , rabbit anti-ETV4 , rabbit anti-Ki67 , rabbit anti-SMA , rabbit anti-p53 , rabbit anti-Nkx3.1 , mouse anti\u2013\u03b2-actin , and mouse anti\u2013glyceraldehyde-3-phosphate dehydrogenase . Tissue paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining were performed by the MSKCC core facility. IHC was performed by the MSKCC molecular cytology core using a Ventana Discovery XT. To generate lysates for Western blotting, tissue was homologized in radioimmunoprecipitation assay buffer using the FastPrep-24 system with Lysing Matrix A .Immunofluorescence staining of Tacstd2/Trop2 , Col17a1 , and Ck5  was performed on frozen sections. Sections were permeabilized with 0.5% Triton X-100 for 10\u00a0min and blocked with 10% normal donkey serum for 30\u00a0min at room temperature. Primary antibodies were incubated overnight. Secondary antibodies with Alexa Fluor 647 and Alexa Fluor 555 (Thermo Fisher Scientific) conjugation were applied on the second day. Image were taken with Leica TCS SP5 upright confocal microscope.Intraperitoneal injection of TAM was administered in 8-week-old mouse. Two weeks after TAM treatment, mouse prostate was digested 1 hour with collagenase/hyaluronidase  and then 30\u00a0min with TrypLE Express Enzyme  at 37\u00b0C to isolate single prostate cells. The prostate cells were stained with phycoerythrin/cyanine 7\u2013conjugated anti-mouse CD326 (EPCAM) antibody , and then, CD326 and EYFP double-positive cells were sorted out by flow cytometry, which are luminal cells mainly from anterior prostate and dorsal prostate. The mRNA or genomic DNA was extracted from these double-positive cells and then was used for ATAC-seq and RNA-seq analysis.https://github.com/kundajelab/atac_dnase_pipelines). Briefly, paired-end reads were trimmed, filtered, and aligned against mm9 using Bowtie2  ranges between 28 and 33, suggesting that the libraries have a high quality and were able to capture the majority of regions of interests.ATAC-seq was performed as previously described (P < 0.01 and |log2(FC)|\u00a0>\u00a02. For visualization, coverage bigwig files were generated using bamCoverage command from deepTools2 and were normalized using the size factor generated by DESeq2. The differential ATAC-seq peak density plot was generated with deepTools2 ] \u00d7 [\u2212log] as input for GSEA analysis using \u201cRun GSEA Pre-ranked\u201d with 1000 permutations (The extracted RNA was processed for RNA-seq by the Integrated Genomics Core Facility at MSKCC. The libraries were sequenced on an Illumina HiSeq-2500 platform with 51\u2013base pair (bp) paired-end reads to obtain a minimum yield of 40 million reads per sample. The sequenced data were aligned using STAR v2.3  calculated by DESeq2. P values were estimated using Wilcoxon rank t test and Student\u2019s t test.To assign ATAC-seq peaks to genes, ChIPseeker_v1.12.1 . After euthanasia, the prostates were dissected out and minced with scalpel and then processed for 1\u00a0hour of digestion with collagenase/hyaluronidase  and 30\u00a0min of digestion with TrypLE . Live single prostate cells were sorted out by flow cytometry as DAPI\u2212. For each mouse, 5000 cells were directly processed with 10x Genomics Chromium Single Cell 3\u2032 GEM, Library & Gel Bead Kit v3, according to the manufacturer\u2019s specifications. For each sample, 200 million reads were acquired on NovaSeq platform S4 flow cell.2(X\u00a0+\u00a01)\u2013transformed for analysis of the combined dataset. The top 1000 highly variable genes were found using SCANPY (version 1.6.1) (Reads obtained from the 10x Genomics scRNAseq platform were mapped to mouse genome (mm9) using the Cell Ranger package (10x Genomics). True cells are distinguished from empty droplets using scCB2 package (https://arxiv.org/abs/1802.03426). We then performed Leiden clustering  package  ."}
+{"text": "Euphorbiatithymaloides is one such medicinal plant that hasgained importance but is often confused with other plants of the samespecies. In order to address this issue, this study aimed to conducta conventional and molecular pharmacognostic study for the identificationof the root of E. tithymaloides. Theroot of the plant was studied for the macroscopic observations, andthen, the root was ground into coarse powder for microscopic studiesand to determine the physiochemical properties. The powder was subjectedto extraction with solvents such as ethanol, ethanol/water (1:1),hexane, and ethyl acetate. The extracts were then used for qualitativeand quantitative  phytochemicalanalysis. The molecular study was performed with the DNA barcodingtechnique. The DNA was extracted from the root of the plant, and itspurity was examined by gel electrophoresis (1% w/v). The DNA was thenamplified using an Applied Biosystems 2720 thermal cycler for therbcL, matK, and ITS primers. The amplified primers were sequencedwith a 3130 Genetic Analyzer, and the generated sequences were searchedfor similarity in the GenBank Database using the nucleotide BLASTanalysis. The micro- and macroscopic studies revealed the morphologicaland organoleptic characters as well as the presence of medullary rays,fiber, cork, sclereids, parenchymal cells, and scalariform vessels.The physiochemical properties were found within the limit. The phytochemicalanalysis revealed the presence of terpenoids, flavonoids, saponins,and alkaloids. In addition, the alkaloidal content was high in theethanol extract (63.04 \u00b1 3.08 mg At E/g), while the phenol contentwas high in the hexane extract (10.26667 \u00b1 1.77 mg At E/g), andthe flavonoid content was high in the ethyl acetate extract (41.458\u00b1 1.33 mg At E/g). After the BLAST analysis from the GenBankdatabase, the rbcL, ITS, and matK primers showed a similarity percentageof 99.83, 99.84, and 100. The phylogenetic tree for the species closestto each primer was generated using the MEGA 6 software. The matK locihad the highest percentage similar to the rbcL and ITS loci, indicatingthat the matK loci can be used to identify the root of E. tithymaloides as a standalone. The results fromthis study can be used to establish a quality standard for E. tithymaloides that will ensure its quality andpurity.Adulteration and substitution of medicinal plants havebecome amatter of great concern in recent years.  This might rapidly increase the production ofmedicinal plant-based products such as teas, tinctures, ointments,and other forms of medicine, which will become more accessible toconsumers, leading to improved health outcomes for many individuals.This is important for a variety of reasons, such as being more cost-effectivethan other medicines, being organic and natural, and being readilyavailable in many parts of the world. In addition, these plants oftenhave fewer side effects than other treatments especially syntheticdrugs and heavy metal formulations.2The use of medicinal plantsfor treating various illnesses anddisorders has a long history, stretching back centuries. In recentyears, the use of medicinal plants in modern medicine has seen a rapidincrease due to their potential benefits in treating conditions likecancer, chronic pain, and other prevalent health problems.3 as well as in more recent sources like the British Pharmacopoeia.4 Authentication and identification of herbal ingredientsare necessary to address the problem of adulteration and substitution.With time, plants have become increasingly important to human medicaltreatments and advancements. The extinction of certain medicinal plantscan have a drastic effect on the continued growth of medical treatments,leading to a greater understanding of the importance of these plants.Without them, it is impossible to make progress in many areas of medicalresearch. The regulation of medicinal plants is a very important anddifficult task, which is why it is necessary to have stringent lawsto ensure safety and quality and to distinguish between the adulterationand substitution of medicinal plants.5 Despiteincreased regulations, it is hard to guarantee the safety and qualityof herbal products. This not only affects the people consuming theseplants but also adversely affects the environment as well. The majorpart of quality control (QC) is to check the product for any adulterationor contamination to determine its safety and efficacy. Generally,QC is done with analytical techniques, which involve the use of electrophoretic,6 chromatographic ,7 and hyphenated methods ,8 including specific and non-specific detectorsystems. Some other aspects that are included in QC are physical examination,microscopical examination, chemical examination, microbiological examination,stability testing, toxicological examination, and a combination ofthe aforementioned techniques.9 Despitethese advances, adulteration and substitutions persist in the currentperiod. These conventional pharmacognostic studies and techniques,even the use of modern hyphenated analytical systems, also failedto detect adulterants and substitutions.As the medicinal plant industry continues to expand with betterprofits, substitution and adulteration of herbal ingredients havebecome more and more of an issue. These practices are documented inancient medical texts, such as the Ebers Papyrus,11 There areseveral techniques available, including conventional sequencing, non-Sangersequencing, random amplifiable polymorphic DNA, DNA barcoding, amplifiablefragment length polymorphism, microsatellites, single nucleotide polymorphism,and others. The limitation with a few of these methods is that noideal marker exists for all the species, and sometimes, it may misleadin identifying the taxonomy but could be highly beneficial in populationgenetics.12 Because of its accuracy andreliability to identify variations, DNA barcoding has gained the mostpopular of all methods. Traditional methods of plant identification,such as morphological analysis, can be subjective and prone to error,particularly in cases where species are closely related or have similarphysical characteristics.14 On the other hand,DNA barcoding enables accurate identification of a plant species basedon its distinct genetic make-up. Since every plant has a differentDNA sequence, it includes using a short, standardized DNA sequenceas a \u201cbarcode\u201d to identify a certain species of plant.The chloroplast gene rbcL, matK, and trnH-psbA are the most frequentlyused barcode region, but the ITS  markerhas been also used in recent studies.16 In a study,the ITS marker was used for DNA barcoding of Euphorbiaspecies, and the researchers found that this marker was able to effectivelydistinguish between Euphorbia subgenusand closely related species. It was reported that the ITS marker mightbe used to identify Euphorbia speciesand to verify the authenticity of the plant material used in conventionalmedicine and the pharmaceutical industry.17 Similarly, Akilabindu in 2019 used chloroplast rbcL and matK genefrom Flacourtia inermis Roxb for barcoding.It was reported that rbcL gene showed 100% similarity and matK geneshowed 99.20% similarity with Flacourtia jangomas.18 These studies strongly suggest thatmatK, ITS, and rbcL markers are intensively used in plant DNA barcoding.Despite being a promising technique, employing DNA barcoding for theauthentication and identification of medicinal plants comes with anumber of limitations such as limited reference libraries, inter-and intra-specific variation, and quality of plant material, cost,and complexity.19On the other hand,molecular biology has had a significant impacton plant science research, allowing us to uncover many previouslyunknown genetic and behavioral traits in plants. These genetic traitsare very helpful in identifying plants of particular species in casesof adulteration and substitution.Euphorbia tithymaloides, also knownas \u201cdevil\u2019s-backbone\u201d or \u201ccoast spurge,\u201dis a medicinal plant that belongs to the family Euphorbiaceae. Itis widely distributed in Central and South America, including theAfrican and Caribbean. The plant is found in many other subtropicaland tropical areas as an invasive species. The plant is traditionallyused in folk medicine to treat various ailments, such as inflammatoryconditions, fever, and tumors. Some studies have also shown that extractsof E. tithymaloides have pharmacologicalactivities such as anti-inflammatory, anti-tumor, anti-diabetic, anti-cancer,anti-leishmanial, anti-malarial, anti-helminthic, anti-microbial,anti-oxidant, anti-ulcerogenic, and cytotoxicity.21 Extensive and elaborative research works have been carried out inthe aerial parts of the plant, while the roots are yet to be explored.Hence, this study aimed to carry out the pharmacognostical study of E. tithymaloides root by both conventional and molecularmethods.22.1E. tithymaloides were collected from Jamia Hamdard Herbal Garden, New Delhi, India.Department of Botany, Jamia Hamdard, New Delhi, India, helped to identifyand authenticate the plant. For future reference, a sample of theplant material was deposited in the herbarium under the voucher specimennumber BOT/DAC/2022/01. The roots were cleaned with water to removemud, broken into small pieces, and completely air-dried. The powderwas then pulverized into a coarse powder using an analytical millingmachine and stored in an airtight glass jar for use in the currentwork.In March 2022, root parts of 2.2E. tithymaloides wereexamined using visual perception. The color, odor, and taste of theroot were observed and recorded as organoleptic properties. The macroscopiccharacteristics of the root, such as shape, size, fracture, and othersurface features, were observed using the protocol mentioned in Indianpharmacopeia.22Thefresh roots of 2.3500 mgof a moderately fine (44/85) grounded root powder was immersed in10 mL of water (1:20) and left to stand overnight for 24 h. Subsequently,the contents were put into a Petri-plate, and a slide was preparedby placing the contents with a brush on a clean and dry slide. Thecontents were then examined using a Motic microscope moticam 3.0 MP,AE 2000, and images were taken.2.422 and WHO.23To assess the purity and quality of the crude drug, analytical standardsand physicochemical constants of the root were determined. The ashvalue , foamingindex, foreign matter, swelling index, moisture content, and extractivevalue  of root powderedwere determined using the standard protocol provided by the IndianPharmacopoeia2.5The extractswere prepared by using the Soxhlet apparatus by increasing the polarityof a solvent such as ethanol/water (1:1), ethyl acetate, hexane, andethanol. For each solvent, 2 h was given for extraction with optimaltemperature.2.62.6.124To investigate the existence of different secondarymetabolites in the crude extract, qualitative phytochemical assayswere carried out using established methods. Saponin test with frothtest; terpenoid/steroid test with Liebermann\u2013Burchard reagent;flavonoid test with Shinoda test; Tannin test with ferrous(III) chloride;and alkaloid test with Dragendorff reagents.2.6.2The quantitative phytochemical tests were doneusing standard procedures to detect the number of secondary metabolitessuch as alkaloid, flavonoid, and phenolic compounds in the crude extract.2.6.2.125 Standard quercetin solutions of 20, 40, 60,80, and 100 g/mL were produced in 96% ethanol. The standard extractsolution (1 mg/mL) was prepared. 12 \u03bcL of 10% aluminium chloridesolution, 60 \u03bcL of extract solution, 180 \u03bcL of 96% ethanol,and 12 \u03bcL of 1 M sodium acetate were added to the mixture ina 96-well plate. 96 percent ethanol was used as a reagent blank. Afterbeing mixed, each substance was incubated for 40 min at room temperaturein a dimly lit area. The absorbance at 415 nm was measured with amicroplate reader. Total flavonoid content was calculated as quercetinequivalents per gram of plant extract.Total flavonoid content was determined using a slightly modifiedversion of the Sembiring et al. aluminium chloride colorimetric test.2.6.2.225 Ina flat-bottom microplate, a standard solution of extracts (1 mg/mL)was mixed with the Folin\u2013Ciocalteu reagent (1:4) in a 40 and400 \u03bcL ratio and shaken for 2 min. After 5 min, sodium carbonatesolution (100 g/L) was added (125 \u03bcL) and shaken for 1 min ata medium speed. Absorbance was measured at 765 nm using a VersaMaxAbsorbance Microplate Reader after 3 h at room temperature. The absorbancewas corrected by subtracting the ethanol control reaction from thesample reaction. Gallic acid in dilutions of 10, 50, 100, 150, and200 \u03bcg/mL was used as calibration standards. The total phenoliccontent was expressed in milligrams of gallic acid equivalents (GAE)per gram of plant extract.The Folin\u2013Ciocalteu method for a 96-well microplate wasoptimized based on Sembiring et al.2.6.2.326 Bromocresol green solution wasmade by dissolving 69.8 mg of BCG in 3 mL of 2 N NaOH and 5 mL ofdistilled water and then diluted to 1000 mL with distilled water.Phosphate buffer (pH 4.7) was prepared by adjusting the pH of 2 Msodium phosphate solution to 4.7 with 0.2 M citric acid. Atropinestandard solution was made by dissolving 1 mg of pure atropine in10 mL of distilled water. The extract was dissolved in 2 N HCl, filtered,and washed with CHCl3 (three times) and neutralized with0.1 N NaOH. 5 mL of BCG and 5 mL of phosphate buffer were added andcompletely extracted and diluted with chloroform.Accurately measured aliquots of atropine standard  were mixed with BCG and phosphate buffer, extractedwith chloroform, and diluted with chloroform. The complex absorbancein chloroform was measured at 470 nm using a UV-Spectrophotometer.The blank was prepared similarly but without Atropine.The total alkaloid content was determined using the method by Ajanalet al.2.72.7.1The NucleoSpin PlantII, Macherey-Nagel kit instructions were followed to extract DNA fromthe plant root sample (13817). The gel electrophoresis (1% w/v) methodwas used to ensure the purity of the extracted DNA and the same wasdocumented using the Bio-Rad.2.7.2Master Mix PhirePlant (Thermo Scientific) and Applied Biosystems 2720 thermal cyclerwas used for the amplification of the primers selected 1. PCR miExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher)was usedto clean the PCR product. Prior to sequencing, 2% agarose gel electrophoresiswas used to validate the PCR product\u2019s purity. As a molecularstandard, a 100 bp DNA ladder  was employed. Usingthe BIO-RAD GelDoc-XR gel documentation system, gel images were captured.2.7.3The DNA sequencing wasperformed with the PCR products purified with the ExoSAP. ABI BigDyeTerminator v3.1 Cycle Sequencing reaction kit was used.2.7.4The 3130Genetic Analyzer Automated DNA Sequencing Machine were used to generateDNA sequences in.ab1 and FASTA formats, and Sequencing Analysis 5.1software was used to do further analysis. A contig of the truncatedsequence was created using forward and reverse strand sequences. Consequently,a single FASTA sequence was produced and further investigated. Usingthe nucleotide BLAST analysis tool, the sequencing similarity of thegenerated samples was examined with the sequences in the GenBank Database.The Clustal W alignment was utilized for multiple sequence alignmentand comparing different sequences, and the degree of similarity betweenthem was determined. For all three of the sample sequences, a phylogenetictree was constructed using the MEGA 6 software utilizing the closest-matchingsource sequence data from the database (NCBI GenBank nucleotide sequence).33.1The macroscopiccharacteristics of the roots as shown in 3.2E. tithymaloides (L.) Poit that are diagnostically important.3.3E. tithymaloides were determinedand are reported in Total ash, watercontent, alcohol soluble extractive, water-soluble extractive value,acid insoluble ash value, soluble ash, and moisture content of theroot of 3.4The extracts were studied further to discoverwhich phytochemical compounds were present. Flavonoid, alkaloid, terpenoids,steroid, tannin, and saponins are examples of common phytochemistrycomponents found in the root extract 4.3.53.5.1y = 0.043x + 0.122, R2 = 0.9947. Among the four crude extracts, ethyl acetate containedthe highest amount of total flavonoid content compounds followed byhexane, ethanol, and then hydro alcoholic   4. Among  0.9987) 5.3.5.3y = 0.0302x + 0.171 R2 = 0.996)  5. Of all= 0.996) 5.3.6Pedilanthus tithymaloides chloroplast rbcL gene (AB267959.1)showed a maximum similarity of 99.83 , the proportion of duplicate trees in which the linkedtaxa grouped together is displayed next to the branches.28 The tree is rendered to scale with branch lengthsin the same units as the evolutionary distances used to estimate thephylogenetic tree. The evolutionary distances, which are measuredin terms of the number of base substitutions per site, were calculatedusing the maximum composite likelihood technique.29 Six nucleotide sequences were subject to this investigation.Codon positions 1st + 2nd + 3rd + noncoding were included. For eachsequence pair, all uncertain positions were eliminated (pairwise deletionoption). The final dataset had 671 positions altogether. In MEGA X,evolutionary studies were carried out.30 The closest plant species was used to draw the phylogenetic treefor the matches in the BLAST data .The final dataset had 671 locations altogether. In MEGA X, evolutionarystudies were performed.30 From the BLASThits, the distance matrix showed the closest distance between thenearby species   8. The phtermined 9. The fi of 100% 10, follo plotted 11.3.6.1aDNA sequencingThe following sequences were generated for the sample(13817-R(rbcl)):>13817-R(rbCL-F) ATATTGGATCAAGCTGGTGTTAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.>13817-R(rbCL-R) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATACGTAGAGCGTA.>13817-R(rbCL) (assembled contig) TAAGGCAACCCCAAAACAGAGACTAAAGCAAGTGTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTCCTGAATATGAAACCAAAGATACTGATATCTTGGCAGCATTCCGAGTAACTCCTCAACCTGGAGTTCCACCTGAGGAAGCAGGAGCTGCCGTAGCTGCTGAATCTTCTACTGGTACATGGACAACTGTGTGGACCGATGGGCTTACCAGTCTTGATCGTTATAAAGGACGATGCTACCACATCGAGCCCGTTGCTGGAGAAGAAAATCAATATATTGCTTATGTAGCTTACCCCTTAGACCTTTTTGAAGAAGGTTCTGTTACTAACATGTTTACCTCCATTGTGGGTAATGTATTTGGGTTCAAAGCCCTACGCGCTCTACGTCTGGAGGATTTACGAATCCCTACTTCTTATACTAAAACTTTCCAAGGGCCACCTCATGGCATCCAAGTTGAGAGAGATAAATTGAACAAATATGGTCGCCCTCTATTGGGTTGTACTATTAAACCAAAATTGGGGCTATCCGCTAAGAATTACGGTAGAGCGGTTTATGAATGTCTTCGCGGTGGATTGAATTATTTCCAGA.bBLAST analysisLibrary details: molecule type: DNA, query length: 607bases databasename: nr.cDistance matrixDescription: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.2aDNA sequencing.The following sequences were generated for the sample(13817\u2014ITS).>13817(ITS1) GGGCCGTAGCTACTGCAACGACCCGTGACATGTTCATAAACATGGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGCTTCGAGGGGGGGGGGGAGAGAAAAAA.>13817(ITS4) CTTTTTTTCGGCAGTTCCCGACGCCTTGGGTCGGGTTGGTAGAGACCGACAAGAGGAACACGGTGCACACCGGTCCCGCGCCCGCCCTTGGAGCCGTGCCGGACGATCCCGACCGGAAGCCAGTTTATGAAATGTCCCGGGTCGTTTGCGGGCGGGCCTGACAATGATCCTTTCGTAAGGGGGGGGCTGCGAAGGATCATGTCGAGGCCTGCCTAGCAAAACGACCCGTGAACATGTTCATAAACATGGCTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCCACATTGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGAGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGTCAGGCGGATAGACCA.>13817-ITS(assembled contig) GGGTGCCGGTGCGGGATTCGTCCGGCAACGGCATCCCACATGGGCCGGGCAGCGGGGACGCGGGTGGTGCACCCCGTGTTCTCCTTGTCCGGTTCCTTCTAACCAAACACCGACGCCAAACGCGTCAAGGAACTGCGAAAAAAAGGCAGCTTAGGCCCCGGAAACGGCGGTAACCAATGCTGTTTTGGAATAAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACTTGGTGTGAATTGCAGGATCCCGCGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTCGGCCGAGGGCACGCCTGCCCTGGGTGTCACTCAACTGTCGCCCCGACCCCCTCCTGAAAGGAGGGACGTGAGGGGCGGATGATGGCTTCCCGTGCGCTTTGCAGCCCGCGGTTGGCCCAAAATTCTGGTCCCTGGAACAGAATCCACGGCAATCGGTGGTTGAAAGACCCTCGTAAAATGTCGTGGTTTCACGGAAGCCAGAGGCGGCAATTAGACCCCAGAGCGCATCCAAAGCAGCGCTCGAACGGCGACCCCAGGTCAGGCGGGATTACCCGCTGAGTTTAAGC.bBLAST analysis.Library details: molecule type: DNA, query length: 612bases databasename: nr.cDistance matrix.Description: nucleotide collection (nt) *program:BLASTN 2.2.28+.3.6.3aDNA sequencing.The following sequences were generated for the sample(13817M(matK)):>13817-M(MatK-F) ATAAGAGAAATTTCCGCATTTAATTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTATAGCTAACGATGATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAATGCAATTTTGTAATGCAGTAGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCGAACAATTTTGTGCGTATATGCAGAAATCTTTCTCATTATCTCGGGGGGGGGACTCACCAAAAA.>13817-M(MatK-R) CATCATCAAATATTTCCTTTTTAGAGGACAAATTTCCGCATTAATTATGTATCAGATGTACTAATACTATCCCATCCATCTTGAAAAATGGTCCAAACCCTTCGTTTTTGGGTGACAGACCATCTTCTTGCATTTTTTACGATTCTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATCAGATATATCGACAAATTTTGCCTATAATTCC.>13817-M(MatK)(assembled contig) TTATGTATCAGATGTATTAATACCTTATCCCATCCATCTTGAAAAATTGGTCCAAACCCTTCGTTTTTGGGTGACAGACCCTTCTTCTTTGCATTTTTTACGATTCTTTCTTCATCAGTATTGGAATTGGAACAGTCTTATTATTCCAAAGAAATCAATTTCGATTTTTCGAAAAAATAATCCACGATTTTTCTTGTTCATATATAATATTCATATATATCAATATGAATCCATCTTCTTTTTTCTTCGTAATCAGTCCTTTCATTTACGATCAACATTTTCTCGAGTCTTTCTTGAACGAATTTTTTTCTATGGAAAACTAGAACATTTTGCAGAAGTTTTTGCTAATGATTTTCAGACCATCCTAGGGTTGTTCAAGGAGCCTTTCATGCATTATGTTAGATATCAAGGAAAATCAATTCTGGCTTTAAAAGATAAGCCCTTTCTGATGAAAAAATGGAAATATTACCTTGTCAATTTATGTCAATGTCATTTTTATGTGTGGTTTCAACCAGAAAAGATCTATATCAATTCATTATCCAAAAATTCTCTCTATTTTGGGGGATATCTTTCAAGTGTACAAATCAATCCTTTGGTAGTACGGAGTCAAATGCTAGAAAATTCATATCTAATAGCTAACGATAATACTATGAAGAAACTCGATACAATAGTTCCAATTACTCCTTTAATTAGATTATTGGCAAAAATGCAATTTTGTAATGCAGTAGGACATCCTATTAGTAAACCGATCCGGGCTCATTCATCCGATTCAGATATTATCG.bBLAST analysis.cDistance matrix.Library details: molecule type: DNA, query length: 728bases databasename: nr description: nucleotide collection (nt) *program: BLASTN2.2.28+.4E. tithymaloides, was selected for the pharmacognosticstudy. Primarily, the macroscopic analysis of plants aids in identifyingauthentic materials. The E. tithymaloides root is macroscopically seen to be light brown on the outside andbuff color on the inside, with a fibrous texture and dusty and mildlybitter flavors. Similar to this, one of the essential factors in pharmacopeiais the study of powder microscopy. The powdered roots of E. tithymaloides revealed the presence of scalariformvessels with a pitted bordered wall, radially cut medullary rays,a group of fragmented oval-shaped parenchyma cells with a thin wallcontaining starch, a thick wall with an oval to rectangular-shapedcork, group of sclereids with polygonal wall, elongated, blunt end,and thin wall fibers.A pharmacognostic studyof a plant involves the scientific examinationof the plant\u2019s physical and chemical characteristics, as wellas its traditional uses. The goal of such a study is to establisha set of quality standards for the plant material that can be usedto ensure its authenticity and purity. One such plant, E. tithymaloides. It was reported that the ethanol,ethyl acetate, and hexane extracts showed the presence of steroids,triterpenes, saponins, tannin, and coumarin. It was observed thatthe leaves lack alkaloids, which makes the root of E. tithymaloides more advantageous for therapeuticuse than the leaves.31Preliminary phytochemical investigationof medicinal plants isthe initial step in the process of identifying and characterizingthe phytoconstituents present in a plant. The root powder was subjectedto extraction with solvents such as ethanol, ethanol: water, hexane,and ethyl acetate. These extracts were then used for the physiochemicaland phytochemical analysis, which indicated the presence of alkaloids,tannins, flavonoids, steroids, terpenoids, and traces of saponins.A similar study was conducted by Matisui et al. in 2017, where thephytochemical analysis was performed with the leaves of E.tithymaloides was found to be 7.5%, indicating thetotal amount of inorganic matter present in the plant. The water-solubleash value of 2% revealed the presence of water-soluble compounds suchas inorganic compounds, acids, and sugars. The hexane, alcohol, andhydro-alcohol soluble extractive values show the presence of polar-solublesolvents such as tannins, flavonoids, and alkaloids. Similarly, thefoaming index and moisture content were both found to be <100 and3.33%, indicating that no foaming agents and less moisture were presentin the root samples. The presence of moisture in the plant materialcan have a significant impact on the quality and stability of thephytoconstituents present in the material. Moisture serves as an idealmedium for the growth of bacteria and fungi, which can lead to thedegradation of the plant material and the loss of its medicinal properties.33 Additionally, moisture can also cause the hydrolysis and oxidationof moisture-sensitive phytoconstituents, such as alkaloids, flavonoids,and terpenoids, which can result in a decrease in their concentrationand effectiveness. These findings agree with the phytochemical analysis,which shows the presence of polar-soluble solvents and traces of saponins.Extractivevalues are a measure of the amount of certain activeor inert ingredients present in a drug. They are used to determinethe purity and potency of a drug and can help detect if a drug isexhausted or adulterated. The United States Pharmacopeia (USP) andthe European Pharmacopeia (EP) provide guidelines for the acceptablerange of extractive values for various drugs. Drugs that fall outsideof these ranges may be considered exhausted or adulterated and shouldnot be used. The total ash content of the root of 34 This study examined the total amount of flavonoids,phenols, and alkaloids. The aluminum chloride colorimetric test wasused to evaluate the sample\u2019s total flavonoid content. Theassay revealed that the ethyl acetate extract contains the highestamount of flavonoids, while the hydro-alcoholic extract showed thelowest amount. The ethyl acetate extract\u2019s high concentrationof flavonoids is comparable to the research conducted by Ch\u00e1vezet al. in 2022. It was reported that, when compared to hexane, water,and dichloromethane, the ethyl acetate extract of E.tithymaloides leaves contained a high amount of flavonoids.35 This indicates that E. tithymaloides in general, as a whole plant, possesses a good amount of flavonoids.Likewise, using gallic acid as a reference, the Folin\u2013Ciocalteumethod was used to quantify the total phenol concentration of theroot extracts. It was clear from the assay that the hexane extractshad a higher phenolic content than other extracts. The total alkaloidalcontent assay showed that the ethanol extract had a high amount ofalkaloids.The primary goal of quantitative chemical analysis is to estimatethe amounts of the plant\u2019s major phytoconstituents classes.Flavonoids, phenols, and alkaloids are three important classes ofphytoconstituents that are commonly found in medicinal plants. Theyare known for their medicinal properties and have been used in traditionalmedicine for centuries. Their presence in medicinal plants is oftenused as a measure of the plant\u2019s quality and potency.36 In this study, all three markers were used forthe identification of E. tithymaloides. The sequence match with rbcl loci showed a match of 98.64 to 99.83%with the top five hits from the BLAST analysis. Similarly, the sequencematch with the MatK loci was from 96.25 to 100%. These results arein contrast with the phylogenetic study conducted by El-Banhawy, 2020in the genus Euphorbia.37 It was reportedthat the rbcl was the least successful and matK genes were not significantlydifferent in identifying the Egyptian Euphorbia. However, the resultsof this study reveal a good identity score of 99.84 and 100 for Euphorbia plant grown in India. The difference inthe identification of the genus using the rbcl and matk loci couldbe varied due to the geographical location and environment of theplant growth. These genetic and geography-based changes and theiridentification by rbcl and ITS were expressed by Shawkat and Ahmed,2022 in a comparative study. It was reported that the rbcl and ITS2were able to provide a good resolution among the Euphorbiatirucalli, Euphorbia hirta, and Euphorbia peplus but showedonly a minor change in the evolution taxa and phylogenetic tree.39 These data are comparable to the results observedin this study as the phylogenetic tree and the distance matrix showsa minor change and were able to identify the closest species witha good matching percentage. The ITS is considered to be one of themost informative markers for species identification in plants, asit has a high degree of variation among different plant species. TheITS region is located between the 18S and 28S rRNA coding regionsand is transcribed as part of the rRNA precursor molecule. The ITSregion is highly variable, containing both coding and non-coding regions,which makes it a valuable marker for species identification. Additionally,the ITS region is present in multiple copies in the genome, whichincreases the chances of detecting variations among different species.38 Likewise, the ITS primers ITS1 and ITS4 in thisstudy revealed a good match from 95.11 to 99.84% from the BLAST analysis.The ITS loci results are comparable with the study experimented onby Kim et al., in 2020 where DNA barcoding was performed for KoreanEuphorbiaceae. It was reported that among the rbcl, ITS, and MatKloci, the ITS was the most beneficial and can be used to identifyother Korean Euphorbiaceae plants using ITS as a single barcode.40 Likewise, the results from this study show thatall the three loci were able to efficiently identify the E. tithymaloides. In addition, the 100% true matchwith the MatK loci shows that it can be used as a standalone to identify E. tithymaloides from the subspecies and other Euphorbiaceaeplants as well.The nuclear ribosomal DNA ITS regions ITS1 and ITS2and the chloroplastgenes matK, rbcL, and trnH-psbA are the markers in plant barcodingthat are most often investigated. These markers are highly informativefor identifying and differentiating plant species. Recently, the useof DNA barcoding based on the markers rbcl, ITS, and MatK has gainedsignificant traction in the field of plant species authentication.E. tithymaloides is the high demand for the plantand its medicinal properties. This has led to the collection of theplant from wild populations, which can lead to over-harvesting anddepletion of wild populations. Another reason is that E. tithymaloides is often confused with closely relatedspecies, and this can lead to misidentification and substitution.For example, E. tithymaloides is oftenconfused with Euphorbia lathyris, whichis not used for medicinal purposes and is considered toxic. Adulterationand substitution can have serious consequences for both the consumersand the environment. Consumers may be unknowingly consuming harmfulsubstances or receiving ineffective treatment, while wild populationsof medicinal plants may become endangered due to over-harvesting.To address these issues, it is important to establish and use DNAbarcoding techniques for the identification and authentication ofmedicinal plants, including E. tithymaloides, to ensure that the plant material being sold is authentic and unadulterated.Additionally, conservation measures should be implemented to protectwild populations of medicinal plants from over-harvesting, and regulationsshould be put in place to control the trade of medicinal plants.One of the main reasons for the adulterationand substitution of 5E. tithymaloides has been carriedout in this study and reported for the first time. The results fromthe micro-and macroscopic studies, phytochemical analysis, and DNAbarcoding had shown significant results in identifying the E. tithymaloides. It is important to keep in mindthat DNA barcoding is one tool among many that can be used to identifyand authenticate medicinal plants and it should be used in combinationwith other methods, such as morphological and chemical analysis, toensure accurate identification. The results of this pharmacognosticalstudy can be used to establish a set of quality standards for the E. tithymaloides, which can be used to ensure itsauthenticity and purity. This is important for ensuring the safetyand efficacy of traditional medicine and medicinal products made from E. tithymaloides.The conventional and molecularpharmacognostic study on the rootof"}
+{"text": "The current study aims to investigate the regulatory impact of leptin or melatonin on bone metabolism as well as the underlying mechanism in conjunction with Sema4D .Rats were used to create the osteoporosis model utilizing the OVX (OVariectomize) technique. Rat tibial specimens from each side were collected for three-dimensional reconstruction and Micro-CT scanning examination. The Hematoxylin-osinstaining (HE) staining technique was used to determine the pathological condition of bone tissues. The ELISA (Enzyme-Linked Immunosorbent Assay) assay was used to measure the amount of estradiol present in the serum. In the current study, there were six groups: control, OVX, OVX\u2009+\u2009NL (no load group), OVX\u2009+\u2009Sema4D, OVX\u2009+\u2009Sema4D\u2009+\u2009leptin, and OVX\u2009+\u2009Sema4D\u2009+\u2009MT (melatonin). Rats were given injections of the Sema4D or leptin overexpressing vectors via the tail vein in accordance with the aforementioned classification. By using a high-resolution micro-CT technology, 3D bone structure was discovered. The activity of tartrate-resistant acid phosphatase-5b (TRAP-5b) and bone-derived alkaline phosphatase (BALP) in serum was assessed using an ELISA. The number of osteoclasts in the metaphysis of the upper tibia was determined using TRAP (tartrate-resistant acid phosphatase) staining. Immunohistochemistry was used to find leptin and bone morphogenetic protein-2 (BMP-2) expressions in bone tissue.The BV/TV (Bone volume/Tissue volume), Tb.N (Trabecular number), BMD , and BMC  levels were significantly higher in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups compared to OVX\u2009+\u2009NL, while Tb.Sp (Trabecular separation) levels were significantly lower. In contrast to the OVX group, the bone trabeculae in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups had a relatively complete structure and tended to be organized closely. The amount of bone trabeculae grew drastically, whereas the proportion of TRAP-positive osteoclasts declined dramatically. BMP-2 and leptin were also elevated, while BALP and TRAP-5b activity was reduced.Leptin or melatonin improved Sema4d's role in trabecular bone microstructure, bone production, and repairment of trabecular bone loss in osteoporosis rats. A prevalent age-related illness is osteoporosis. Osteoporosis has recently emerged as a major global public health concern due to the sharp rise in patient numbers . StatistSema4D, a protein involved in brain signaling, belongs to the semaphorin family. It has been established that semaphorins, also known as immunological semaphorins, and immune control have a close association. According to reports, Sema4D is involved in a wide range of signal transduction pathways and performs a wide range of biological functions, including the control of B cells, T cells, and other immune cells as well as the development of the nervous system, platelet function, epithelial cell function, and endothelial cell function . Sema4D Using an OVX rat model, the current work intends to investigate the effects and processes of Sema4D, Sema4D paired with leptin, and Sema4D combined with melatonin on bone metabolism in an effort to identify possible molecular targets for osteoporosis therapeutic treatment.SD (Sprague Dawley) rats, 32 females, 200\u2013220\u00a0g Zhejiang Weilihua Experimental Animal Technology Co., Ltd., License No. SCXK (Zhejiang) 2019-0001), Rearing Environment: Temperature 20\u201326\u00a0\u00b0C, Humidity 40%- 70%. All research was done in compliance with international standards for the treatment and use of laboratory animals, and needless harm to the animals was avoided at all costs .Medicine refrigerator , refrigerated freezer , slicer , electric blast drying oven , thermostatic incubator , electric thermostatic incubator ; Pressure cooker , induction cooker , fume hood; Microscope , microtome , multifunctional enzyme marker ; First antibody: BMP-2 ; leptin; Sema4d ; Second antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H\u2009+\u2009L) ; Tartrate-resistant acid phosphatase staining solution ; Rat tartrate-resistant acid phosphatase 5b (TRACP-5b) ELISA reagent ; Rat bone alkaline phosphatase (BALP) kit ; Melatonin (S20287) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a relative molecular weight of 232.28 and a purity of more than 99%.Construct the PCR primer for the target fragment based on the sequence of the target gene Sema4D  and Leptin ; As the carrier for digestion, the suitable restriction enzyme was chosen, and the pure linear carrier was extracted from the agarose gel; Then, perform PCR on the target fragment and recover the agarose gel to acquire the right fragment size; Link the linearization vector to the target fragment utilizing homologous recombination or the T4 technique; Transformed responsive DH5a or stbl3, covered with bacterial fluid and cultured for 12 to 16\u00a0h; Choose the moving monoclonal colony for validation; Choose the most appropriate positive clone for colony verification prior to sequencing; The extraction of plasmids was carried out on cloned samples with the right sequence. Anhui General Biology Co., Ltd. is responsible for the adenovirus packaging of the vector after the sequencing verification. The specific information on the viral vector is as follows :ATGAAGATGTGTGCCCCCGTCAGGGGGCTGTTCTTGGCCCTGGTGGCTGTGTGGAGGACCGCGGTGGCATTCGCCCCTGTGCCTCGGATCACCTGGGAGCACGGAGAGGTAGGTCTGGTGAAGTTTCACGAGCCAGGCATCTTTAACTACTCTTCCTTGCTGATGAGTGAAGACAAAGACACTCTGTATGTGGGTGCCCGGGAAGCTGTCTTTGCAGTGAATGCGCTGGACATCTCTGAGAAGCAACATGAGGTATACTGGAAGGTCTCTGAAGACAAAAAATCCAAGTGCGCAGAGAAGGGGAAATCAAAGCAGACGGAGTGCCTTAACTACATCCGAGTGCTGCAACCGCTTAGCAGCACTTCCCTCTACGTGTGTGGGACCAATGCGTTCCAGCCCACCTGTGACCACCTGAACTTGACCTCTTTCAAGTTTCTGGGGAAAAGCGAAGATGGCAAAGGAAGATGCCCCTTCGACCCCGCCCATAGCTACACATCCGTCATGGTCGGGGGAGAGCTCTACTCTGGGACTTCATATAATTTCTTGGGCAGCGAACCCATCATCTCTCGAAACTCTTCCCACAGTCCCCTGAGGACAGAGTACGCCATCCCTTGGCTAAACGAGCCTAGCTTCGTCTTTGCTGACGTGATCCACAAGAGCCCAGATGGTACAGAGGCTGAGGATGACAAGGTCTACTTCTTCTTTACGGAGGTGTCCGTGGAGTACGAGTTCGTCTTCAAGTTGATGATCCCGCGAGTTGCCAGGGTGTGCAAGGGCGACCAGGGCGGCCTGCGGACTTTGCAAAAAAAGTGGACCTCCTTCCTAAAGGCCAGACTGATCTGCTCCAGGCCAGACAGTGGCCTGGTCTTCAACATTCTTCAAGATGTGTTTGTGCTGAGGGCCCCGGGCCTCAAGGAACCTGTGTTCTATGCGGTCTTCACCCCACAGCTGAACAACGTGGGTCTGTCAGCGGTCTGTGCCTACACGCTGTCCACGGTGGAGGCCGTCTTCTCCCGAGGAAAGTACATGCAGAGTGCCACAGTGGAGCAGTCTCACACCAAGTGGGTACGCTACAATGGCCCAGTGCCCACTCCCCGGCCTGGAGCGTGTATCGACAGTGAGGCCCGGGCAGCCAACTACACCAGCTCCTTGAATCTCCCAGACAAAACGCTGCAGTTTGTCAAAGACCACCCTTTGATGGACGACTCGGTGACGCCAATAGACAACAGGCCGAAACTGATCAAAAAAGATGTCAACTACACCCAGATAGTGGTAGACAGGACCCAGGCCCTGGATGGGACCTTCTACGACGTCATGTTCCTCAGCACAGACCGGGGCGCTCTGCATAAAGCTGTCATCCTTGCAAAAGAGGTACACGTGGTTGAGGAGACCCAACTCTTCCAGGACTTCGAACCGGTCCTGTCTCTGCTGCTATCATCAAAGAAGGGGAGGAAGTTTGTCTATGCTGGCTCCAACTCAGGAGTGGTCCAAGCTCCCCTGGCCTTCTGCGGAAAGCACAGTAGCTGTGAAGACTGTGTGCTAGCACGGGACCCCTACTGCGCCTGGAGCCCAGCCATCAAGGCCTGTGTTACCTTGCACCAGGCAGAGGGCTCTAGCAGGGGCTGGATTCAGGACATGAGTGGCGACACGTCCTCGTGCCTGGATAAGAGTAAAGAAAGTTTCCATCAGCATTTTTTCAAGCACGGCGGCACAGCAGAACTCAAGTGTTTCCAAAAGTCCAACCTGGCCCGGGTGGTGTGGAAGTTCCAGAACGGCGAGTTGAAGGCTGTGAGTCCCAAGTATGGCTTTGTGGGCAGGAAGCACCTGCTCATCTTTAACCTGTCAGACGGAGACAGCGGTGTGTACCAGTGCCTGTCAGAGGAAAGGGTCAGGAATAAAACGGTCTCCCAGCTGCTCGCCAAGCACATCCTGGAAGTGAAAATGGTAGCTCGGATCCCCCCATCACCTACCTCACAGACTGCTCAGACAGAAGGTAGTAGGATCACATCCAAAATGCCTGTGGCGTCTACCCAGGGGTCCTCTCCCCCTACCCCGGCTCTGTGGGCAACCTCCCCCAGGGCTGCCACCCTACCTCCCAAGTCCTCCTCCACCGGCACGTCCTGTGAACCAAAAATGGTCATCAACACGGTCCCACAGCTCCACTCGGAGAAGACAGTGTATCTCAAGTCCAGTGACAACCGCCTGCTCATGTCTCTCCTCCTCTTCCTCTTTGTCCTCTTCCTCTGCCTCTTTTCCTACAACTGCTACAAGGGCTACCTGCCCGGACAGTGCTTAAAGTTCCGCTCAGCCCTGCTGCTCGCAAAGAAAAAACCCAAGTCAGAGTTCTCTGACCTGGAGCAGAGTGTGAAGGAGACGCTGGTAGAACCTGGGAGCTTCTCGCAGCAGAACGGCGACCAGCCCAAGCCAGCCTTGGATACCGGCTATGAAACCGAGCAGGACACTATCACCAGCAAGGTCCCCACCGATCGAGAGGACTCGCAACGTATCGACGAGCTCTCCGCCAGGGACAAACCGTTTGATGTCAAGTGTGAACTCAAGTTTGCAGACTCGGATGCCGACGGGGACTGA.ATGTGCTGGAGACCCCTGTGCCGGTTCCTGTGGCTTTGGTCCTATCTGTCCTATGTTCAAGCTGTGCCTATCCACAAAGTCCAGGATGACACCAAAACCCTCATCAAGACCATTGTCACCAGGATCAATGACATTTCACACACGCAGTCGGTATCCGCCAGGCAGAGGGTCACCGGTTTGGACTTCATTCCCGGGCTTCACCCCATTCTGAGTTTGTCCAAGATGGACCAGACCCTGGCAGTCTATCAACAGATCCTCACCAGCTTGCCTTCCCAAAACGTGCTGCAGATAGCTCATGACCTGGAGAACCTGCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGAGCTGCTCCCTGCCGCAGACCCGTGGCCTGCAGAAGCCAGAGAGCCTGGATGGCGTCCTGGAAGCCTCGCTCTACTCCACAGAGGTGGTGGCTCTGAGCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGACCTTAGCCCTGAATGCTGA.The following is the map of the Semad4 adenovirus overexpression vector:The vector map of Leptin adenovirus overexpression is as follows:The occurrence and progression of osteoporosis are significantly influenced by the estrogen released by the ovaries. Rats' decreased estrogen secretion following ovariectomy results in a weaker suppression of osteoclasts. Bone production is outpaced by bone absorption. Because the bone is in a high stage of transition, the mass of the bone cannot be balanced, which causes more bone to be lost. The best model to research postmenopausal osteoporosis is the ovariectomized osteoporosis model , 20; AccBai et al.  describeObtain serum samples and separate the supernatant for testing using a centrifuge. After treatment, BALP, TRAP-5b, and estradiol were identified according to the processes outlined in the instructions for the respective ELISA kit \u201327. Use Following the collection of tibia tissues from each animal, the tissues were washed in water for two hours. After dehydration with various concentrations of ethanol solution, tissues were dehydrated with xylene until translucent, followed by embedding for one hour and slicing. The slides were then roasted, dewaxed, hydrated, bathed in distilled water, colored in a hematoxylin aqueous solution for three minutes, and then differentiated using a hydrochloric acid ethanol differentiation solution for fifteen seconds. After being cleaned briefly with water and blue-returning solution for 15\u00a0s, slides were rinsed with water and stained with eosin  for three minutes. The inverted microscope was then utilized to capture images .Sections of paraffin were deparaffinized for 5\u00a0min, then progressively incubated in 100% ethanol for 5\u00a0min, 90% ethanol for 2\u00a0min, and 70% ethanol for 2\u00a0min. The TRAP staining solution was then added for fixation at 4\u00a0\u00b0C for 60\u00a0s. After being washed with water, slices were immersed in TRAP incubation solution at 37\u00a0\u00b0C for 45\u201360\u00a0min, then stained with methyl green for 2 to 3\u00a0min. Pictures were captured using a microscope .The slides were washed in PBS for one hour before being incubated overnight with 10% goat serum. The slides were then incubated with primary antibodies against BMP-2  or leptin  at 4\u00a0\u00b0C for 24\u00a0h, after which they were washed with PBS. The secondary antibody  was then added, followed by a 24-h incubation at 4\u00a0\u00b0C, rinsing, and DAB staining. Finally, photos were captured with an inverted microscope .For statistical analysis, SPSS 20.0 software was used. Each experiment was conducted three times. The mean standard deviation was used to express the quantitative outcomes (X As shown in Fig.\u00a0As demonstrated in Fig.\u00a0As shown in Fig.\u00a0TRAP staining was used to evaluate the number of osteoclasts in metaphysis at the upper end of the tibia. Compared to control, the number of TRAP positive osteoclasts was dramatically increased in the OVX, OVX\u2009+\u2009NL, and OVX\u2009+\u2009Sema4D groups, among which the highest number of osteoclasts was observed in the OVX\u2009+\u2009Sema4D group. Furthermore, compared to the OVX group, dramatically declined TRAP positive osteoclasts were observed in the OVX\u2009+\u2009Sema4D\u2009+\u2009leptin and OVX\u2009+\u2009Sema4D\u2009+\u2009MT groups (Fig.\u00a0As shown in Fig.\u00a0As shown in Fig.\u00a0Sema4D plays a crucial function in the regulation of osteoclast formation. Sema4D expression is increased during osteoclast proliferation ; NegishiIn the present study, OVX was used to construct an osteoporosis rat model. Pathological results showed that the number of tibial trabeculae in OVX rats was significantly reduced, and the bone trabeculae were thin and fractured with low density, accompanied by an increased serum E2 level. These results were consistent with the research reports of Zhao  and WangLeptin is reported to promote the proliferation of osteoblasts and the transformation of bone marrow stromal cells into osteoblasts. In previous experiments, an extremely repressed long-bone growth, reduced bone surface area, and decreased mineral and bone density level were observed in leptin deficient mice which were dramatically rescued by the injection of leptin, indicating that leptin played an important role in the differentiation and maturation of osteoblasts . It is rThe effect of melatonin on primary osteoporosis has been widely reported. St Hilaire et found that melatonin levels were lower in premenopausal women and were accompanied by increased bone resorption . Maria rIn conclusion, leptin and melatonin reversed the activation of osteoclasts caused by overexpression of Sema4D, which intensified the pathological process of osteoporosis. The target molecules for the clinical therapeutic development of osteoporosis were offered by the current study as potential and trustworthy candidates."}
+{"text": "Pythium flevoense was diagnosed as the cause of dermatitis in a young adult female harbour porpoise (Phocoena phocoena) that had been trapped in a pound net in a temperate saltwater environment. Disease from Pythium sp. infection\u2014pythiosis\u2014is infrequently diagnosed in humans, horses, dogs, cattle, and few other mammalian species. Pythiosis is typically associated with exposure to tropical or subtropical freshwater conditions, and typically caused by Pythium insidiosum. However, until now, pythiosis has been reported in neither marine mammals nor temperate saltwater conditions, and P. flevoense is not known as a cause of pythiosis in mammals. This porpoise developed generalised dermatitis despite treatment and euthanasia was necessary. Histopathological evaluation revealed a chronic active erosive dermatitis, with intralesional hyphae morphologically consistent with a Pythium sp. PCR analysis and sequencing of affected skin matched Pythium flevoense with a 100% similarity to the reference strain. Additional diagnostics excluded other pathogens. Based on this case report, P. flevoense needs to be considered as a mammalian pathogen. Furthermore, harbour porpoises and possibly other marine mammals may be at risk of infection with P. flevoense, and pythiosis should be included in the differential diagnosis of dermatitis in marine mammals.The oomycete The online version contains supplementary material available at 10.1186/s13567-023-01226-1. Pythium species are oomycetes, fungus-like filamentous eukaryotic organisms belonging to the kingdom Stramenopila, phylum Oomycota, class Oomycetes, order Pythiales, family Pythiaceae, which may reproduce both sexually and asexually [Pythium insidiosum in humans and other animals may cause disease, called pythiosis (insidiosi), typically characterized by necrotising and granulomatous inflammation [sexually \u20133. Most sexually . Howeverammation , 3. The ammation , 3, suppammation .P. insidiosum occurs typically in (sub)tropical stagnant freshwater containing motile zoospores that enter skin lesions and invade tissues. Zoospores show tropism for skin and hair [Infection with and hair . Pythiosand hair \u20138. Pythiand hair , 3.Depending on the site of infection and involved species, cutaneous, lymphonodular, osseous, vascular, ocular, visceral, and disseminated forms of pythiosis may occur . VasculaP. insidiosum is morphologically discernible as hyaline, pauci-septate, nonparallel thin-walled (5\u201310\u00a0\u03bcm) hyphae  with infrequent and irregular branching. As routine H&E stains may fail to stain these hyphae, enhancement of discernibility requires silver stains like Grocott silver stain [Histologically, er stain , 15.Pythium flevoense was isolated and identified for the first time in soil samples from the province Flevoland, The Netherlands in 1968 [Parabroteas sarci) in Argentina [Plecoglossus altivelis) in Japan [P. flevoense, and simultaneously the first case of pythiosis in a marine mammal, a harbour porpoise (Phocoena phocoena). in 1968 . Sporadirgentina  and ayu in Japan . Here wend of May 2018, trapped in a pound net in the coastal seawaters near the town Kors\u00f8r in Denmark. It was freed and brought to the Marine Biological Research Center, University of Southern Denmark, and intended to be part of the porpoise collection at the neighbouring sea aquarium Fjord & B\u00e6lt at Kerteminde Bay which is permitted by Danish law to house bycaught harbour porpoises for research and conservation purposes. It was named Idun and kept alone in a quarantined saltwater basin. A separated basin housed another normal healthy harbour porpoise. Both basins were circulated continuously with the same fresh seawater from the bay. Despite treatments for a progressive generalised erosive dermatitis, the animal did not recover and was euthanised after 3\u00bd months . The carcass was stored on wet ice directly after euthanasia until autopsy was performed, 24\u00a0h after death.A female harbour porpoise, estimated to be 14 months of age, was found alive on the 22During autopsy the porpoise\u2019s external and internal organs were examined. The skin lesions were photographed and sampled for histology, bacteriology and molecular analysis. Internal organs and tissues were similarly sampled. Samples for histology of skin, lung, trachea, heart, kidney, spleen, lymph nodes, urinary bladder, liver, pancreas, stomach, small and large intestines were fixed by immersion in 10% neutral-buffered formalin. These fixed samples were routinely processed and embedded in paraffin. Tissue sections of 4\u00a0\u03bcm thick were mounted on glass slides, deparaffinised with xylene, rehydrated using graded alcohols, and stained according to routine histochemical protocols  with haeSamples from affected skin were biopsied from the live animal on day 34 after rescue and taken during autopsy on day 105 after rescue. Samples for molecular analyses were stored in lysis buffer until processing. Tissue samples for bacteriology including affected skin were taken during autopsy on day 105, stored and frozen at \u221220\u2009\u00b0C and subsequently thawed a week later for further analyses. Fluorescent microscopy using Blancophor was performed on the skin samples (taken on day 34), to visualize fungal(-like) organisms. Fungal cultures from the same samples were performed using SGA agar  and YGC agar  with incubation at 35\u201337\u00a0\u00b0C for 2 weeks. Species identification included classical techniques including macro- and micromorphology and thermotolerance testing. Molecular analysis included internal transcribed spacer (ITS)-PCR and sequencing of which the results were compared with online sequence databases in July 2018, NCBI (BLAST) and CBS* database. . The sequence analysis was repeated in August 2021 specifically for this study.For bacteriological examination, samples of skin, lung, kidney, liver, spleen, uterus, tracheobronchial lymph node and adrenal gland were frozen at \u221220\u2009\u00b0C\u00a0and after thawing, cultured according to an in-house established standard protocol, including classical methods and commercial API identification kits , for bacterial species from marine mammals, as described previously . For virnd of May 2018 into the Marine centre (day 0), blood samples were collected. Results from standard haematological and clinical biochemical analyses on these samples were all within normal ranges, without any indication of immunosuppression, anaemia, or inflammation  , days 3 to 29: enrofloxacin , days 20 to 104: tramadol , days 25 to 31: itraconazole , days 27 to 37: miconazol , days 29 to 49: terbinafine , days 31 to 45: posaconazole , days 45 to 101: voriconazole , days 45 to 104: amoxycillin with clavulanic acid , days 100 to 102: posaconazole , days 100 to 103: terbinafine , days 101 to 103: enilconazole (14.4% w/w baths), days 103 to 104: enrofloxacin .Macroscopic examination showed a female non-pregnant harbour porpoise in moderate physical condition , with a generalised dermatitis. The dermatitis was characterised by multifocal to coalescing lesions with an irregular rough surface , threadlike structures were discernible. These structures did not resemble true hyphae. They lacked the usual characteristics of fungal hyphae since they were of significantly variable wideness, had no distinct regular septa, and the tips were cut rather than rounded hyphal ends and fluorescence was not distinct as is usual for fungal hyphae.Aspergillus niger on YGC agar only. As A. niger is found ubiquitously as cause of black mold on certain fruits and vegetables, this single culture growth is considered more likely as a contaminant rather than a primary pathogen.After 2\u2009weeks, fungal culture of one of four skin samples (taken on day 34) grew Mucorales sp. (zygomycetes) and for Aspergillus sp. were all negative (likely confirming contaminated growth of Aspergillus niger in culture), but all four samples were positive for Pythium sp. The results of ITS sequencing in 2018 matched Pythium pectinolyticum with a similarity of >\u200999.5% compared with the reference strain CBS* 122643 . Sequence analysis was rerun in August 2021 for the current study, and this time showed a 100% sequence similarity to CBS strain 234.72 of Pythium flevoense, which had been submitted to the database after 2018, and thus the aetiological species was reclassified as P. flevoense in this study. The fasta of the sequenced porpoise skin samples were:Specific fungal PCRs on four skin samples (taken on day 34) for >ITS1&2GCCCGTCGCACCTACCGATTGAATGACTCGGTGAGAAATCGGGACCGTGAATCCGTTTGCTTCATTGCGAGTGGACTTATGGGAACTTTTTCTAACCTCGCCATTTAGAGGAAGGTGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCACACCCTAAAACTTTCCACGTGAACCGTTGTAAATATGTTCTGTGCTCTCTCTCGGGAGAGCTGAACGAAGGTGGCCTGCTTAATTGTAGACTGCCGATGTACTTTTAAACCCATTAAACTAATACTGAACTATACTCCGAAAACGAAAGTCTTTGGTTTTAATCAATAACAACTTTCAGCAGTGGATGTCTAGGCTCGCACATCGATGAAGAACGCTGCGAACTGCGATACGTAATGCGAATTGCAGAATTCAGTGAGTCATCGAAATTTTGAACGCACATTGCACTTTCGGGTTATGCCTGGAAGTATGCCTGTATCAGTGTCCGTACATCAAACTTGCCTTTCTTTTTTTGTGTAGTCAAGATTAGAAACGGCAGACTGTGAGGTGTCTCGCTGACTCCCTCTTCGGAGGAGAAGACGCGAGTCCCTTTAAATGTACGTTCGCTCTTTCTTGTGTTTAAGTAGAAGTGTGACTTTCGAACGCAGTGATCTGTTTGGATCGCTTTGCTCGAGTAGGCGACTTCGGTTAGGACATTAAAGGAAGCAACCCTATTGGCGGTATGTTAGGCTTCGGCCCGACTTTGCAGCTGACGGTGTGTTGTTTTCTGTTCTTTCCTTGAGGTGTACCTGTCTTGTGTGAGGCAATGGTCTaGGCAAATGGTTATTGTGTAGTAGGAAGTTGCTGCTCTTGAACGCCCTGTttTCGGATAGGGTAAAGGAGGCAACACCAATTTGGGATAGTCTTTGATTTATCATTGGCGCTCTTTCTAATTGGACCTGATATCAGGCAAGACTACCCGCTGAACTTAAGCATATTAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACGGCGAGTGAAGCGGGATGAGCTCAAGCTTAAAATCTCTGTGCCAGTTTGGCATGGCGAATTGTAGTCTATGGAGGCGCTATCAGTGCGATTGTTCGGGG.Vibrio parahaemolyticus, considered a bacterial commensal of harbour porpoise\u2019s skin flora. Bacteriological cultures (of samples taken during autopsy on day 105) were unremarkable, with no growth obtained after 14 days of culture in all sampled sites, except for a few Pseudomonas sp. from the tracheobronchial lymph nodes and also from skin, along with a few Acinetobacter sp.Bacteriological PCR analysis of the affected skin samples (taken on day 34) were positive for Virological PCR analysis on samples from brain, lung, kidney and urinary bladder collected during autopsy on day 105 tested negative for the presence of morbilliviral RNA.Pythium insidiosum as the causative pathogen [P. flevoense infection as the cause of dermatitis in a harbour porpoise. To our knowledge, it is the first time both for the diagnosis of pythiosis in a marine mammal and for P. flevoense as a cause of disease in a mammalian species. P. flevoense infections were reported previously in non-mammalian species only; ayu fish larvae [Pythium species as the causative pathogen was indicated by histopathological evaluation and confirmed by DNA sequencing that identified this particular, pathogenic species. Histological examination, especially in silver stains, revealed intralesional presence of fungus-like hyphae of approximately 7\u201310\u00a0\u03bcm diameter with rare septations and irregular branching  were detected increasingly at warmer freshwater temperatures\u2009>20\u00a0\u00b0C in Argentina [Pythium species have been isolated from seaweeds and algae in temperate coastal saltwater environments [P. flevoense infection was reported in ayu fish larvae reared in artificial saltwater conditions at 12\u201315\u2009\u00b0C in a fishery in Japan [Another unusual aspect of this case was the temperate saltwater environment, which is in contrast with the subtropical stagnant freshwater conditions, such as swamps and rice fields, where infections with ntil now , 29, 30.rgentina . Nonetheronments , 32. Morin Japan . Even soP. flevoense in the water. Skin wounds in animals and humans are reported to serve as potential port d\u2019entr\u00e9e for the infectious motile zoospores [P. flevoense was isolated from soil samples previously, another possible explanation is that the seawater in the bay and basin may have been contaminated via freshwater runoff from land. However, another harbour porpoise that was kept in a separated adjacent basin circulated with the same seawater remained healthy without any signs of dermatitis or skin lesions.As pythiosis is not known to be contagious between mammals, infection in our case most likely occurred at sea or in the marine centre, presumably from infectious zoospores of oospores . In our oospores , 34. Furoospores , 32. TheImmunosuppression and hemopathies are risk factors for contracting pythiosis. For example, there are scientific reports regarding pythiosis with haemoglobinopathies, anaemia and leukaemia as risk factors for infection in humans \u201312, and Phoca vitulina) and several species of cetaceans [Rhizopus sp. [Cryptococcus gatti [Candida albicans [Initial clinical examination and diagnostics implicated a fungal organism as a cause of disease in this case, although a specific identification at that time was not made. Marine mammals are known to be susceptible to fungal diseases. In particular, respiratory aspergillosis has been reported in harbour porpoises , harbouretaceans , 39. Raropus sp. , Cryptocus gatti  and Candalbicans  as causaDespite various antifungal and antibiotic treatments in this case, the clinical course subsequently developed into a progressive severe dermatitis. Retrospectively, such a clinical course, with difficulties in initial diagnosis and resistance to antifungal therapy, is typical for pythiosis, which is not a true fungal disease , 43, 44.P. flevoense as cause of this dermatitis was evidenced by its intralesional presence in combination with histological absence and microbiological exclusion of other potential pathogens, such as bacteria, true fungi, protozoa, and viruses. Also, the possibility of this particular Pythium species being an opportunistic infection was considered and excluded because: (1) there was no evidence from haematology, histopathology, or (molecular) microbiology to suggest underlying immunosuppression, and (2) infection occurred in a non-pregnant initially healthy young female harbour porpoise in good physical condition without co-morbidities. Furthermore, our findings of this new case of pythiosis in a harbour porpoise are consistent with observations from previous investigators that the host range of pythiosis is increasing [Pythium species, we recommend including P. flevoense as a differential diagnosis for dermatitis in marine mammals, and more specifically, in harbour porpoises that have been entangled in fishing nets.In conclusion, the diagnosis of ion, and  infectiocreasing , 5. So gAdditional file 1. Results of blood analyses for haematologic and biochemistry parameters from harbour porpoise Idun. Reference intervals from free-ranging harbour porpoises from Danish waters with a healthy clinical appearance  according to Siebert et al. [th and 90th percentiles for each blood parameter with their 95% bootstrapped confidence intervals, respectively. NA:\u00a0not available.t et al. . The low"}
+{"text": "Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage. Between 60 and 99% of global populations carry HCMV, depending on demographics; in part, this prevalence is made possible by the ability of the virus to establish lifelong persistence in the infected host. After primary lytic infection, HCMV can establish a quiescent or latent infection, which helps it avoid immune clearance . The lytDuring latent infection of undifferentiated myeloid cells, the MIEP is known to be targeted for repression to suppress the expression of key lytic IE genes by an overall balance of MIEP repressors ,7,8. HowAnother viral gene that has been shown to be expressed during latency is LUNA. It was one of the first genes identified to be transcribed during natural latency  and has In this manuscript, we further investigate this transcriptional phenotype and demonstrate that LUNA is important for the expression of multiple latency-associated transcripts, which correlates with increased levels of activatory epigenetic marks at latent promoters. In silico analyses reveal that multiple latent promoters encode binding sites for the hematopoietic transcription factor GATA-2, and interestingly, we show that LUNA interacts with transfected GATA-2 by co-immunoprecipitation (Co-IP). Finally, we link GATA-2 binding and the formation of an open chromatin conformation at latent viral promoters with the deSUMOylase motif of the LUNA protein. Together, these data argue that LUNA contributes to the regulation of expression of latency-associated HCMV gene expression in myeloid cells through an interaction with an important hematopoietic transcription factor, GATA-2.2 at 37 degrees. CD14+ cells were isolated from venous blood as described previously [THP1 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FCS and 5% penicillin/streptomycin in 5% COeviously ,26. TB40TB40E-SV40-GFP BAC was used to generate the LUNA mutant, LUNA knockout, and revertant viruses. For this, gBLOCKs from IDT  were utilised with primers for recombineering using the GalK/2-DOG selection method as described previously , using tTACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGCCTGTTGACAATTAATCATCGGCA 3\u2032 GalK insert LUNA (primer) GGTCTCTTTCCACGGAGCAACGTCATGCGCGGCGCCGTCTCCGAGTTTCTTCAGCACTGTCCTGCTCCTT LUNA G81A stop (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGAGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC LUNA G233C (gBLOCK)TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTCCGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.LUNA C234G (gBLOCK) TACCGCTTCGACGTCTTTGTCCGGTCAGGATCAGTGCCCGGGACAGTCCGGCTTGGGTGTCCGAGTCCTCGTCGCCGCTGGCCTCCTCGAAGCCGGCAAACATGGCTTCGGACAGGGGGGTCGGCGTCGGTGTGGATGAGAGGTCATCTTCGTCGTCCTCTTCCTCTTCTTCCTCCTCTTCCTCGGTGGGTGGTAATCCGGGGGACTGGGGGAGAAACTCGGAGACGGCGCCGCGCATGACGTTGCTCCGTGGAAAGAGACC.5\u2032 LUNA seq (60.4) (5\u2032 primer for sequencing the mutants) GCG TGT TGC ACG CTC ACC 3\u2032 LUNA seq (59.8) (3\u2032 primer for sequencing the mutants) CCG CCG TGG GTT TTG GAC 5\u2032 amp LUNA gB (55) TACCGCTTCGACGTCTTTG.3\u2032 amp LUNA gB (55.5) GGTCTCTTTCCACGGAGC.\u00ae Green RT-qPCR kit according to the manufacturer\u2019s instructions and the samples were amplified and detected using an ABI 7500 Fast Real-Time PCR machine (95 \u00b0C for 15 s and 60 \u00b0C for 45 s), as described previously [Chromatin immunoprecipitations were carried out as described previously . After sImmunoprecipitation analysis was carried out as described previously  using anIsopeptidase inhibitor, G5 (Sigma), was used as described previously . First, Quantification of viral and cellular mRNAs was carried out by SYBR green detection using primers described previously , except Genome copy number was determined using droplet digital PCR as described previously ,30. BrieTo investigate directly whether LUNA is important for latency-associated viral gene expression, we analysed the levels of UL138 and vIL-10 RNA during latent infection in the absence of LUNA. Recombinant viruses were generated in which a premature stop codon was engineered at the start of LUNA, resulting in viruses unable to express LUNA protein but which did not disrupt the UL82 gene on the complementary DNA strand (TB40E-LUNAmut), which was then controlled for through production of a revertant virus with LUNA repaired (TB40E-LUNArev). It is worth pointing out that this equivalent mutation of LUNA in the context of the Merlin clinical isolate of HCMV had little impact on the growth of Merlin in fibroblasts and the expression of viral gene products, including UL82 . The datTo further understand the mechanisms important for these differences in levels of UL138 and vIL-10 gene expression in the presence or absence of LUNA, we analysed the chromatin structure around the promoters of these latency-associated genes . ConsistWe previously identified that the hematopoietic transcription factor GATA-2, which plays an important role in host gene expression in myeloid cells, is also important for the expression of both LUNA and UL144 mRNAs during latency by GATA-2 binding to their promoters . IntriguThere is no evidence to suggest that LUNA can function as a general transcription factor; thus, we hypothesised that LUNA could regulate latent gene expression via interaction with GATA-2. To investigate whether LUNA might enhance expression from GATA-2-bearing promoters by a direct interaction between LUNA and GATA-2, we carried out interaction assays using co-immunoprecipitation in cells overexpressing GATA-2 and LUNA, which showed a clear interaction between GATA-2 and LUNA . FurtherWe observed that LUNA expression resulted in the formation of active chromatin on viral latency-associated promoters, that all these promoters contained GATA-2 transcription factor binding sites, and that LUNA interacted with GATA-2 in a deSUMOylase-dependent manner. Thus, we hypothesised that the chromatin signature observed with a LUNA deletion virus should be phenocopied with a LUNA deSUMOylase mutant. To test this, we generated a LUNA mutant virus in which we introduced a point mutation in the deSUMOylase domain of LUNA, rendering LUNA devoid of deSUMOylase activity (TB40E-LUNApoint), and tested whether the loss of the deSUMOylase activity from LUNA affected the transcription of viral genes. An analysis of infected cells shows that the expression of both vIL-10 and UL138 is reduced in cells latently infected with the TB40E LUNApoint compared to the control A,B. NextFinally, we wished to determine whether these LUNA-mediated changes could have any impact on the biology of latency. It is well established that HCMV infection extends the lifespan of classically short-lived CD14+ monocytes, which could be important for virus dissemination in vivo. Furthermore, we have observed that the expression of vIL-10 enhances the survival of infected CD34+ cells in long-term culture. Thus, we hypothesised that the defect in latency-associated gene expression observed in the absence of LUNA could have an impact on monocyte survival. To this end, CD14+ cells were infected with wild-type or a LUNA deletion virus and cultured for 10 days, and monocyte viability was assessed. The data show that viral infection improves cell survival compared to mock cells A, consisPrevious studies have demonstrated a clear role for the latency-associated gene product, LUNA, in latent carriage and reactivation from latency ,22. HereWhilst the absence of LUNA does not prevent the establishment of latency in CD14+ monocytes, we show here that it significantly enhances the expression of latency-associated genes, which we hypothesise is at least in part via the myeloid transcription factor GATA-2; this included the expression of vIL-10, which was significantly reduced in the absence of LUNA.We have previously shown that vIL-10 drives the expression of cellular IL-10 in monocytes ,31, and It is important to highlight that vIL-10 has pleiotropic roles in HCMV infection, with key roles in immune regulation. Thus, the enhancement of vIL-10 production by LUNA during latency could also be important for immune evasion during latency in vivo. For example, vIL-10 directly modulates the immune response  and the Our previous work has shown that LUNA is a functional deSUMOylase , and thiPML is generally repressive for viral IE gene expression, which would be advantageous during latency to help repress lytic gene expression. However, it is at present unclear how the virus maintains a repressive chromatin structure around the MIEP whilst at the same time allowing expression of latency-associated genes. The dispersal of repressive PML bodies during latency would be predicted to be disadvantageous for maintaining the general repressive chromatinization of the viral genome observed during HCMV latency; PML disruption is known to lead to activation of HCMV IE gene expression and gene expression of other viruses ,41. For In contrast, reactivation is likely regulated by the increase in differentiation-dependent transcriptional activators of the MIEP, coupled with a decrease in MIEP repressive factors, which can be enhanced by inflammatory signals that then allow viral IE gene expression  and progTaken together, we believe that the deSUMOylation function of LUNA not only ensures that reactivation from latency is primed to go, by removing repressive PML bodies in latently infected cells, but is also crucial for latent carriage by enhancing the transcription of latency-associated genes."}
+{"text": "Nature Communications 10.1038/s41467-022-32395-w, published online 13 August 2022Correction to: The original version of this Article contained an error in Table 1. The correct version of the 3rd row (Circle part 2) of the 3rd column (Sequence (5\u2019 -> 3\u2019)) states \u2018CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGACG\u2019instead of the original, incorrect \u2018CGAGGTGCTTTTAGCACCTCGAAGTAAAGCTATCCACTGTCACCAACTACTAGATAAACGTCACACTTTTCGTGTGAC\u2019\u00a0where the final \u2018G\u2019 was omitted.This has been corrected in both the PDF and HTML versions of the Article."}
+{"text": "Plant probiotics bacteria are live microbes that promote soil health and plant growth and build the stress-tolerant capacity to the plants. They benefit the plants by increasing nutrient absorption and release of stress-related phytohormones. These plant probiotic bacteria serve a better purpose to the plant when compared to chemical fertilizers. Use of chemical fertilizers such as arsenic and cadmium can lead to soil acidification and even release of harmful gases such as methane which further pollutes the environment.Corynebacterium spp., Bacillus spp., Lactobacillus spp., and Cytobacillus spp. The results were also examined using various bioinformatics tools for accuracy in their phylogenetic pattern.Different bacterial species were isolated from the agricultural fields of Tattiannaram, Telangana, and identified as the efficient rhizosphere bacteria with the essential qualities of plant growth promotion by evaluating the nitrogen-fixing ability on a selective media and various other methods. Upon the molecular characterization of the isolates, they were identified as The recognized species of plant probiotics have established roles in promoting plant growth and strengthening plant immunity. This research introduces an innovative methodology for evaluating and investigating recently identified bacterial isolates, focusing on their distinctive plant probiotic attributes. Through harnessing the potential of advantageous microorganisms and comprehending their interaction with plants and soil, our objective is to formulate inventive approaches to elevate crop productivity, enhance soil richness, and foster environmentally sustainable and robust agricultural methodologies. These characteristics exhibit promising potential for future incorporation into plant systems, fortifying growth and development, and underscoring their distinctive significance within the realm of agriculture.The online version contains supplementary material available at 10.1186/s43141-023-00615-5. Soil, the organic geological skin of the earth, is essential for life and habitat to an incredible microbial community. Microbes have mostly been regarded as stewards of the biosphere . Soil baBacilli and Pseudomonas being the most common genera reported [Plants interact with a range of microbes in both aerial and subsoil tissues, establishing associations that can be harmful (pathogenic), neutral, or advantageous to the host plant. Several studies have shown evidence of the ability of microbes to boost plant development in various cultivated species. One such criterion is the development of plant growth-promoting bacteria. Plant growth-promoting bacteria (PGPB) are one of the most generalized findings of plant probiotic microbes (PPM) in soil, having reported \u201317. Planreported . Upon coreported . Severalreported .Fusarium oxysporum is treated by Bacillus subtilisssp [Probiotics are living microbial species utilized to improve the health and vitality of the host. Microorganisms employed for probiotic purposes can be found in nature, but they must be correctly identified, isolated, and studied for virulence . Plant ptilisssp .. Plant tilisssp , 27.Haas and Keel coined the term plant probiotic bacteria (PPB) to describe a group of microorganisms that benefit plants and meet three necessary attributes that when combined result in improved plant protection: (i) niche colonization effectiveness and competitiveness, (ii) ability to induce systemic resistance (ISR) in their hosts, and (iii) existence of specific antagonistic traits on pathogens .Agriculture and forestry are constantly being impacted by increasing population, soil degradation, environmental degradation, and global warming . This haIn long-term agricultural production, nitrogen fertilizers are frequently used to boost early crop output. Long-term administration of nitrogen fertilizers, on the other hand, can alter the plant soil microbe system by altering the composition of vegetation and soil microbial populations \u201336. NitrThe entire methodology followed during the work is represented in Fig.\u00a0Soil samples were gathered from the Tattiannaram fields in Telangana, India Fig.\u00a0. TattianThe rhizosphere is considered an important plant nutrient source where most of the microbes make up this as a resident club. The first step in developing a producer strain is the isolation of concerned microorganisms from their natural habitats. A set of highly selective procedures which allows the detection and isolation of microbes producing the desired metabolite constitute primary screening. However, this is possibly the most critical step since it eliminates the bulk of unwanted useless isolates, either nonproducers or producers of known compounds. As a part of the primary screening technique, the isolation of the bacteria was done using basic lab equipment and certain media. To analyze for the various microbes within the collected soils, the basic medium such as nutrient agar was used, believing that majority of microbes can be screened. Each soil sample was serially diluted and plated as per the standard protocols of microbial isolation techniques. After the growth observed on basic media, the isolates with different morphological features were subcultured and further tested for biochemical properties. The biochemical properties were analyzed by performing IMViC tests, catalase, coagulase, oxidase, starch hydrolysis, organic acid production tests and blood haemolysis test, etc. In detail, the IMViC test helps to narrow down the group of bacteria and provide information about their metabolic capabilities. Plant probiotic bacteria often produce organic acids through their metabolic processes. These organic acids can help in solubilizing minerals, making them more available to plants. The ability to produce organic acids like citric acid and malic acid can be a desirable trait in plant probiotic bacteria. The purpose of performing various biochemical tests was tabulated in Table 2PO4 0.1%, MgSO4.7H2O 0.05%, NaCl 0.05%, FeSO4 0.01%, Na2Mo 0.0005%, CaCO3 0.2%, agar 1.5%). First, the isolates were inoculated on solid medium. Later to estimate their growth levels, the isolates were re-examined on liquid medium. This growth was analyzed for 24\u00a0h and 48\u00a0h by colorimetric analysis at 600\u00a0nm. Bacteria that thrive on Jensen\u2019s media might have the ability to fix nitrogen, making them potentially beneficial for plants. The organisms that have shown positive with Jensen\u2019s media were further examined if they can also utilize and tolerate to different chemical exposures such as zinc, phosphorus, and iron using modified zinc solubilizing medium , modified phosphate-solubilizing medium 2 0.02%, MgSO4.7H2O 0.010%, FeSO4 0.00001%, pH 7), and modified iron-solubilizing media , respectively. The growth rate was analyzed by measuring the OD  which is set to 540\u00a0nm (nanometers) and 580\u00a0nm using a colorimeter. Plants often face challenges in accessing essential nutrients like zinc, iron, and phosphorus due to their limited solubility in the soil. Bacteria capable of solubilizing these minerals can enhance nutrient availability for plants. The organisms which have shown good growth rate and minimum standard deviation upon all the triplicate values were selected as the best isolates and moved for further examination. The selected isolates were inoculated to Ashby\u2019s liquid medium  to confirm the nitrogen-fixing property [Later, the studies were carried out to screen out the nitrogen-fixing property using certain specific media such as Jensen\u2019s medium \u201d), inputting the 16S rRNA sequence into the query box, adjusting parameters, and subsequently initiating the BLAST search . SubsequLater, the bootstrapping technique was employed as it is crucial for making well-informed evolutionary interpretations, as it addresses the inherent noise in genetic data and the complexities of evolutionary processes . Using tUpon screening the isolates by considering the basic traits, a total of 101 isolates were found initially. Colony morphological testing, gram staining, and biochemical tests  were performed. The organisms with similar traits were re-examined and eliminated. For the second stage of screening, 36 isolates were selected. The selected isolates were highlighted in Table Later, these 36 isolates were subjected to test their nitrogen-fixing capacity. Jensen\u2019s media was used to detect the presence of nitrogen-fixing bacteria. This media was prepared in broth consistency and inoculated with all the selected 36 isolates. All the isolates were grown as triplicates, and the average of all the readings was tabulated as the results in Table The seven best isolates which proved their ability of nitrogen fixation was further moved to examination such as zinc solubilizers, phosphate solubilizers, and zinc solubilizers. The result was tabulated in Table The isolates that shown best within chemical analysis tests were further examined using Ashby\u2019s nitrogen liquid medium in order to compare their main purpose to be a plant probiotic. Surprisingly, the isolate nos. KL-015, KL-076, and KL-089 have shown positive results indicating their potentiality for nitrogen fixation. Along with these isolates, remaining screened out isolates were subjected to the exposure to liquid medium of Ashby\u2019s. The isolate which had shown growth in liquid medium  technique. Based on the gel amplicon picture, the isolates have shown the bands at app. 1000\u00a0kb Fig.\u00a0. The posCTGCACTTCGGGATAAGCTTGGGAAACTGGGTCTAATACCGGATAGGAACCATCTTTAGTGTGATGGTTGGAAAGTTTTTTCGGTGTAGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTTGTGACGGTACCTAGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATTCTGGGGCTTAACTCCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGTAACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGTTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGAGGGTCTTTCTACGACTTTCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGG.AGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATC.ACCCGCGGTGCATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCATTGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAAGATAGAGAGAGTAACTGATCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAGATAAGTCTGATGTGAAAGCCCTCGGCTCAACCGAGGAACTGCATCGGAAACTGTCTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGCGCAATCCCAAGAGATTGGGAGTTCCCTTCGGGGACGCTAAGACAGG.GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTCCTGACAACCCTAGAGATAGGGCGTTCCCCTTCGGGGGACAGGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAA.The above sequences were used to run the BLAST separately, and later on, the top 10 sequences were used to construct the cladogram using Clustal Omega tool as shown in Fig.\u00a0To select a bacteria as a potent plant probiotic, it must be beneficiary to humans also as the end result of this concept is growth of a healthy plant. As we know, the greatest ecological and agronomical advantages derived by plants from their contact with microbes are biological nitrogen fixation by bacteria. It is believed that the bacterial isolates which were finally identified within this work are the one which can convert the atmospheric nitrogen to ammonia using its nitrogenase enzyme, thus making it available for plant absorption. The presence of the nitrogenase enzyme decides the plant\u2019s maturity and adds economic value too. The bacteria may also be capable to utilize various chemicals such as zinc which stimulates enzymes that are involved in the creation of some proteins. It aids in the production of chlorophyll and certain carbohydrates, the transformation of starches to sugars, and its availability in plant tissue aids in the plant\u2019s resistance to low temperatures . Zinc is\u201cPlant probiotics\u201d is an emerging and intriguing concept within the realm of agriculture and plant science. This concept revolves around the idea of using beneficial microorganisms to enhance plant health and growth. This innovative approach delves into the analysis of plant immunity as well, aiming to better understand how these microorganisms can interact with plants\u2019 natural defense mechanisms. Numerous studies have drawn attention to the significant role that plant probiotics can play. These studies have identified specific microorganism isolates that have demonstrated particularly promising results. These promising isolates are then subjected to a thorough screening process to assess their potential. This assessment involves employing techniques such as biopriming, which entails treating seeds with these beneficial microorganisms to facilitate their growth and establishment in the plant\u2019s environment. Moreover, we are simultaneously working on optimizing the growth conditions and parameters for these beneficial microorganisms. This involves creating an environment that allows these microorganisms to thrive and interact harmoniously with the plant. The ultimate goal is to develop a technique that can reliably enhance plant health and performance. One aspect of research in this area focuses on investigating the specific compositions of microbial communities in the soil. Different combinations of microorganisms can have varying effects on soil fertility and subsequently impact the development of plants that grow in that soil. Through careful analysis, researchers seek to uncover the intricate relationships between these microorganisms and the overall health of both the soil and the plants. Early findings suggest that these microbial communities possess an adaptive capability, allowing them to adjust and thrive under different chemical conditions present in the soil. This adaptability opens up avenues to cultivate these microorganisms into potent plant probiotics that can potentially withstand a range of environmental challenges. In summary, the emerging field of plant probiotics holds immense promise for revolutionizing agriculture and plant cultivation.Additional file 1. BLASTN Results."}
+{"text": "Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip. Summary: Myosin-X transports lamellipodin (RAPH1) to filopodia tips to regulate filopodia functions. Cell migration is essential during embryonic development, immune surveillance and wound healing. Misregulation of cell migration is implicated in multiple diseases, including inflammation and cancer. One hallmark of cell motility is a high degree of plasticity, allowing cells to adopt different morphologies and migration modes . A shareFilopodia are small and dynamic finger-like actin-rich protrusions (1\u20135\u2005\u00b5m in length and 50\u2013200\u2005nm in width) and are often the first point of contact between a cell and its immediate surroundings. Filopodia contain cell surface receptors, such as integrins, cadherins and growth factor receptors, interacting with and interpreting various extracellular cues. Filopodia assembly is primarily driven by the linear polymerization of actin filaments with their barbed ends facing the plasma membrane . These fHere, we set out to identify novel MYO10 cargo molecules. Using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10-binding partner. Using structured illumination microscopy, we report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips; thus, RAPH1 is likely an MYO10 cargo. We map the RAPH1 MYO10-binding site to a previously uninvestigated RAPH1 sequence, and demonstrate that RAPH1 is a critical positive regulator of filopodia formation and stability in cells. Our results indicate that, in filopodia, RAPH1 is not required for integrin activation. Instead, RAPH1 regulates MYO10 filopodia formation and stability.FERM, or GFP\u2013TLN1FERM , followed by mass spectrometry analysis , the main cargo-binding site in MYO10 . We perfanalysis A,B. TLN1analysis . We idenanalysis . Interesanalysis .FERM and that RAPH1 is biotinylated in cells expressing GFP\u2013MYO10\u2013BioID .RAPH1 is a member of the Mig-10/RIAM/lamellipodin (MRL) protein family, with MIG-10 being the of RAPH1 . RAPH1 wof RAPH1 , but itsof RAPH1 . In addiof RAPH1 ; Movie\u00a01\u0394F) in cells  within RAPH1. RAPH1 comprises several conserved domains, including a Ras-association (RA) and a pleckstrin homology (PH) domain. RAPH1 also contains known profilin-, VASP- and multiple putative SH3-binding sites A. Furthe\u0394F filopodia  showed that this construct could accumulate at the tip of MYO10-containing filopodia  MYO10 is required to target RAPH1 to filopodia tips, and (2) the MYO10\u2013RAPH1 interaction contributes to formation of filopodia containing MYO10. We propose that MYO10 transports RAPH1 to filopodia tips, contributing to filopodia stability via yet unknown mechanisms, possibly involving RAPH1 interactions with other proteins such as VASP. However, our data do not fully exclude the possibility that RAPH1 simply diffuses to filopodia and that MYO10 only contributes to RAPH1 accumulation at filopodia tips without direct transport. Testing this would require performing two-color single-molecule imaging of MYO10 and RAPH1 to see whether these proteins move toward filopodia tips together. However, we find that RAPH1 is not very abundant in filopodia when the MYO10 FERM domain is missing, suggesting that RAPH1 is likely to be actively transported by MYO10.RAPH1 is presumably in a complex with MYO10, VASP and actin at filopodia tips. In this scenario, MYO10 could tether RAPH1 to filopodia tips using its motor domain, providing resistance against the retrograde actin flow in filopodia . Once tein vivo.Interestingly, both MYO10 and RAPH1 have been implicated separately as positive regulators of cancer cell migration and invasion in similar contexts . In addi\u22121 final concentration), and sorted for green fluorescence using a fluorescence-assisted cell sorter (FACS). All cell lines tested negative for mycoplasma. Cells were not authenticated.U2-OS osteosarcoma cells and MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FCS) . U2-OS cells were purchased from DSMZ . MDA-MB-231 cells were provided by ATCC. The U2-OS MYO10-GFP lines were generated by transfecting U2-OS cells using Lipofectamine 3000 (Thermo Fisher Scientific), selected using geneticin , 1:1000], anti-His tag , and anti-tubulin . Rabbit polyclonal antibodies used in this study were anti-RAPH1 , anti-MYO10 , and anti-GFP . Biotinylated proteins were detected using Streptavidin conjugated with Alexa Fluor\u2122 555  or Alexa Fluor\u2122 647 , both provided by Thermo Fisher Scientific (S21381 and S21374). The bovine plasma fibronectin was provided by Merck (341631). DAPI  was provided by Thermo Fisher Scientific (D1306).U2-OS and MDA-MB-231 cells were transfected using Lipofectamine 3000 and the P3000TM Enhancer Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.47608 . The mScarlet-I-MYO10\u0394F (MYO10\u0394F-RFP) construct was previously described (145139). The GFP-VASP  plasmid was Addgene plasmid #54297 (deposited by Michael Davidson). The GFP-RIAM(1-666) construct was Addgene plasmid #80028  plasmid was Addgene plasmid #escribed  and is aThe GFP\u2013MYO10\u2013BioID construct was generated as follows. Flanking XbaI sites were introduced into BioID by PCR  and the resulting amplicon was then inserted into a unique XbaI site in the EGFPC1-hMyoX plasmid resulting in an EGFP-MYO10-(stop codon)-BioID fusion gene. The stop codon between MYO10 and BioID was then replaced with a codon encoding valine (GTA) using a quick-change mutagenesis kit from Agilent and following the manufacturer's instructions.\u0394TBS [RAPH1 with amino acids (aa) 2\u201392 deleted] construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the XhoI/HindIII sites. The sequence of the gene block is provided below:The GFP\u2013RAPH15\u2032-ATTAGACTCGAGCCGCGATGTGCTCTATAGAGCAGGAGCTCAGCAGCATTGGTTCAGGAAACAGTAAGCGTCAAATCACAGAAACGAAAGCTACTCAGAAATTGCCTGTTAGCCGACATACATTGAAACATGGCACCTTGAAAGGATTATCTTCTTCATCTAATAGGATAGCTAAACCTTCCCATGCCAGCTACTCCTTGGACGACGTCACTGCACAGTTAGAACAGGCCTCTTTGAGTATGGATGAGGCTGCTCAGCAATCTGTACTAGAAGATACTAAACCCTTAGTAACTAATCAGCACAGAAGAACCGCGTCAGCAGGCACAGTGAGTGATGCTGAAGTACACTCTATTAGTAATTCCTCCCATTCCAGCATCACTTCCGCAGCCTCCAGCATGGACTCTTTGGATATTGATAAAGTAACACGCCCTCAAGAGCTGGATTTGACACATCAAGGGCAGCCAATTACTGAGGAAGAACAGGCAGCAAAATTGAAAGCTGAGAAGATCAGAGTTGCCCTAGAGAAAATTAAAGAGGCACAAGTGAAAAAGCTGGTGATCAGAGTCCACATGTCTGATGACAGTTCTAAAACAATGATGGTGGATGAGAGGCAGACAGTAAGACAAGTACTGGATAACCTGATGGACAAATCCCACTGCGGTTATAGTTTAGACTGGTCACTGGTAGAAACCGTTTCTGAATTACAAATGGAGAGAATCTTTGAAGACCATGAAAACTTGGTTGAAAATCTTCTTAATTGGACAAGAGATAGCCAAAACAAGCTTATTAGA-3\u2032.F2 construct (RAPH1 aa 535\u2013868) was purchased from GenScript. The gene fragment was synthesized using gene synthesis and cloned into pGEX-4T-1 using the BamHI/XhoI sites.The RAPH1 fragments F1 (RAPH1 aa 1\u2013535), F2 (RAPH1 aa 535\u2013868), F3 (RAPH1 aa 868\u20131062), F4 (RAPH1 aa 1062\u20131250) and F5 (RAPH1 aa 536\u2013587) constructs were purchased from GenScript. Briefly, the gene fragments were synthesized using gene synthesis and cloned into pcDNA3.1(+)-N-eGFP using the BamHI/XhoI sites. The GST\u2013RAPH1\u0394536-587 (RAPH1 aa 536-587 deleted) construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the HindIII/KpnI sites. The sequence of the gene block is provided below:The GFP\u2013RAPH15\u2032-AAGCTTATATTTATGGAGCGTATAGAAAAATATGCACTTTTCAAAAACCCACAGAATTATCTTTTGGGGAAAAAGGAAACAGCTGAGATGGCAGATAGAAACAAAGAAGTCCTCTTGGAGGAATGTTTTTGTGGAAGTTCTGTAACTGTACCAGAAATTGAAGGAGTCCTTTGGTTGAAGGATGATGGCAAGAAGTCCTGGAAAAAGCGTTATTTTCTCTTGCGAGCATCTGGTATCTACTATGTTCCCAAAGGAAAAGCAAAGGTCTCTCGGGATCTGGTGTGCTTTCTCCAGCTGGATCATGTCAACGTTTATTATGGCCAGGACTATCGGAACAAATACAAAGCACCTACAGACTATTGTCTGGTGCTGAAGCATCCACAAATCCAGAAGAAATCTCAATATATCAAATACCTTTGTTGTGATGATGTGAGGACACTGCATCAGTGGGTCAATGGGATCCGCATTGCAAAGTATGGGAAGCAGCTCTATATGAACTACCAAGAAGCCTTGAAGAGGACAGAGTCAGCCTATGATTGGACTTCCTTATCCAGCTCCAGCATTAAATCGGAAGAGTCCAGCAAGGCCAGAATGGAGTCTATGAATCGGCCCTACACTTCACTTGTGCCCCCTTTATCCCCGCAACCTAAGATAGTCACCCCCTACACTGCTTCACAGCCTTCACCACCTCTACCTCCTCCGCCACCCCCACCTCCTCCTCCACCACCCCCTCCACCACCCCCTCCTCCCCCACTCCCCAGCCAGTCTGCACCTTCTGCAGGCTCAGCAGCCCCAATGTTCGTCAAGTACAGCACAATAACACGGCTACAGAATGCGTCTCAGCATTCAGGGGCCCTGTTTAAGCCGCCAACACCCCCAGTGATGCAGTCACAGTCAGTGAAGCCTCAGATCCTGGTACC-3\u2032.The expression of RAPH1 was suppressed using 83\u2005nM siRNA and Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. siRNAs used were AllStars Negative siRNA control (cat. no. 1027418), RAPH1 siRNA #2  and RAPH1 siRNA #5  provided by Qiagen.Protein extracts were separated under denaturing conditions by SDS-PAGE and transferred to nitrocellulose membranes using a Mini Blot Module . Membranes were blocked for 30\u2005min at room temperature using 1\u00d7 StartingBlock buffer . After blocking, membranes were incubated overnight with the appropriate primary antibody (1:1000 in blocking buffer), washed three times in PBS, and probed for 1\u2005h using a fluorophore-conjugated secondary antibody diluted 1:5000 in the blocking buffer. Membranes were washed three times using PBS over 30\u2005min and scanned using an iBright FL1500 imaging system (Invitrogen).g for 5\u2005min at 4\u00b0C. Clarified lysates were incubated with GFP-Trap magnetic or agarose beads for 1\u2005h at 4\u00b0C. Complexes bound to the beads were isolated by centrifugation, washed three times with ice-cold lysis buffer, and eluted in Laemmli reducing sample buffer for 10\u2005min at 95\u00b0C.Cells transiently expressing bait GFP-tagged proteins were lysed in a buffer containing 20\u2005mM HEPES pH 7.4, 75\u2005mM NaCl, 2\u2005mM EDTA, 1% NP-40, as well as a cOmplete\u2122 protease inhibitor tablet , and a phosphatase inhibitor mix (Roche cat. no. 04906837001). Lysates were then centrifuged at 15,000\u2005Escherichia coli strain  was transformed with plasmids encoding the relevant His-tagged or GST-tagged proteins. Bacteria were grown at 37\u00b0C in LB medium supplemented with ampicillin . Protein expression was induced with IPTG  at 20\u00b0C. After 5\u2005h, bacteria were harvested by centrifugation (20\u2005min at 6000\u2005g) and resuspended in resuspension buffer . Bacteria were then lysed by adding BugBuster  and a small spoonful of lysozyme . The suspension was mixed at 4\u00b0C for 30\u2005min. Cell debris were then pelleted by ultracentrifugation  at 4\u00b0C for 1\u2005h. His-tagged MYO10 FERM was purified using a Protino Ni-TED 2000 packed column  according to the manufacturer's instructions. The protein was eluted in multiple 1\u2005ml fractions, supplemented with 1\u2005mM AEBSF , and kept at 4\u00b0C until needed (for up to 1 week). For GST-tagged proteins, 600\u2005\u00b5l of equilibrated glutathione\u2013Sepharose 4B beads  was added to the supernatant and agitated for 1\u2005h at 4\u00b0C. Beads were collected and washed four times with TBS supplemented with PMSF (1\u2005mM). Protein-bound beads were stored at \u221280\u00b0C until needed.The BL-21(DE3) F2 Sepharose beads were incubated with 10\u2005mM His-tagged MYO10FERM, and the mixture was rotated overnight at 4\u00b0C. Beads were then washed four times with TBS supplemented with 1\u2005mM PMSF. Proteins bound to beads were then eluted in 2\u00d7 Laemmli sample buffer at 80\u00b0C. Results were then analyzed by western blotting.GST and GST\u2013RAPH1g, +4\u00b0C, 2\u2005min). Biotinylated proteins were then incubated with streptavidin beads  for 1\u2005h with rotation at +4\u00b0C. Beads were washed twice with 500\u2005\u03bcl wash buffer 1 [10% (w/v) SDS], once with 500\u2005\u03bcl wash buffer 2 , and once with 500\u2005\u03bcl wash buffer 3 . Proteins were eluted in 40\u2005\u03bcl of 2\u00d7 reducing sample buffer  for 10\u2005min at 90\u00b0C.U2-OS cells stably expressing GFP\u2013MYO10 or GFP\u2013MYO10\u2013BioID were plated on fibronectin-coated plates in a medium containing 50\u2005\u03bcM biotin for 24\u2005h. After washing cells with cold PBS, cells were lysed, and debris were removed by centrifugation , gel lanes were sliced into five 2-mm bands. The slices were washed using a solution of 50% 100\u2005mM ammonium bicarbonate and 50% acetonitrile until all blue color vanished. Gel slices were washed with 100% acetonitrile for 5\u201310\u2005min and then rehydrated in a reducing buffer containing 20\u2005mM dithiothreitol in 100\u2005mM ammonium bicarbonate for 30\u2005min at 56\u00b0C. Proteins in gel pieces were then alkylated by washing the slices with 100% acetonitrile for 5\u201310\u2005min and rehydrated using an alkylating buffer of 55\u2005mM iodoacetamide in 100\u2005mM ammonium bicarbonate solution . Finally, gel pieces were washed with 100% acetonitrile, followed by washes with 100\u2005\u03bcl 100\u2005mM ammonium bicarbonate, after which slices were dehydrated using 100% acetonitrile and fully dried using a vacuum centrifuge. Trypsin  was used to digest the proteins (37\u00b0C overnight). After trypsinization, an equal amount of 100% acetonitrile was added, and gel pieces were further incubated at 37\u00b0C for 15\u2005min, followed by peptide extraction using a buffer of 50% acetonitrile and 5% formic acid. The buffer with peptides was collected, and the sample was dried using a vacuum centrifuge. Dried peptides were stored at \u221220\u00b0C. Before liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, dried peptides were dissolved in 0.1% formic acid. The LC-ESI-MS/MS analyses were performed on a nanoflow HPLC system  coupled to the Orbitrap Fusion Lumos mass spectrometer  equipped with a nano-ESI source. Peptides were first loaded on a trapping column and subsequently separated inline on a 15\u2005cm C18 column . The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile/water [80:20 (v/v)] with 0.1% formic acid (solvent B). Peptides were eluted with 40\u2005min method: from 8% to 43% of solvent B in 30\u2005min, from 43% to 100% solvent B in 2\u2005min, followed by a wash for 8\u2005min at 100% of solvent B. MS data was acquired automatically by using Thermo Xcalibur 4.4 software (Thermo Fisher Scientific). A data-dependent acquisition method consisted of an Orbitrap MS survey scan of mass range 350\u20131750\u2005Raw data from the mass spectrometer were submitted to the Mascot search engine using Proteome Discoverer 1.5 (Thermo Fisher Scientific). The search was performed against the human database SwissProt_2021_02, assuming the digestion enzyme trypsin, a maximum of two missed cleavages, an initial mass tolerance of 10\u2005ppm (parts per million) for precursor ions, and a fragment ion mass tolerance of 0.020 Dalton. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation was set as a variable modification.FERM and TLN1FERM datasets, two biological replicates were combined. Proteins enriched at least twofold in MYO10FERM over GFP and over TLN1FERM  and detected with more than ten spectral counts (across both repeats) were considered putative MYO10-binding proteins. The fold-change enrichment and the significance of the association used to generate the volcano Plots  and detected with over five spectral counts  were considered putative MYO10-binding proteins. To generate the MYO10no Plots A,B were The spinning-disk confocal microscope used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 . Images were acquired using either an Orca Flash 4 sCMOS camera  or an Evolve 512 EMCCD camera . The objective used was a 100\u00d7 oil  objective.The structured illumination microscope (SIM) used was DeltaVision OMX v4  fitted with a 60\u00d7 Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases\u00d7three rotations). Emitted light was collected on a front-illuminated pco.edge sCMOS  controlled by SoftWorx.The confocal microscope used was a laser scanning confocal microscope LSM880 (Zeiss) equipped with an Airyscan detector (Carl Zeiss) and a 40\u00d7 water (NA 1.2) or 63\u00d7 oil (NA 1.4) objective. The microscope was controlled using Zen Black (2.3), and the Airyscan was used in standard super-resolution mode.For the filopodia formation assays, cells were plated on fibronectin-coated glass-bottom dishes (MatTek Corporation) for 2\u2005h. Samples were fixed for 10\u2005min using a solution of 4% PFA, then permeabilized using a solution of 0.25% (v/v) Triton X-100 for 3\u2005min. Cells were then washed with PBS and quenched using a solution of 1\u2005M glycine for 30\u2005min. Samples were then washed three times in PBS and stored in PBS containing SiR-actin  at 4\u00b0C until imaging. Just before imaging, samples were washed three times in PBS. Images were acquired using a spinning-disk confocal microscope (100\u00d7 objective). The number of filopodia per cell was manually scored using Fiji .2. Filopodia lifetimes were then measured by identifying and tracking all MYO10 spots using the Fiji plugin TrackMate . All live-cell imaging experiments were performed in normal growth medium, supplemented with 50\u2005mM HEPES, at 37\u00b0C and in the presence of 5% COrackMate . In TracrackMate . The traU2-OS cells transiently expressing the constructs of interest were plated on high tolerance glass-bottom dishes  pre-coated first with poly-L-lysine  and then with bovine plasma fibronectin . After 2\u2005h, samples were fixed and permeabilized simultaneously using a solution of 4% (w/v) PFA and 0.25% (v/v) Triton X-100 for 10\u2005min. Cells were then washed with PBS, quenched using a solution of 1\u2005M glycine for 30\u2005min, and, when appropriate, incubated with the primary antibody for 1\u2005h (1:100). After three washes, cells were incubated with a secondary antibody for 1\u2005h (1:100). Samples were then washed three times and incubated with SiR-actin  at 4\u00b0C until imaging . Just before imaging, samples were washed three times in PBS and mounted in Vectashield (Vector Laboratories).To map the localization of each protein within filopodia, images were first processed in Fiji , and datThe preferential recruitment of protein to filopodia tips or shafts was assessed by calculating an enrichment ratio where the averaged intensity of the signal at the filopodia tip (bin 1\u20136) was divided by the averaged intensity at the filopodia shaft (bin 7\u201340).https://huygens.science.uva.nl/PlotsOfDifferences/) (https://github.com/guijacquemet/FiloMAP).Randomization tests were performed using the online tool PlotsOfDifferences (rences/)  preprintrences/) . Volcanorences/) . Superplrences/) . All numClick here for additional data file.10.1242/joces.260574_sup1Supplementary informationClick here for additional data file."}
+{"text": "The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 . Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions. An anti-inflammatory enzyme fused with a tissue-anchoring protein and injected into inflamed tissues ameliorates local inflammation without causing systemic immune suppression, as shown in multiple rodent models of inflammatory diseases. A major challenge in the treatment of chronic inflammatory diseases is the development of therapeutics to safely and specifically direct resolution of inflammation in a site-specific manner1. Anti-inflammatory drugs such as glucocorticoids are pleiotropic, non-specifically affecting numerous pathways1 and are consequently accompanied by issues of toxicity, resistance and a wide array of serious adverse effects such as infection and defective wound healing3. Additionally, systemic immune modulation leads to disease states such as hypertension, osteoporosis, obesity, cataracts and diabetes3. Biologic immunosuppressive drugs functioning through either cytokine blockade, cell depletion or cell surface receptor blockade provide improved specificity and can effectively modulate immune responses to halt disease progression in certain, but not all patients4. However, such treatments can also increase susceptibility to infections and disrupt tissue homoeostasis, leading to cancer, exacerbation of congestive heart failure and neurologic events, among other pathologies4. Critically, each of these therapeutics require life-long continual use and clinical options to resolve chronic inflammation and restore tissue homoeostasis remain to be developed5.Chronic inflammation, characterized by professional immune cell and resident tissue cell interactions, irreversibly damages tissues of the body and is an associated risk factor for a host of diseases such as cardiovascular disease, diabetes and cancer6. Catabolism of the essential amino acid tryptophan (Trp) by the cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO) and the resultant production of kynurenine metabolites is a general regulator of inflammation in response to sterile and pathogenic inflammatory stimuli, acting on both innate and adaptive immune cells8. IDO catabolism of Trp is also a contributing factor to promoting fetal tolerance in pregnancy, warding off autoimmunity and avoiding immune elimination in some forms of cancer9. Trp insufficiency via IDO activates the metabolic stress sensor general control nonderepressible 2 (GCN2) to regulate immune cell cycle10, while kynurenine pathway metabolites activate anti-inflammatory programmes, such as kynurenine binding to the aryl hydrocarbon receptor (AHR)11. IDO expression in immune cells such as macrophages and dendritic cells has been demonstrated to suppress T cell activation and proliferation while activating and promoting phenotypic maintenance of regulatory T cells14. Additionally, the terminal product of the kynurenine pathway, nicotinamide adenine dinucleotide (NAD+), regulates innate immunity function in macrophages during aging and inflammation15. In total, actions of IDO serve as a key mechanism maintaining homoeostasis, suppressing autoimmunity and shutting down excess inflammation10. Informed by these data, we envisioned therapeutic delivery of exogenous IDO as a regulator of chronic inflammatory diseases.The capability to direct cellular metabolism to programme immune responses has recently emerged as a new avenue for therapeutic immunomodulation19, we recently demonstrated the utility of model enzymes fused to galectin-3 , a carbohydrate-binding protein, as a generalizable means to restrict enzyme diffusion via binding to tissue glycans20. Gal3 binds N-acetyllactosamine and other \u03b2-galactoside glycans, as well as glycosaminoglycans, which are highly abundant in mammalian tissues22, collectively representing a more universal target than a specific extracellular matrix protein. Thus, Gal3 fusion to enzymes represents a promising approach to retain local enzymatic function at an intended site of action. Building upon this success, we engineered the IDO-Gal3 fusion with the expectation of creating a tissue-anchored IDO administered as a localized anti-inflammatory therapeutic  to serve as positive feedback to further elevate expression9, endogenous Ido1 transcripts were quantified after treatment with IDO-Gal3 in both the LPS and psoriasis models  could also potentially contribute to the resolution of inflammation. In sum, s.c. administration of IDO-Gal3 ameliorated skin inflammation both prophylactically and therapeutically, using two different inflammatory insults acting through different specific receptors (CD14/TLR4 for LPS and TLR7 for imiquimod)26.Given the capacity for endogenous cellular IDO gene expression  and hard (bone) tissue destructionl29 Fig. , submandl29 Fig. . In vivol29 Fig. , whereasl29 Fig. . Retentil29 Fig. . The pril29 Fig. . Specifil29 Fig.  and thicl29 Fig. , were prl29 Fig.  and IL-1l29 Fig. , and chel29 Fig. . In contl29 Fig. , wherebyl29 Fig. , where tl29 Fig. . IDO-Gall29 Fig. . Further31 was utilized to test for the ability of IDO-Gal3 to reduce load-induced inflammatory joint damage. Intra-articular injection of IDO-Gal3 was locally retained, suppressed inflammation and spared joint tissue destruction , a form of osteoarthritis (OA), commonly arises after a ligament or meniscus tear, or following repeated overloading of a joint. A cyclic mechanical overloading PTOA mouse modelion Fig. , with meion Fig.  and day ion Fig.  and quanion Fig.  by ex viion Fig. . Weekly ion Fig.  and draiion Fig. , whereasion Fig. . Criticaion Fig.  and artiion Fig. . Additioion Fig.  and 20. We note that multiple injections of human IDO-Gal3 in mice led to the emergence of some anti-IDO-Gal3 antibodies 34. Intra-articular injection of IDO-Gal3 was locally retained, suppressed joint inflammation, reduced OA-associated pain and improved rat gait  vectors between NcoI and XhoI sites. IDO-Gal3 genetic and protein sequences are provided in E. coli (ThermoFisher), selected on Luria-Bertani (LB) ampicillin (50\u2009\u03bcg\u2009ml\u22121) agar and incubated overnight at 37\u2009\u00b0C. Isolated colonies from the plates were subcultured in 5\u2009ml LB ampicillin broth overnight at 37\u2009\u00b0C with orbital shaking. Successful transformants were confirmed by Sanger sequencing (Genewiz). Positive DNA sequences were then transformed into the kanamycin B resistant expression strain, Origami B (DE3) E. coli (Novagen) and selected on LB ampicillin with kanamycin B (15\u2009\u03bcg\u2009ml\u22121) agar. Positive clones were picked, banked in LB with 10% w/v glycerol and used to preculture 5\u2009ml of LB ampicillin and kanamycin. Overnight precultures were used to inoculate 1l 2\u00d7 TY media  with ampicillin and kanamycin B at 37\u2009\u00b0C and 225\u2009r.p.m. in an orbital shaker until approximate exponential growth phase 600\u2009nm\u2009=\u20090.6\u20130.8). IDO-Gal3 expression cultures were supplemented with 500\u2009\u03bcM \u03b4-Aminolevulinic acid (Sigma) at the time of inoculation. Recombinant protein expression was triggered using 0.5\u2009mM isopropyl \u03b2-d-1-thiogalactopyranoside (ThermoFisher) and incubated for 18\u2009h in an orbital shaker at 18\u2009\u00b0C. Bacteria were washed and pelleted with PBS via centrifugation  with a superspeed centrifuge (ThermoFisher). Afterwards, the pellet was weighed, resuspended in 4\u2009ml PBS per gram pellet with protease inhibitor (ThermoFisher) and disrupted by sonic dismembration . Following dismembration, lysates were treated with 20 units per gram pellet DNAse I (ThermoFisher) and 800\u2009\u03bcg\u2009g\u22121 pellet lysozyme (ThermoFisher). The lysate was centrifuged  to remove the insoluble fraction and the supernatant was collected by decanting. Supernatant containing soluble recombinant protein was loaded into an \u03b1-lactose agarose (Sigma) affinity column pre-equilibrated with PBS. Columns were washed with 20 column volumes PBS and recombinant proteins were eluted with 100\u2009mM \u03b2-d-lactose (Sigma) prepared in PBS. A final polishing step was performed by 200\u2009kDa size exclusion chromatography on AKTA pure chromatography system (GE Life Sciences) to remove \u03b1-lactose and further isolate IDO-Gal3. Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS\u2013PAGE) and Coomassie staining. Endotoxin contaminants were removed by endotoxin removal solution (Sigma) following manufacturer instructions. Endotoxin content was analysed using Chromo-LAL kinetic chromogenic endotoxin quantification assay (Associates of Cape Cod) and determined to be below 0.1\u2009EU\u2009ml\u22121 in all stocks.NanoLuc is the tradename of an engineered deep-sea shrimp luciferase variant developed by Promega CorporationE. coli was purchased from R&D Systems  as measured by its ability to oxidize l-tryptophan to N-formyl-kynurenine (NFK)). The specific activity of both proteins, IDO and IDO-Gal3, was measured before experiments to ensure maximal effect at the beginning of the assay, following the IDO manufacturer\u2019s protocol. IDO-Gal3 was reacted in equimolar amounts to IDO in the standard protocol, and activities were compared using the unit pmol\u2009NFK\u2009min\u22121\u2009pmol\u22121 IDO to more accurately compare activity.Recombinant human IDO expressed in \u03b1-lactose agarose affinity resin (Sigma-Aldrich). Proteins were eluted with a linear gradient of \u03b2-lactose (Sigma-Aldrich) in phosphate buffer.Affinity of IDO-Gal3 for lactose was determined using affinity chromatography in an AKTA Pure chromatography system (GE Life Sciences) equipped with consumer-packable glass column (GE Life Sciences) packed with CCATGGCGCACGCGATGGAAAACAGCTGGACCATCAGCAAAGAGTACCACATTGACGAGGAAGTTGGTTTCGCGCTGCCGAACCCGCAGGAAAACCTGCCGGACTTCTATAACGATTGGATGTTTATCGCGAAGCACCTGCCGGATCTGATTGAGAGCGGCCAGCTGCGTGAGCGTGTGGAAAAACTGAACATGCTGAGCATCGACCACCTGACCGATCACAAGAGCCAACGTCTGGCGCGTCTGGTTCTGGGTTGCATTACGATGGCGTACGTGTGGGGCAAAGGTCACGGCGACGTGCGTAAGGTTCTGCCGCGTAACATCGCGGTTCCGTACTGCCAACTGAGCAAGAAACTGGAACTGCCGCCGATTCTGGTGTATGCGGACTGCGTTCTGGCGAACTGGAAGAAGAAGGACCCGAACAAACCGCTGACCTATGAGAACATGGATGTGCTGTTCAGCTTTCGTGACGGTGATTGCAGCAAGGGCTTCTTTCTGGTGAGCCTGCTGGTTGAAATCGCGGCGGCGAGCGCGATCAAAGTGATTCCGACCGTTTTCAAGGCGATGCAGATGCAAGAGCGTGACACCCTGCTGAAAGCGCTGCTGGAAATCGCGAGCTGCCTGGAGAAGGCGCTGCAGGTGTTTCACCAAATTCACGATCACGTTAACCCGAAAGCGTTCTTTAGCGTGCTGCGTATCTACCTGAGCGGTTGGAAGGGCAACCCGCAGCTGAGCGACGGTCTGGTTTATGAGGGCTTCTGGGAAGATCCGAAAGAGTTTGCGGGTGGCAGCGCGGGTCAGAGCAGCGTGTTCCAATGCTTTGACGTTCTGCTGGGCATTCAGCAAACCGCGGGTGGCGGTCATGCGGCGCAGTTCCTGCAAGATATGCGTCGTTACATGCCGCCAGCGCACCGTAACTTCCTGTGCAGCCTGGAAAGCAACCCGAGCGTGCGTGAGTTTGTTCTGAGCAAAGGTGACGCGGGCCTGCGTGAAGCGTATGATGCGTGCGTGAAGGCGCTGGTTAGCCTGCGTAGCTACCACCTGCAGATCGTTACCAAATATATCCTGATTCCGGCGAGCCAGCAACCGAAAGAAAACAAGACCAGCGAGGACCCGAGCAAACTGGAGGCGAAGGGTACCGGCGGTACCGATCTGATGAACTTTCTGAAGACCGTGCGTAGCACCACCGAGAAGAGCCTGCTGAAAGAGGGTGGATCCGGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGAATTCGCGGACAACTTCAGCCTGCACGATGCGCTGAGCGGTAGCGGTAACCCGAACCCGCAGGGTTGGCCGGGTGCGTGGGGTAACCAACCGGCGGGTGCGGGTGGCTACCCGGGTGCGAGCTATCCGGGTGCGTATCCGGGTCAGGCTCCGCCGGGTGCGTACCCGGGCCAAGCTCCGCCGGGTGCTTATCCTGGTGCGCCGGGCGCGTACCCGGGTGCGCCGGCGCCGGGCGTGTACCCGGGTCCGCCGAGCGGTCCGGGCGCGTATCCGAGCAGCGGCCAGCCGAGCGCGCCGGGTGCGTATCCGGCGACCGGCCCGTATGGTGCGCCGGCGGGTCCGCTGATTGTTCCGTATAACCTGCCGCTGCCGGGTGGCGTGGTTCCGCGTATGCTGATCACCATTCTGGGCACCGTGAAGCCGAACGCGAACCGTATCGCGCTGGACTTCCAACGTGGTAACGATGTTGCGTTCCACTTTAACCCGCGTTTTAACGAGAACAACCGTCGTGTGATTGTTTGCAACACCAAACTGGACAACAACTGGGGCCGTGAGGAACGTCAGAGCGTGTTCCCGTTTGAGAGCGGCAAGCCGTTCAAAATTCAAGTGCTGGTTGAACCGGACCACTTTAAGGTGGCGGTTAACGATGCGCACCTGCTGCAGTACAACCACCGTGTTAAGAAACTGAACGAAATCAGCAAACTGGGCATCAGCGGTGACATTGATCTGACCAGCGCGAGCTATAACATGATTCTCGAG, with restriction sites underlined.The sequence used was GSGGGSGGSGGSGGEFADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYNMILEHHHHHHMAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG\u22121 lipopolysaccharide (LPS) in 40\u2009\u03bcl, after 24\u2009h or 120\u2009h post IDO-Gal3 treatment, to assess local and distal modulation of inflammation. At 2\u2009h after LPS administration, animals were euthanized and the injection site collected.B6 mice were administered 2.1\u2009\u03bcg IDO-Gal3 in 40\u2009\u03bcl PBS subcutaneously  at the region of the hock and challenged with 2\u2009ng\u2009g\u03b2, IFN-\u03b3 and IL-6) using Applied Biosystems QuantStudio 12K Flex Real-Time PCR System. Results are presented as the ratio of gene expression to glyceraldehyde 3\u2010phosphate dehydrogenase (GAPDH) expression determined by the relative quantification method. Treatment groups were normalized to PBS only group.Soft tissue was separated from bone and stored in RNAlater RNA stabilization reagent (Qiagen) in preparation for qPCR. Soft tissues were homogenized and RNA purified using RNeasy Protect mini kit (Qiagen). Complementary DNA was synthesized from RNA using the High-Capacity cDNA Reverse Transcriptase kit (ThermoFisher) for use in qPCR in accordance with manufacturer instructions. qPCR analysis was run with primers specific for pro-inflammatory cytokines  overnight. After fixation, tissues were washed in deionized water and decalcified by storing in 10% ethylenediaminetetraacetic acid (EDTA) at 4\u2009\u00b0C for 3 weeks. Samples were assessed every 2\u20133\u2009d for stiffness and EDTA solution replenished. Tissues were submitted in 70% ethanol to the University of Florida Molecular Pathology Core for processing, paraffin embedding, sectioning, mounting and staining with haematoxylin and eosin (H&E). Tissues were imaged using a Zeiss Axiovert 200M with a \u00d720 objective lens through the multidimensional acquisition module. Eleven images were taken per tissue and scored by two blinded independent individuals on the basis of cellular infiltration  and epidermis hypertrophy .n\u2009=\u20093). At 30\u2009min after injection, the mice were euthanized, the hock and tibia tissue regions excised and flash frozen using liquid nitrogen. These samples were then submitted to the Southeast Center for Integrated Metabolomics at the University of Florida for mass spectrometric analysis of kynurenine levels.IDO-Gal3 (2.2\u2009\u03bcg) in 40\u2009\u03bcl PBS or 40\u2009\u03bcl PBS alone was injected into the hock site of B6 mice  was collected in microtainer serum separator tubes and centrifuged at 1,500\u2009r.p.m. for 5\u2009min at room temperature. The serum was collected and stored at \u221220\u2009\u00b0C. Cytokine analysis was performed using a Luminex 200 system running xPONENT 3.1 software (Luminex) following manufacturer instructions.37. Briefly, mice were anaesthetized and their backs were shaved, followed by application of depilatory cream to remove any remaining fur. Each day for 14\u2009d total, 5% IMQ cream  was applied to the backs of the mice. On the 3rd day of IMQ application, mice were subcutaneously injected with five 10\u2009\u03bcg doses of IDO-Gal3 in sterile saline distributed evenly throughout the back, a molar equivalent of NL-Gal3 delivered in the same volume, or a sterile saline control (n\u2009=\u200912 per group). Disease severity was measured each day using a modified version of the Psoriasis Area and Severity Index (PASI) where area of effect is not taken into account. Erythema (redness), scaling and thickening were scored independently and assigned a score on a scale of 0 to 4: 0, none, 1: slight, 2: moderate, 3: marked, 4: very marked. The cumulative score was reported as a measure of the severity of inflammation .Pre-clinical modelling of psoriasis was carried out in 8\u201312-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. The model used was a modified version of a previously reported modelIn vivo imaging of fluorescently tagged IDO-Gal3 was carried out in 8\u201312-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the IACUC at the University of Florida. Before injection, IDO-Gal3 was incubated with IRDye 680RD NHS Ester  following manufacturer instructions. Mice received a 40\u2009\u03bcl  subcutaneous injection of fluorescently labelled IDO-Gal3 to the hock. Immediately following injection and every subsequent 24\u2009h following injection, mice were imaged using an IVIS Spectrum in vivo imaging system (PerkinElmer). Fluorescent images were captured using the AF680 emission filter, subject size 1.5\u2009cm, 0.2\u2009s exposure time, field of view B (6.6\u2009cm), medium binning (factor of 8) resolution and a 1F/Stop aperture. Relative fluorescent intensities were represented by a pseudo colour scale ranging from red (least intense) to yellow (most intense).\u22121\u2009cm\u22122\u2009sr\u22121). The specific amount of protein in tissue was determined by comparison with a standard curve of NanoLuc-Gal3 activity.NanoLuc-Gal3 (164\u2009pmol) in 40\u2009\u03bcl PBS was injected subcutaneously into the hock of B6 mice. At the prescribed time points, animals were euthanized in accordance with approved protocols. Organs and tissues of interest were collected, weighed, processed, incubated with furimazine and bioluminescence quantified by a luminometer. Bioluminescence images were acquired using an IVIS Spectrum in vivo imaging system. Living Image software v4.3.1 (PerkinElmer) was used to acquire the data immediately after furimazine administration. Exposure time for the bioluminescence imaging was 1\u2009s. Regions of interest (ROIs) were quantified as the average radiance (photons\u2009sListeria monocytogenes InIAM (strain 10403s). Tissue bacteria burdens in the liver and spleen were used as a metric for infection where the liver is local for gut infection and the spleen represents systemic spread of infection. On the day before infection, 10-week-old na\u00efve C57BL/6 mice (Taconic Biosciences) received IDO-Gal3 injection subcutaneously (hock), while control mice received sterile saline injection. The following day, mice were orally infected with 2\u2009\u00d7\u2009109 colony-forming units (c.f.u.) of Listeria monocytogenes per mouse. To prepare for infection, bacteria were cultured overnight in BHI broth at 37\u2009\u00b0C, shaking at 220 r.p.m. Before infection, a subculture of 2\u2009ml of the bacteria and 18\u2009ml BHI broth was cultured under the same conditions until reaching an OD600\u2009=\u20090.8. Bacteria were pelleted, resuspended in 500\u2009\u03bcl of sterile PBS, and 50\u2009\u03bcl of bacteria was pipetted onto a small square of white bread and fed to each mouse individually. Once the entire piece of bread was eaten, mice were returned to their cage. At day 7 post infection, mice were euthanized, followed by collection of the spleen and liver. Tissue was homogenized, suspended in 1% saponin for 1\u2009h, then plated at serial dilutions from undiluted to 1:1,000. BHI Agar plates were treated with streptomycin to limit non-specific bacterial growth, as this strain of L. monocytogenes is streptomycin resistant. C.f.u.s were counted at 24\u2009h post-plating.Immune suppression from IDO-Gal3 was evaluated in response to oral infection with 9P. gingivalis strain 381 and 2.5\u2009\u00d7\u2009109Aggregatibacter actinomycetemocomitans strain 29522 (ATCC) resuspended in 2% low-viscosity carboxy-methyl-cellulose (Sigma-Aldrich). Oral lavage was repeated every week for 5 weeks. Each week, 1\u2009d before the first day of infection (prophylactic) or 1\u2009d after the last day of infection (therapeutic), 10\u2009\u03bcl of IDO-Gal3 was injected into the submandibular space using a 30-gauge insulin syringe (Becton Dickenson). Each week of infection, on the first day of infection, before infection, microbial sampling of the oral environment was performed with calcium alginate swabs (ThermoFisher). One week following the last infection, the mandibles were collected to evaluate soft tissue soluble mediator expression and bone morphometric analysis.All mice were lavaged with 25\u2009\u03bcl 0.12% chlorhexidine gluconate (3\u2009M) for 3\u2009d. On days 4, 5, 6 and 7, mice received a 25\u2009\u03bcl oral lavage with 2.5\u2009\u00d7\u200910\u03b2, IL-10 and MCP1 according to manufacturer protocols. Data were acquired on a Luminex 200 system running xPONENT 3.1 software (Luminex) and analysed using a 5-paramater logistic spline-curve fitting method using Milliplex Analyst v5.1 software (Vigene Tech). Data are presented as pg\u2009ml\u22121 normalized to total protein .Mandibles with both soft tissue and bone were subjected to bead beating at two 2\u2009min intervals with 2\u2009min of cooling in between using 1.0-mm-diameter zirconia silica beads (BioSpec) in cell extraction buffer (ThermoFisher). The buffer was prepared with a protease inhibitor cocktail  and PMSF protease inhibitor (Abcam) to allow for dissociation and lysis of all soft tissue while leaving the hard tissues intact. MILLIPLEX Multiplex assays (EMD Millipore) were used to probe resulting lysates for IL-6, IL-13 ROI was set with standardized dimensions of 1.5 \u2009\u00d7\u20094.0 \u2009\u00d7\u20090.9\u2009mm . Anatomical landmarks were used for the standardized positioning of the ROI: frontal plane, the roof of the furcation area between mesial and distal roots of the upper first molar; sagittal plane, anterior limit was the distal aspect of the mesial root of the first molar. The thickness of the ROI on the transversal plane was set to 50 slices (900\u2009\u03bcm) and counted towards the palatal /medial direction beginning from the image that included the centre of the upper first molar in its transversal width. A standardized threshold was set to distinguish between non-mineralized and mineralized tissues where total volume and total thickness of the ROI were calculated. Mean trabecular bone volume (BV) and thickness (BT) analysis assessed the percentage of mineralized tissue  within the total volume/thickness  of the ROI and is presented as the BV/TV ratio (mean trabecular bone volume) or BT/TT ratio (mean trabecular thickness). Vertical bone loss is the distance from the cementoenamel junction (CEJ) to the alveolar bone and was calculated at 12 sites over three molars and averaged to calculate the average vertical bone loss in mm.Mandibles were fixed in 4% buffered formalin for 24\u2009h, stored in 70% alcohol and scanned at 18\u2009\u03bcm resolution using a micro-CT system (Skyscan). Three-dimensional images were reconstructed and the resulting images re-oriented spatially using anatomical landmarks with the NRecon and DataViewer software (Skyscan). A standardized 5.4\u2009mmP. gingivalis 16S, A. actinomycetemocomitans 16S and total 16S using real-time PCR. The percentage of A. actinomycetemocomitans 16S and P. gingivalis 16S within the total 16S compartment was calculated using the following formula: Ct value of total 16S/Ct value of A. actinomycetemocomitans or P. gingivalis 16S. 16s rRNA For: AGA GTT TGA TCC TGG CTC AG; Rev: ACG GCT ACC TTG TTA CGA CTT; Pg For: CTT GAC TTC AGT GGC GGC AG; Rev: AGG GAA GAC GGT TTT CAC CA; Aa For: GTT TAG CCC TGG CCG AAG; Rev: TGA CGG GCG GTG TGT ACA AGG.Genomic DNA was isolated from microbial sampling of the oral environment using a DNeasy kit (Qiagen) according to manufacturer instructions. The gDNA was then probed for 30 that applies cyclic mechanical loading to the knees of aged (6 months) mice, causing mechanical damage and consequent inflammation and cartilage degradation31. The mice were anaesthetized and placed in a fixture with the knee in flexion; loading (9\u2009N) was applied axially for 500 cycles and loading sessions were done on the mice 5 times per week during the experiment. IDO or IDO-Gal3 was prepared at 143\u2009\u03bcM, and 20\u2009\u03bcl of each treatment was injected intra-articularly at the start of each week of the study, with each knee receiving 4 total treatments. At the end of the 4-week study, mice were euthanized for analysis of gene expression and histopathology.IDO-Gal3 activity was assessed in a post-traumatic osteoarthritis mouse modelPharmacokinetics of IDO and IDO-Gal3 retention after local injection at the disease site was assessed by intravital imaging over the course of 7\u2009d. Both proteins were labelled with Li-Cor IRDye 680RD NHS ester (Li-Cor Biosciences) to visualize and measure protein knee retention. Mice were subjected to mechanical loading for 2 weeks before each treatment was administered via intra-articular injection. Intravital imaging was performed  immediately following injection and every subsequent 24\u2009h. The fluorescence signal measured at the joint over time was normalized to the initial measurement for each knee and an exponential decay was individually fit for each specimen. The AUC/bioavailability for each joint was calculated from the best-fit line of exponential decay. After 7\u2009d, the mice were euthanized and an ex vivo image was taken of each joint with the surrounding skin removed to increase measurement sensitivity.TaqMan qPCR in the knee joint and the popliteal lymph node that drains the knee joint. Following euthanization, knees and popliteal lymph nodes were excised. Combined joint tissue from the synovial wall and articular surface  and the popliteal lymph nodes were homogenized with bead pulverization in Qiazol. RNA was extracted and purified using the RNeasy Plus mini kit from Qiagen and quantified using NanoQuant plate from Tecan in a microplate reader . The RNA was converted to cDNA using the iScript Synthesis kit from Bio-Rad (Hercules). Gene expression was calculated by the \u0394\u0394Ct method, normalizing to GAPDH and beta-actin (ACTB). TaqMan reagents were purchased from ThermoFisher and used according to provided protocols, using appropriate primers .Gene expression was evaluated by 38. OARSI scoring was based on medial and lateral tibial plateaus 39, and a generic score was concurrently assigned on the basis of H&E features and the safranin O staining of the tibial plateaus according to the DJD methodology .Tissue samples were fixed in 10% formalin and decalcified in 20% EDTA for 7\u2009d. A standard 8\u2009h cycle of graded alcohols, xylenes and paraffin wax was used to process tissues before embedding and sectioning at 5\u2009\u03bcm thickness. Sections were mounted on positively charged glass slides and stained with H&E using the Gemini autostainer (ThermoFisher). Safranin O staining was performed using the StatLab staining kit. Sections were imaged using Leica SCN400 slide scanner. Each joint was evaluated by at least two mid-frontal sections for both H&E and safranin O stains. A board-certified veterinary pathologist conducted histopathologic interpretations under blinded conditionsSixteen male Lewis rats  were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. After acclimation, rats underwent baseline gait and von Frey behavioural testing. Following baseline behavioural testing, all rats received medial collateral ligament plus medial meniscus transection (MCLT\u2009+\u2009MMT) surgery to their right hind limb. Gait data were collected on weeks 3, 5 and 7 post-surgery using a Phantom Miro Lab320 (Phantom Camera Control 3.0), while von Frey testing was conducted weekly after surgery. At 8 weeks post-surgery, rats received a unilateral saline or IDO-Gal3 injection. Rats underwent von Frey testing the day following injection and gait testing 2\u2009d after injection. Both gait and von Frey data were collected weekly until euthanasia.\u22121) (Patterson Veterinary) intra-operatively and every 12\u2009h for 48\u2009h. Rats were grouped into a saline injection cohort (n\u2009=\u20097) or IDO-Gal3 injection cohort (n\u2009=\u20098). At 8 weeks after OA induction, rats received 30\u2009\u00b5l unilateral injections of either sterile saline or IDO-Gal3 (33\u2009pg\u2009\u00b5l\u22121) in the operated knee using sterile allergy syringes  (Becton Dickinson). First, rats were anaesthetized using 2.5% isoflurane (Patterson Veterinary) and the operated knee was aseptically prepped as described above. Then the needle was inserted through the patellar ligament following the patellar groove into the joint space. The knee was flexed and the injection site was cleaned with sterile gauze and 70% ethanol.MCLT\u2009+\u2009MMT surgery rats were anaesthetized in a 2.5% isoflurane (Patterson Veterinary) sleep box. Rats were then aseptically prepped with betadine surgical scrub (Purdue Products) and 70% ethanol in triplicate, and transferred to a sterile field with anaesthesia maintained via mask inhalation of 2.5% isoflurane. During MCLT\u2009+\u2009MMT surgery, a 1\u20132\u2009cm midline skin incision was made along the medial aspects of the rat knee and the skin was retracted to reveal the medial collateral ligament. The medial collateral ligament was transected and the knee was placed in the valgus orientation to stretch the medial compartment and expose the medial meniscus. The medial meniscus was cut radially, and absorbable 5\u20130 vicryl braided sutures (Ethicon) were used for muscle closure and 4\u20130 ethilon nylon monofilament sutures (Ethicon) were used for skin closure. Rats recovered post-operatively in a warming box until weight bearing on all limbs. For pain management, rats received a subcutaneous injection of buprenorphine  was applied to the plantar region of each hind foot. First, the 4.0\u2009g von Frey filament was applied. A less-stiff filament was applied if a paw withdrawal occurred, and a stiffer filament was applied if a paw withdrawal did not occur. Using these data, the force at which rats were equally likely to withdraw or tolerate was calculated via Chaplan\u2019s approximation1.Tactile sensitivity was assessed by measuring the 50% paw withdrawal threshold determined using the Chaplan up-down method for von Frey filaments\u22121 anti-CTXII, 8.9\u2009ng\u2009\u00b5l\u22121 anti-IL-6 or 357\u2009ng\u2009\u00b5l\u22121 anti-MCP1 and then moved to static incubation at 4\u2009\u00b0C overnight. Particles were then washed three times in PBS containing 2% BSA and 2\u2009mM EDTA (capture buffer), with final antibody amounts per particle measured to be 0.33\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-CTXII), 0.33\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-IL-6) and 13.0\u2009ng\u2009Ab\u2009\u00b5g\u22121 particle (anti-MCP1).Commercially available streptavidin-functionalized particles  were coated with biotinylated antibodies for CTXII , IL-6 and MCP1 . Here, particles were washed 3 times in PBS, incubated for 2\u2009h on a tube revolver at room temperature in antibody mixes that contained either 8.9\u2009ng\u2009\u00b5l40. Briefly, 300\u2009\u00b5g of antibody-conjugated magnetic particles  were suspended in 10\u2009\u00b5l of saline, then injected in the operated and contralateral knee. After 2\u2009h incubation in the knee, particles were collected via 5 repeated 50\u2009\u03bcl PBS washes of the knee, with collected fluid combined and particles in the fluid isolated via a magnetic separator. Collected particles were washed twice with capture buffer, then incubated for 15\u2009min in the 100\u2009mM Glycine-Tris buffer (pH 3.1) containing 2% BSA and 2\u2009mM EDTA (release buffer). Following biomarker release, magnetic particles were isolated by magnetic separation and the pH of the supernatant was adjusted to 8.3 for enzyme-linked immunosorbent assays (ELISA). CTXII was then measured in the supernatant using the Cartilaps CTXII ELISA kit  according to manufacturer instructions, and CCL2 was quantified using a rat CCL2 ELISA kit (KRC1012 Life Technologies) according to manufacturer instructions and previously described modifications41. IL-6 was quantified using ELISA developed in the laboratory using biotin anti-rat IL-6 antibody (517703) and purified (coating) anti-rat IL-6 antibody . Coating antibody was diluted in 100\u2009mM NaHCO3 and 34\u2009mM Na2CO3 (pH 9.5), placed in coated microwells , incubated for 30\u2009min on a plate shaker then overnight at 4\u2009\u00b0C, washed 5 times with PBS with 0.5% Tween-20 (wash buffer), blocked for 1\u2009h with PBS containing 2% BSA and 10% heat-treated bovine serum, and finally washed 5 times with wash buffer. Samples and standards were pre-incubated with anti-IL-6 detection antibody (30\u2009min at room temperature and then overnight at 4\u2009\u00b0C), then added to ELISA plate and incubated for 3\u2009h. The plate was then washed 5 times, and 100\u2009\u03bcl of avidin-HRP  was added to the plate and incubated for 30\u2009min. The plate was again washed 5 times, with 100\u2009\u03bcl of tetramethylbenzidine substrate added for 15\u2009min, followed by 100\u2009\u03bcl of stop solution (diluted sulfuric acid). Absorbance was read at 450 and 650\u2009nm. Particles were quantified in 60\u2009\u03bcl of capture buffer, with particle suspensions read for absorbance at 450\u2009nm using Synergy 2 Multi-Mode microplate reader, as previously described40.Following euthanasia via exsanguination, magnetic capture was performed in both the operated and contralateral knee, as previously describedFollowing magnetic capture, operated and contralateral knees were dissected and placed in 10% neutral buffered formalin (ThermoFisher) for 48\u2009h at room temperature. Following fixation, knees were decalcified using Cal-Ex (ThermoFisher) for 5\u2009d at room temperature, dehydrated through an ethanol ladder and embedded in paraffin wax via vacuum infiltration. Then, 10\u2009\u00b5m frontal sections were acquired, with at least one section taken at every 100\u2009\u00b5m through the loading region of the medial meniscus. Slides were stained with toluidine blue.n\u2009=\u20098) or bilateral injections of NL-Gal3  (n\u2009=\u20098) in both operated and contralateral knees. (Surgeries and injections were conducted the same way as described above). Knees were flexed, then injected with 50\u2009\u00b5l furimazine . Immediately after injection, knees were flexed again and luminescence was measured with IVIS using a 1\u2009s (and 60\u2009s) exposure time in field of view D. Furimazine injections and IVIS imaging were repeated at 1, 2, 4, 8, 12, 16, 20, 24 and 28\u2009d post NL or NL-Gal3 injection. For analysis, a ROI was drawn around the largest luminescent signal and copied to create an identical sized ROI for all knees.Sixteen male Lewis rats  were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. Then rats underwent MCLT\u2009+\u2009MMT surgery. After 8 weeks, rats received a 50\u2009\u00b5l injection of either Nano-Glo Luciferase (NL) or Nano-Glo Luciferase Galectin-3 . Rats were IVIS imaged immediately following injection, then at 1, 2, 4, 8, 12, 16, 20, 24 and 28\u2009d post-injection. After imaging on day 28, rats were euthanized, and both operated and contralateral knees were dissected for joint tissue distribution analysis via IVIS. In vivo joint retention was measured in the operated and contralateral knees of 16 male Lewis rats. At 8 weeks post MCLT\u2009+\u2009MMT surgery, knees were aseptically prepped and rats received bilateral injections of NL   at room temperate for roughly 1\u2009min. After incubation, tissues were removed from the 24-well plates and luminescence was measured with IVIS using auto-exposure in field of view C. For assessment, individual ROIs were drawn around the patellar tissue, tibial tissue, femoral tissue and meniscus. The ROI for each tissue was copied to create an identical sized ROI for the respective tissue.U-tests on psoriasis data. Study-specific analyses are reported in figure captions.Statistical analyses were performed using GraphPad Prism 8 and 9, with the following exceptions. Surgically induced osteoarthritis data were analysed using R Analytics 4.0.4 with RStudio 2022, and MATLAB 2020b was used for Mann-Whitney Further information on research design is available in the Supplementary InformationSupplementary methods and figures.Reporting Summary"}
+{"text": "Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling show an increased defecation cycle period. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system in  Glycoprotein hormones are key neuroendocrine factors that control diverse physiological processes, such as development, reproduction, energy homeostasis and growth , 2. In vDrosophila melanogaster and the mosquito Aedes aegypti  and thyrotropin-releasing hormone (TRH), have been identified in C. elegans and were found to have conserved physiological roles, such as in the control of reproduction and growth , respectively. GPLA-1 and GPLB-1 resemble vertebrate GPA2 and GPB5 and are widely conserved in nematodes  and gplb-1 (ibt4) knockout mutants display a significantly shorter body length compared to wild-type animals  mutant carries a substitution of the glycine residue between the second and third cysteine (G63E), which is highly conserved and important for proper cysteine knot formation (gpla-1 (ok2127) mutant contains a large deletion, removing the start codon and the complete first exon of the gene  cells. We co-expressed FSHR-1 with a cAMP response element (CRE)-luciferase reporter in HEK cells and quantified its activation by assessing bioluminescence levels in the presence of luciferin substrate  and the enteric muscles of the hindgut, which are involved in defecation  or hmc and the enteric muscles (gplb-1).To gain further insight into the role of thyrostimulin-like signaling in growth regulation, we investigated the expression patterns of  defects 2E, show defects , 49, 50, elegans . These i elegans \u201355. We afecation \u201357. Takefshr-1 under its endogenous promoter recapitulated the reported expression of the receptor in the intestine and in multiple neurons in the head  and exp-1(ox276) mutants are also significantly smaller than wild-type animals  have a carboxytail extension, referred to as the \u201cseatbelt\u201d, that wraps around the alpha subunit to stabilize heterodimer configuration , 79, 80.erodimer , 28, 82.erodimer , 38, 82.solution . Howeversolution . Based osolution . Severalsolution , in whicC. elegans FSHR-1, is abundantly expressed in the alimentary canal  superfamily that is required for intestinal calcium and pH oscillations  plates seeded with Escherichia coli OP50 bacteria. All experiments were performed using 1-day adult hermaphrodites, unless mentioned otherwise. Wild-type (N2-Bristol), IBE7 flr-2 (ut5), IBE24 flr-2 (ok2127), IBE1 fshr-1 (ok778) and EG1285 lin-15B&lin-15A (n765) oxIs12 [unc-47p::GFP + lin-15(+)] strains were obtained from the Caenorhabditis Genetics Center . CB156 unc-25 (e156) and EG276 exp-1 (ox276) strains were a kind gift from the lab of W.R. Schafer . Deletion alleles for gpla-1 (ibt1) and gplb-1 (ibt4) were obtained by CRISPR/Cas9 genome editing, as described below. A full list of strains used in this study can be found in All fshr-1 (a isoform), gpla-1, and gplb-1 (a isoform) were cloned in front of a SL2 trans-splicing site and fluorescent reporter gene.All fluorescent reporter and rescue constructs were made using the MultiSite Gateway Three-Fragment cloning system (Invitrogen). Genomic DNA or cDNA sequences of fshr-1 (3110 bp), gpla-1 (4058 bp), and gplb-1 (3589 bp) were cloned from genomic DNA of wild-type C. elegans. Tissue-specific rescue transgenes were generated using promoter regions of rab-3 (1208 bp) (rgef-1 (3660 bp)  (mir-228 (2217 bp)  , 66, rge3660 bp) , ges-1  \u201363, and 2217 bp) . All confshr-1 in HEK cells was obtained by directionally cloning the fshr-1 cDNA into the pcDNA3.1/V5-His-TOPO vector (Invitrogen). The cDNA sequence of the fshr-1a gene isoform was amplified by PCR using cDNA from mix-staged wild-type C. elegans as template. The forward primer included a \u2018CACC\u2019 sequence at the 5\u2019 end that introduced a partial Kozak sequence for increased translation efficiency in mammalian cells.The plasmid for heterologous expression of gpla-1(ibt1) and gplb-1(ibt4) knockout alleles were generated by CRISPR/Cas9-mediated deletion of the gpla-1 or gplb-1 open reading frames. For each gene, two crRNA sequences were designed by looking for PAM sites (NGG) that were in the vicinity of the double stranded break site. After analysis of putative off-target sites (http://crispor.tefor.net/), we selected the highest scoring sequences based on their predicted on-target activities. Repair templates were designed to be in-frame and were codon-optimized for C. elegans (https://www.genscript.com/tools/codon-frequency-table). To simplify selection of the successfully edited worms, we used a Co-CRISPR technique in which the dpy-10 gene was also knocked out by CRISPR editing (The  editing . We used editing  for the  editing Table S2gpla-1 (ibt3 [His::GPLA-1]) knockin allele was made by CRISPR/Cas9 mediated insertion of a His-tag at the N-terminus of the gpla-1 open reading frame, using a similar strategy as described above and the crRNA and repair template listed in The Below, the sequences of the gene knockouts and knockins are shown with flanks on the (-) strand. Exons are in grey, with start and stop codons in red, the remaining gene sequences after CRISPR gene editing are underlined, and with the inserted gene sequence in blue.gpla-1(ibt1) deletion:ATGGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgtcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaattctttaaattctatttttaacttttcagcctataccttctcaaatggplb-1(ibt4) deletion:tgttgacgttaagctcttttgaaaaaaATGCTTATATTTCCCGTGATCACTATACTTCATATATTTTTGgtaactttttgaaatatcttctaaaatgaatttaatatgtaacaatattctaacaaaatttttaatattgatcagatagtttttccaaatttaagtattaaccaggcgcgaaattttccgaattttaggccaaaaatacggtgcccggtctcgacacgaatttttttattaggtaaaaatgggtgtgtgcctttaaagagtactgtaactttaaactttcgttgctgcggaacttttgtcgacttttcatagctattagataaaaataaaaaaatattcaatattttcaacaaatctttagaaaaactatgtaaaatcgataaaaattctgcaacaaaaatttgaagttacagtactctttaaaggcacacactcgattgtatttaacaaaaaaagtcgtgtcgagaccggttaccgtaatttttgcgcaaatcggaataatttcgcgcttgggtaataagcatcacatctccaactaatttaaaagcaaaagtgtgatttttaaattcagATTTCGGTTGAATCTGGAAAAGAATGCGAGTTTGCAATGCGATTGGTCCCAGGATTCAATCCACTTCGTCAAGTTGATGCAAATGGAAAAGAATGCCGAGGAAACGTGGAATTGCCATTTTGCAAGGGTTACTGTAAGACTAGCGAGgtgaatttccttttttttccgattcaaaaataatccaattaaatttcagAGTGGCACCCATGGCTTTCCACCACGAGTTCAAAATAGTAAAGTGTGCACATTGGTCACCACTTCAACTCGAAAAGTAGTTCTTGATGATTGTGATGATGGAGCCGATGAGAGTGTCAAGTTTGTAATGGTTCCACATGGAACTGATTGTGAATGTTCTGCAGTTCCACTTGAACAACATCATTCATAAattatcatacattcattcaaaaattcatcgaataaataaaagttttgtggpla-1(ibt3 [His::GPLA-1]) insertion:ttctttaaattctatttttaacttttcagcctataccttctcaaatgATGCATCACCATCACCATCACGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgektcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaamyo-2p::mCherry, unc-122p::dsRed, or unc-122p::GFP) and 1-kb DNA ladder (Thermo Scientific) as carrier DNA.Germline transformations were carried out by injecting constructs into the syncytial gonad of young adult worms together with a co-injection marker  and resulting Z-stack projections were created and analyzed with ImarisViewer (v9.7.2) software. Adult worms were immobilized by mounting in 50 mM Sodium Azide solution in M9 buffer on a 2% agarose pad and covered with a glass cover slip. Expression patterns were confirmed in at least two independent transgenic strains. DVB and RME expression was confirmed by crossing with marker strain EG1285, which marks GABAergic neurons . Other chttps://github.com/jwatteyne/WormSizer). Synchronized L1 larvae were placed onto freshly seeded NGM plates one day post-synchronization and kept at 20\u00b0C until assaying. After 65 hours on food, 20 to 30 well-fed day one adults were transferred from the plates and anesthetized in 20 \u00b5L of 10 mM tetramisole hydrochloride solution (Sigma-Aldrich) in Milli-Q water on a 2% agarose pad. Images were captured with a ZEISS Axio Observer.Z1 at 5x magnification. Body size was measured from images of anesthetized day 1 adult worms on at least two independent days. After skeletonization, delineating the worm\u2019s outline and midline, the worm\u2019s total length and middle width were calculated using the calibration factor (pixels/\u00b5m) of the pictures. For the volume estimation, the midline of the worm was separated in 30 segments of the same size. The volume for each segment was determined by using the formula for the frustum of a cone, taking the natural shape of a worm into consideration. The summation of all these volumes was described as the worm\u2019s total volume. To assess growth throughout adulthood, body size measurements were executed for 5 subsequent days (day 1 to day 5 adulthood) for at least two independent time periods.Body size parameters  of day one adults were measured using the custom WormSizer MATLAB script , luminal width was determined by measuring the average width (in \u00b5m) of the intestine at two different points in the posterior lumen. Body size parameters and luminal widths were plotted, and significance levels were calculated with GraphPad Prism 8 software.C. elegans N-terminal 6xHis-tagged GPLA-1 and non-tagged GPLB-1. Recombinant proteins were purified from cell lysate by affinity chromatography in PBS buffer  and analyzed by Tricine-SDS-PAGE on a 8 \u2013 20% gel in Tris-Glycine running buffer and by subsequent Western blot using anti-His monoclonal antibodies. A similar approach was used to synthesize individual GPLA-1 and GPLB-1 proteins. N-terminal 6xHis-tagged GPLA-1 was expressed in HEK293 cells and purified by affinity chromatography. Non-tagged GPLB-1 was expressed in HEK cells and purified using ion exchange chromatography. Both recombinant GPLA-1 and GPLB-1 protein samples were analyzed by Tricine-SDS-PAGE.Recombinant proteins were synthesized and purified by OriGene\u2122 Technologies . To generate recombinant GPLA-1/GPLB-1, HEK293 cells were transfected with expression vectors encoding 2, in Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM)/Nutrient F-12 Ham (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum  and 1% Penicillin/Streptomycin (Gibco). Co-transfection with the cAMP indicator CRE(6x)-luciferase and pcDNA3.1/fshr-1a was done in a 1:1 ratio when cells reached 60-70% confluency. Transfection medium contained Opti-MEM , CRE(6x)-luciferase and receptor plasmid DNA, Plus Reagent (Invitrogen) and Lipofectamine LTX (Invitrogen). One day post-transfection, fresh culture medium was added to the transfected cells. Two days post transfection, each well of a Bio-One CELLSTAR\u2122 96-well, Flat Bottom Microplate (Greiner) was loaded with a dilution series of recombinant protein compounds in 200 \u00b5M 3-isobutyl-1-methylxanthine (IBMX), which inhibits cAMP hydrolysis. Compound buffer (PBS with 10% glycerol) without hormone was added to the IBMX medium as a ligand-free negative control. Cells were detached, counted, pelleted and resuspended to a concentration of 10E-06 cells/mL in IBMX medium. Each well of the compound plate was supplemented with 50 \u00b5L of the cell suspension (50 000 cells/well) and the plate was incubated at 37\u00b0C for 3.5 h. Cells were then loaded with 100 \u00b5L SteadyLitePlus substrate (PerkinElmer) and incubated on a shaking plate for 15 minutes under dark conditions at RT. Finally, luminescence was measured twice for 5s (at 0s and 5s) per well at 469 nm on a Mithras LB 940 luminometer (Berthold Technologies). Measurements were performed in triplicate in at least 4 independent experiments. Luminescence values were plotted and significance levels were calculated with GraphPad Prism 8 software.The cAMP bioluminescence assay quantifies ligand-induced bioluminescent responses by measuring changes in intracellular cAMP levels after receptor activation. HEK293T cells were cultured in monolayer at 37\u00b0C in a humidified atmosphere with 5% COhttps://github.com/NathanDeFruyt/DefecationAnalysisBeetsLab). The length of one DMP cycle was defined by the time elapsed between two subsequent contractions of the posterior body wall muscles (pBocs). The average cycle length was calculated over 10 defecation cycles for each worm. aBoc frequency was defined as the ratio of aBoc over pBoc. Defecation parameters were plotted and significance levels were calculated with GraphPad Prism 8 software.The defecation motor program (DMP) was analyzed as previously described . AnimalsC. elegans GPLA-1 and GPLB-1 sequences were used as queries to identify sequences with highest similarity in selected model systems representative for their phylum by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) database. The multiple sequence alignment program Clustal Omega  was used to align sequences, and conserved residues were identified using BoxShade .The H. Sapiens GPA2 and GPB5 and The original contributions presented in the study are included in the article/SK, MI, JW, LS and IB designed the research. SK, MI, SD and EV performed the experiments. SK, MI and IB analyzed data. IB and LS supervised the project and were responsible for funding acquisition. SK and IB wrote the paper. All authors contributed to the article and approved the submitted version."}
+{"text": "The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster ( C-terminal tail threads through the macrolactam ring formed by the N-terminal amino group and the carboxylic acid side chain of an aspartate or a glutamate located at position 7\u20139 of the core peptide via an isopeptide bond (D), which is responsible for excretion and self-resistance. B genes from actinobacteria and firmicutes are usually split into two genes: B1 encodes the N-terminal RiPP precursor recognition element (RRE) domain with homology to PqqD family proteins, while B2 encodes the C-terminal protease domain that cleaves the leader peptide of the precursor, after which the remanent core peptide is macrolactamized to form the mature lasso peptide  with a characteristic lariat topology, in which the ide bond . The numide bond . Generalide bond . Many la peptide .Beyond the class-defining leader peptide removal and core peptide macrolactamization, nine unique post-translational modifications (PTMs) are reported in total in the biosynthesis of lasso peptides up to now , among wBradymonas sediminis FA350 is a Gram-negative, rod-shaped, and facultatively prey-dependent bacterium isolated from coastal sediments in Weihai, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to a novel bacterial order Bradymonadales and a novel family Bradymonadaceae in the class Deltaproteobacteria (B. sediminis FA350 (accession No. NZ_CP030032.1) and subsequent search for natural products BGCs allowed the identification of a lasso peptide BGC, which we subsequently named bsf (bsfA), a discrete RRE (bsfB1), a leader peptidase (bsfB2), a lasso peptide cyclase (bsfC) and an ABC-transporter (bsfD), two additional genes encoding a putative kinase (bsfK) and a predicted nucleotidyltransferase (bsfN) have been found in this BGC as well. Herein, we characterized the activities of BsfK, BsfB1, and BsfB2.bacteria . Sequencamed bsf . The canApexHF HS DNA polymerase for high-fidelity amplification from Accurate Biology Co. Ltd. (China). Chemical compounds and reagents were purchased from Bide Pharmatech Ltd. (China), Macklin Biochemical Technology Co., Ltd. (China) and J&K Scientific Ltd. (China) unless stated otherwise. Gene synthesis and codon optimization were performed at GENEWIZ, Inc. (China). Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech Co. Ltd. (China). Primers used in this study are summarized in Biochemicals and media were purchased from Sinopharm Chemical Reagent Co. Ltd. (China) or Oxoid Ltd. (United Kingdom) unless stated otherwise. Enzymes were purchased from New England Biolabs Ltd. (United Kingdom) except 2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3\u2009mL/min over a 25\u2009min period as follows: T\u2009=\u20090\u2009min, 5% B; T\u2009=\u20093\u2009min, 5% B; T\u2009=\u200918\u2009min, 95% B; T\u2009=\u200922\u2009min, 95% B; T\u2009=\u200923\u2009min, 5% B; and T\u2009=\u200925\u2009min, 5% B (mAU at 220\u2009nm). ESI-MS was performed on a Bruker AmaZon SL Ion Trap LC/MS spectrometer , and the data were analyzed using Bruker Daltonics DataAnalysis. ESI-HRMS analysis was carried out on a Bruker High Resolution Q-TOF mass spectrometry  and the data were analyzed using Bruker Daltonics DataAnalysis. Tandem MS analysis was performed by collision-induced dissociation (CID) using He as collision gas.HPLC analysis was carried out on a Thermo Fisher Dionex UltiMate 3,000 UHPLC system  using an Acclaim TM RSLC 120 C18 column  by gradient elution of solvent A  and purified by ethanol precipitation. The digested genomic DNA, p15A vector with homologous arms, T4 polymerase, and buffer were mixed and reacted in a thermocycler with the following program: 25\u00b0C, 60\u2009min; 75\u00b0C, 20\u2009min; 50\u00b0C, 60\u2009min. The mixture was then electroporated into the l-arabinose induced E. coli GB05-dir competent cells and plated on appropriate Luria-Bertani (LB)-agar plates to incubate at 37\u00b0C overnight. The bsf region (approximate 22.67\u2009kb) was cloned into the corresponding p15A-cm vectors in E. coli, and several constitutive promotors such as PTn5-Km, Papra, or the inducible promoter Ptet were subsequently inserted upstream to bsfC, respectively, leading to the constructions of p15A-cm-Tn5-km-bsf, p15A-cm-Papra-bsf, and p15A-cm-Ptet-km-bsf. Then, the plasmids were electroporated into the heterologous hosts E. coli BL21(DE3) or E. coli GB2005. In order to heterologous express the BGC in Burkholderiales hosts, the chloramphenicol resistance gene was then replaced with phiC31-site-specific integrase by recombineering, resulting the construction of p15A-phiC31-amp-Papra-bsf. This plasmid was then electroporated into Schlegelella brevitalea DSM 7029 or introduced into the specific chromosomal site of Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264 with the help of donor strain E. coli WM3064, which is auxotrophic for diaminopimelic acid , inserted into the BamHI-HindIII site of the plasmid pRSFDuet-1ATGGAAATGAAAAAAGCTAAAAACAAAATCTCTCGTAAACTGATCTACAAAAAACCGCAGCTGACCTACCACTCTACCCTGGCTGCTATCATCCGTGGTACCTCTGGTAACGAAGTTGACGGTTCTACCCCGGCTGACCGTGGTAACAACCCGGGTGACTAAbsfB1 (accession No. WP_162687378.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGCGTGAAACCCCGATGACCGAAAACCTGTTCCCGCGTGACGCTGTTTTCTCTATCCGTGAAGACCTGGTTGTTGAACAGGTTGACGACGAATTCCTGGTTCTGGACCTGCGTGGTAACGAATACTTCGGTCTGAACGCTGTTGCTCGTCACATCTGGGCTGCTATCGACGCTGGTGACTCTCTGGCTGCTATCGCTGACTCTGTTTGCGAACGTTTCGAAGTTGAACGTGAACGTGCTGCTACCGACGTTGCTGACTTCATCGCTAACCTGCTGGAACAGCGTCTGGTTTCTCGTGTTGACGCTTAAbsfB2 (accession No. WP_111331306.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGAAACCGATCCACCCGATCGCTCAGGCTGAAATGCTGGTTGAAGTTGCTATCGCTCGTGTTCTGTCTGAATTCCTGTCTATCGAAGACCTGCTGCGTCTGGTTGGTGAAGCTGCTCGTATCGCTCAGCAGTGGCCGGCTCGTTCTTGCGTTGCTTCTCCGTCTGCTGAAGCTATCAACGAAACCGCTGCTCTGTCTGAACTGCTGGCTCACCGTGCTTCTCGTCTGGTTCCGGGTGCTCAGTGCCTGCAGCGTGCTCTGGCTGGTCGTGTTTGGCTGGCTCGTCGTGGTATCGCTTCTGAAATCGTTGTTGGTTTCCGTAAACGTGGTGTTCTGGAAGGTCACGCTTGGCTGGAAGTTATGTCTCCGGACGGTCTGATCGAACTGTTCAACAACGCTGACGACGGTTACCGTGAATCTTTCCGTGAAGTTGCTGCTTAAbsfK (accession No. WP_204355063.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGGAAAACACCGACCGTCTGGTTCGTTACCGTGCTTTCGGTCGTGACATCGACGGTCCGGCTGGTCTGTCTCTGTCTCCGGCTCCGGCTGAAGCTGCTCCGGGTGAAGTTCCGGTTCTGCGTCTGCAGCCGGACGCTTCTCTGCGTGTTTTCATCGACGAAAACCTGCCGCCGCTGCTGCACAACGTTGAAGACTACCCGGGTGGTCCGAAATTCTACGTTTGGCAGGAAGGTGAAGCTGTTGGTGTTCAGTACGACCGTTGGCGTACCCGTCTGATCCCGGGTGAAGGTCGTATCGACTTCGCTGAACTGCCGCCGTCTGGTCCGAAACGTGTTGAAGACGCTGGTGACGAATACGGTCGTTTCCGTTTCTCTCTGGCTATGGAACGTGTTTTCCTGCCGCTGTACGCTCTGTTCTCTATGCCGGACGCTGTTGCTCTGCACGGTTCTGCTGTTGTTCTGAACGGTGAAGCTTTCCTGTTCATCGGTCGTTCTGGTGCTGGTAAATCTACCACCGCTTACGAATTCGTTCGTCGTGGTGCTACCCTGCTGGCTGACGACCTGATCGTTGCTGACGTTGCTCGTGGTATCGCTCTGGGTGGTGCTCCGACCCTGCGTCTGTGGAAAGGTGAAGGTGCTCTGCCGGAAGCTCAGGAAGACCGTTCTCTGTGGCGTCACGACGCTTCTAAACGTTGGTTCCGTATCCCGGCTGAACGTGGTGCTGCTTCTGCTGTTCCGATCGCTGCTATCGTTATGCTGGACCCGGACACCCTGGGTGGTCAGCGTGACGTTCTGCCGGGTCTGGAAACCTCTCCGCAGCGTAAAGCTCTGACCGACCTGCTGGGTCAGACCTTCGACCTGTCTCACGGTACCCCGGAATGGATGGTTGCTCGTTTCCGTAACACCGCTCGTCTGATCCGTGAATACCCGTTCTACCACTTCCGTTACGTTAAATCTGCTGACGGTAAACCGACCCACATGGACGCTCTGTACCAGGCTATCGTTGGTCTGGCTTCTAAATAAbsfK was amplified by PCR with pET28a\u2009+\u2009bsfK as the template. After purification by agarose gel electrophoresis and restrictive digestion, the bsfK fragment was inserted into the NdeI-XhoI site of the plasmid pRSFDuet-1\u2009+\u2009bsfA to yield the recombinant plasmid pRSFDuet-1\u2009+\u2009bsfA\u2009+\u2009bsfK.The DNA fragment containing bsfB1 or bsfB2 was amplified by PCR, respectively. The fragments were inserted into the NcoI-HindIII site of pACYCDuet-1 and the NdeI-XhoI site of pCold-TF to yield the recombinant plasmids pACYCDuet-1\u2009+ bsfB1 and pCold-TF\u2009+ bsfB2, respectively.The DNA fragment containing bsfA was transferred into E. coli BL21 (DE3) for expression. BsfA, fused to an N-terminal 6\u2009\u00d7\u2009His tag was expressed at 20\u00b0C for 24\u2009h with 100\u2009\u03bcM isopropyl-\u03b2-d-thiogalactopyranoside  induction and shaking at 200\u2009rpm. Cells were harvested by centrifugation at 4\u00b0C and re-suspended in buffer A containing 6 M guanidine hydrochloride, 20\u2009mM NaH2PO4 (pH 7.5), 500\u2009mM NaCl, 0.5\u2009mM imidazole. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000\u2009g and 4\u00b0C for 1\u2009h. The soluble fraction was subjected to purification using a Ni Bestarose FF column . Briefly, the column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with elution buffer containing a low concentration of imidazole (25\u2009mM) to remove the impurities. The sample was further eluted with elution buffer containing a high concentration of imidazole (1\u2009M) to obtain the purified BsfA peptide. The eluted fractions were desalted by reversed phase (RP) HPLC on a Shimadzu LC-20AT system  equipped with a ReproSil 300 C4 column . The solvent used for RP-HPLC separation was deionized water with 0.1% trifluoroacetic acid , and acetonitrile with 0.1% TFA (solvent B). A gradient was applied: 5% B for 15\u2009min, ramp up to 100% B over 50\u2009min, hold at 100% B for 7\u2009min, and ramp to 5% B over 5\u2009min. Eluted fractions were monitored by UV absorbance at 220\u2009nm. Desalted precursor peptides were lyophilized and stored at \u221220\u00b0C. The peptide was validated by HPLC-ESI-(HR)MS analysis on an Acclaim TM RSLC 120 C18 column  by gradient elution of solvent A (H2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3\u2009mL/min over a 25\u2009min period as follows: T\u2009=\u20090\u2009min, 5% B; T\u2009=\u20093\u2009min, 5% B; T\u2009=\u200918\u2009min, 95% B; T\u2009=\u200922\u2009min, 95% B; T\u2009=\u200923\u2009min, 5% B; and T\u2009=\u200925\u2009min, 5% B (mAU at 220\u2009nm).The plasmid pRSFDuet-1\u2009+\u2009bsfA\u2009+\u2009bsfK was electroporated solely or co-electroporated with pACYCDuet-1\u2009+\u2009bsfB1  into E. coli BL21 (DE3) for expression. The precursor peptides were purified, desalted, lyophilized, and validated according to the procedures described above for unmodified BsfA precursor peptide.The plasmid pRSFDuet-1\u2009+\u2009bsfK was transferred into E. coli BL21(DE3) and expressed according to the procedures described above for BsfA. Cells were harvested by centrifugation at 4\u00b0C and re-suspended in lysis buffer containing 50\u2009mM Tris\u2013HCl (pH 8.0), 300\u2009mM NaCl, 5\u2009mM imidazole and 10% (v/v) glycerol. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000\u2009g and 4\u00b0C for 1\u2009h. The soluble fraction was subjected to purification using a Ni Bestarose FF column. The column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with binding buffer. The sample was further eluted with elution buffer using a stepwise gradient containing different concentrations of imidazole (10\u2013500\u2009mM). The elution fractions were determined by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the fractions containing the recombinant protein were desalted using a PD-10 Desalting Column  into storage buffer containing 50\u2009mM Tris\u2013HCl (pH 8.0), 100\u2009mM NaCl, 10% (v/v) glycerol, and 1\u2009mM DTT. The resulting protein was concentrated and stored at \u221280\u00b0C. The purity of the protein was determined by SDS-PAGE analysis, and the concentration was determined by the Bradford assay using bovine serum albumin (BSA) as the standard.The plasmid pET28a\u2009+\u2009bsfB1 and pCold-TF\u2009+\u2009bsfB2 were transferred into E. coli BL21 (DE3) for expression, respectively. The expression temperature was 15\u00b0C for TF-BsfB2 instead of 20\u00b0C for BsfK and BsfB1. The proteins were purified to homogeneity, and concentrated according to the procedures described above for BsfK.The plasmids pET28a\u2009+\u2009bsfA or pRSFDuet-1\u2009+ bsfA\u2009+\u2009bsfK as the template. Then, 1\u2009\u03bcL of DpnI enzyme was added to the PCR system and incubated at 37\u00b0C for 3\u2009h to remove the template. And the PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting precursor peptide variants were expressed individually, or co-expressed with BsfK and BsfB1 in E. coli BL21 (DE3), and purified to homogeneity, desalted, and lyophilized according to the procedures described above for the native precursor peptides.Plasmids containing site-directed mutations were generated by PCR amplification with pRSFDuet-1\u2009+ bsfK as the template. Then, 1\u2009\u03bcL of DpnI enzyme was added to the PCR system and incubated at 37\u00b0C for 3\u2009h to remove the template. The PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting BsfK variants were expressed in E. coli BL21 (DE3), purified to homogeneity and desalted according to the procedures described above for the native protein.Plasmids containing site-directed mutations were generated by PCR amplification with pET28a\u2009+ 4HCO3 buffer (pH 7.8) to a concentration of 500\u2009\u03bcM. 20\u2009\u03bcg trypsin  was dissolved in 20\u2009\u03bcL resuspension buffer (50\u2009mM acetic acid) and heated at 30\u00b0C for 15\u2009min before utilization for maximum activity. 10\u2009\u03bcL resuspended trypsin was added to a total volume of 50\u2009\u03bcL BsfA reaction system, followed by incubation at 37\u00b0C for 2\u2009h. The reaction was quenched by adding an equal volume of CH3OH. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-HRMS analysis according to the procedure described above for BsfA precursor peptide.The (phosphorylated) precursor peptide BsfA (or the BsfA variants) was dissolved in 50\u2009mM NH2, 5\u2009mM ATP, 200\u2009\u03bcM BsfA  in the presence of 20\u2009\u03bcM BsfB1 and 20\u2009\u03bcM BsfK (or the BsfK variants). ATP was added last to initiate the reaction. The assays were quenched by adding an equal volume of CH3CN. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for BsfK  were performed at 30\u00b0C for 5\u2009h in 100\u2009mM Tris\u2013HCl buffer (pH 8.0) containing 5\u2009mM MgCl2HPO4 buffer (pH 7.5) containing 100\u2009mM NaCl, 2\u2009mM MgCl2, 2\u2009mM ATP, 1\u2009mM DTT, 100\u2009\u03bcM BsfA in the presence of 20\u2009\u03bcM BsfB1 and 20\u2009\u03bcM TF-BsfB2. ATP was added last to initiate the reaction. After similar termination and centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for TF-BsfB2  were performed at 30\u00b0C for 12\u2009h in 100\u2009mM Kbsf cluster is prone to be silent under laboratory conditions, and the Bradymonas sediminis FA350 strain is difficult to genetically manipulate. Thus, heterologous expression of the bsf cluster has been tried in other hosts. Briefly, the DNA fragment containing all the bsf genes and the flanking regions was removed from the genomic DNA by restriction digestion and further connected to the vector p15A via Red/ET recombination. The obtained plasmid (p15A-bsf) was then transformed into E. coli BL21(DE3), E. coli GB2005 and Schlegelella brevitalea DSM 7029, or conjugated into Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264, respectively, for heterologous expression in different culture media. However, no mass corresponding to the predicted lasso peptide has been observed. Moreover, several constitutive promotors such as PTn5-Km, Papra and the inducible promoter Ptet have been inserted upstream to bsfC, respectively, but the mature lasso peptide has never been detected. This results in difficulty to demarcate the boundary between the leader and the core peptide in BsfA. It was reported that the conserved Tyr(-17), Pro(-14), and Leu(-12) residues in leader peptides were required for the recognition by the B1 proteins , Microvenator marinus V1718 (mmv), Lujinxingia litoralis B210 (llb), L. sediminis SEH01 (lss) and L. vulgaris TMQ2 (lvt), et al.  motif  motif , indicatocessing . Neverthpeptides .N-terminally hexaHis-tagged BsfA (BamHI\u2009+\u2009HindIII) and untagged BsfK (NdeI\u2009+\u2009XhoI). After purification, high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (HPLC-ESI-HRMS) was used for the detection of PTMs in the N-terminally tagged BsfA. Apart from the dominant unmodified BsfA , a small component with a mass increase of 79.9640\u2009Da was also detected, which was barely visible when bsfK was omitted  with other proteins in the National Center for Biotechnology Information (NCBI) database.A BLASTP search using the BsfK protein sequence as input revealed that it was a hypothetical protein with a very low identity (<35%) with histidine containing phospho carrier protein (HPr) kinases . To vali omitted . This inNext, untagged BsfB1was introduced into the co-expression system above. In comparison with the BsfA and BsfK co-expression experiment , the monC-terminal Ser residues of the precursors , indicative of a distinct phosphorylated position from the known lasso peptides. Trypsin digestion and subsequent tandem MS analysis of phosphorylated precursor peptide pointed to the Ser3 residue as the modification site by BsfK . It was observed that while BsfA S3T was phosphorylated efficiently, no phosphorylation was observed with BsfA S3Y, surmising that BsfK is somewhat substrate tolerant . This fiN-terminally hexaHis-tagged BsfK was characterized in vitro in the presence of the BsfA-precursor, ATP and magnesium chloride. In accordance with the in vivo characterization, BsfK phosphorylated the precursor peptide in a very low yield, whereas no conversion was observed in the absence of ATP, magnesium chloride, or with heat-treated BsfK  tag , which was confirmed by MS/MS fragmentation analysis . A similar result was obtained when unphosphorylated precursor peptide was used instead of phosphorylated precursor. The mass of unphosphorylated BsfA core peptide  was detected and its identify was further verified by MS/MS analysis (Since the (TF) tag . HPLC-ES(TF) tag . The foranalysis . The latanalysis . These ranalysis , which iAcidobacteriaceae bacterium TAA 166  residue is also observed in a list of Sphingomonadales derived lasso peptide precursors I substitutions were still accepted as substrates from the processing machinery, albeit with lower turnover , 2014. I TAA 166 , and a n TAA 166 . Besidesecursors , suggestecursors . These aC-terminal serine residues were reported for ThcoK and SyanK (We were curious to know whether the leader peptide Ile(-2) residue in BsfA also plays a role in phosphorylation and leader peptide removal. This residue was targeted for replacement with other amino acids. Ile(-2) was first exchanged with the congener leucine residue in BsfA, which was then co-expressed with BsfB1 and BsfK. HPLC-ESI-HRMS analysis of the BsfA variant after purification and desalting revealed that only part of the precursor peptide was phosphorylated , comparend SyanK , whereasBradymonadales derived B1 proteins (The BsfA Ile(-2) substitution variants were furthermore utilized for the characterization of TF-BsfB2 activity. In contrast to the wild type BsfA that was cleaved almost completely, I(-2)T as well as I(-2)A exchanges completely hindered the removal of the leader peptide even in the presence of BsfB1 , 8B. In proteins . The intAla-replacements of Tyr(-17), Pro(-14), and Leu(-12) were also performed and tested for their effects on phosphorylation and leader peptide excision. The Y(-17)A and L(-12)A exchanges did not have any effect on the phosphorylation of BsfA, as the masses of unphosphorylated precursors are barely detected in HRMS analyses. Nevertheless, the P(-14)A variant precipitates immediately after addition to the BsfK assay, and no mass of the precursor is observed. Thus, double (Y(-17)A&P(-14)A) and triple (Y(-17)A&P(-14)A&L(-12)A) exchange variants of BsfA were generated. Surprisingly, neither the double nor the triple Ala substitution affects precursor phosphorylation . MoreoveAcidobacteriaceae and Sphingomonadales.Our results demonstrate that the kinase BsfK specifically catalyzes the phosphorylation of the Ser3 residue in BsfA, while BsfB1 performs dual functions to accelerate the post-translational phosphorylation and to assist BsfB2 in leader peptide removal. Moreover, it is noteworthy that the penultimate residue in the leader peptide is isoleucine rather than the conserved threonine in all other so far investigated lasso peptide precursors. This Ile(-2) residue has a profound effect on the monophosphorylation of Ser3 and leader peptide removal, both of which are decreased in Ile(-2) substitution variants. The presence of a penultimate Ile/Val residue was also observed in putative lasso peptide precursors from Paenibacillus dendritiformis C454, ThcoB2 from Thermobacillus composti KWC4, BaceB2 from Bacillus cereus VD115, PapoB2 from Paenibacillus polymyxa CR1, SyanB2 from Sphingobium yanoikuyae ATCC 51230 and PsmB2 from Bacillus pseudomycoides DSM 12442 are the only B2 proteins reported to be involved in the biosynthesis of phosphorylated lasso peptides  can be found in the article/in-silico analyses. D-SM and Z-JD assisted with data analysis and manuscript preparation. GZ and YD analyzed the data and wrote the manuscript. GZ, XB, and YZ designed the work. All authors contributed to the article and approved the submitted version.YD carried out the experiments. YD, WN, and LP performed the This work was supported by the National Natural Science Foundation of China (21907057 and 32170038), the Natural Science Foundation of Jiangsu Province, China (BK20190201), the Future Plan for Young Scholars, and the Fundamental Research Funds (2019GN032) of Shandong University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Numerous circular RNAs (circRNAs) have been recently identified in porcine tissues and cell types. Nevertheless, their significance in porcine spleen development is yet unelucidated. Herein, we reported an extensive overlook of circRNA expression profile during spleen development in Meishan pigs.CD226, MBD2, SAMD3, SIT1, SRP14, SYTL3 gene expressions via acting as miRNA sponges. Moreover, the circRNA_21767/miR-202-3p axis regulated SIT1 expression in a ceRNA manner, which is critical for the immune-based regulation of spleen development in Meishan pigs.Overall, 39,641 circRNAs were identified from 6,914 host genes. Among them, many circRNAs are up- or down-regulated at different time points of pig spleen development. Using WGCNA analysis, we revealed two essential modules for protein-coding genes and circRNAs. Subsequent correlation analysis revealed 67 circRNAs/co-expressed genes that participated in immnue-associated networks. Furthermore, a competing endogenous RNA (ceRNA) network analysis of circRNAs revealed that 12 circRNAs modulated Overall, our results demonstrated that the circRNAs were differentially expressed during different stages of porcine spleen development, meanwhile the circRNAs interacted with immune-related genes in a ceRNA-based fashion. Moreover, we presented biomedical researchers with RNAseqTools, a user-friendly and powerful software for the visualization of transcriptome profile data.The online version contains supplementary material available at 10.1186/s12864-023-09612-x. Sus scrofa) are an incredible resource for agriculture and food production, as well as biomedical research for the examination of human development, congenital diseases, and pathogen response networks [Pigs  is a newly discovered form of non-coding RNA (ncRNA) known for its enclosed configuration, generated via precursor mRNA back-splicing . In contSpleen is a secondary lymphoid organ involved in older erythrocyte elimination, iron recycle, capture and destruction of pathogens, and induction of adaptive immune responses , 18. BeiTo explore the presence of circRNAs during spleen development, we assessed circRNAs expression in the spleen tissues of Meishan pigs at various developmental stage. We prepared and sequenced ribo-depleted total RNA-seq libraries, as shown in the flow chart Fig.\u00a0. Table STo characterize the circRNAs in the Meishan pig spleen tissues, we evaluated their sequences. In all, 39,641 candidate circRNAs were identified from 6,914 host genes, using\u2009\u2265\u20091 unique back-spliced read Fig.\u00a0A. This iEmerging evidences support the presence of DE circRNAs during porcine skeletal muscle development , howeverp\u2009=\u20092e-08). The heatmap shows the expression pattern of red module, where the expression of genes in the red module tends to increase with time  . As depicted in Fig. 05) Fig. C. Moreov05) Fig. D. TherefBased on the above results, we identified 36 circRNAs in the red module, and 508 mRNAs in the darkturquoise module. We performed correlation analysis on the circRNAs/mRNAs co-expression  using the Shiny framework  and immunomodulation of several genes were significantly enriched. Cell adhesion molecules, which express on the cell surface, facilitate activities of several biological processes, including, immune response, inflammation, and embryogenesis [SIT1 was strongly modulated by the circRNA_21767/miR-202-3p axis. Based on a prior investigation, signaling threshold regulating transmembrane adaptor 1 (SIT1), encoded by the SIT1 gene, is a disulfide-linked homodimeric glycoprotein that interacts with the lymphocyte-specific transmembrane adaptor protein family to modulate the immunologic process [CircRNAs are known to modulate numerous physiological processes using distinct signaling networks . Althougogenesis . CD226 iogenesis . Herein, process . It is iSIT1, which may modulate immune activities. Our findings provide candidate ncRNA-based targets for future in vivo animal study on the molecular regulation of immune function within the porcine spleen. Finally, we designed a user-friendly software named RNAseqTools that is available for use for the visualization of transcriptomic data using the Shiny app (https://github.com/ChaoXu1997/RNAseqTool).In conclusion, this study presented the first reported catalog of circRNAs expression profile in the spleen tissues from Meishan pigs. We characterized numerous DE circRNAs that were relevant to the porcine spleen development. We also generated a comprehensive expression profile of various RNAs, particularly mRNAs and circRNAs, which, together, modulate a myriad of physiological processes within the Meishan pig spleen. Moreover, we identified a ceRNA network involving circRNA_21767/miR-202-3p/Overall, 24 healthy Meishan pigs were acquired from the Kunshan Conservation Ltd.  (Permission No. JS-C-05). Our examination involved 8 developmental stages, namely, 1d, 7d, 14d, 21d, 28d, 35d, 120d, and 180d after birth. All piglets were provided with the same standard diet, with housing at an environmentally regulated facility. Three piglets were sacrificed per treatment via an intravenous administration of pentobarbital sodium to minimize suffering. The spleen was immediately extracted and frozen in liquid nitrogen. Our animal care and treatment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University (No. SYXK(Su)2021-0026).Splenic total RNA extraction employed TRIZOL  following kit directions. RNA quality assessment employed an Agilent 2100 Bioanalyzer . Ribosomal RNA elimination from extracted RNA utilized the Ribozero\u2122 rRNA Removal Kit . Subsequently, linear RNA was eliminated via an RNAse R kit . Lastly, sequencing libraries were generated with rRNA-free and linear RNA-free RNA using the NEBNext\u00ae Ultra\u2122 Directional RNA Library Prep Kit .Sscrofa11.1). We uploaded the resulting RNA-seq information in the Gene Expression Omnibus (accession codes GSE228936). To construct the SAM file, we employed BWA [Sscrofa11.1 genome.Sequencing of generated libraries was done via the Illumina HiSeq 2500 platform . TopHat2 (v2.1.1) mapped the clear data against the porcine reference genome  website, and the mRNA 3\u2019UTR sequences from BioMart  [We retrieved the miRNA sequences from the miRbase (er 2022) . To expler 2022)  predicteer 2022)  to examiWe performed the GO term and KEGG pathway \u201349 enricGAPDH (for mRNA and circRNA) and U6 (for miRNA) served as controls for normalization purpose, and all qRT-PCR reactions were performed three times, and the average adjusted value was used for analysis. Relative gene expressions were computed using the 2\u2212\u0394\u0394Ct formula. All data analysis employed the unpaired 2-tailed Student\u2019s t-tests; *p\u2009<\u20090.05, **p\u2009<\u20090.01, and data are provided as mean\u2009\u00b1\u2009SD (standard error of mean).qPCR employed an Applied Biosystems qPCR apparatus and SYBR green master mix , as per kit protocols. The reaction mixture was composed of 5\u00a0\u00b5l SYBR Green Mixture , 1\u00a0\u00b5l cDNA template, 0.2\u00a0\u00b5l primer (each), and 3.6\u00a0\u00b5l deionised water, and the conditions were set as follows: 95\u00a0\u00b0C for 5\u00a0min, 40 cycles of 95\u00a0\u00b0C for 10\u00a0s, and 60\u00a0\u00b0C for 30\u00a0s. All employed primer sequences were summarized in Table SIT1-3\u2019UTR or circRNA_21767 downstream of the pmirGLO Dual-Luciferase vector. Next, we plated cells into a 24-well plates, and co-transfected the cells with the WT (SIT1-WT or circRNA_21767-WT), or Mut , and miR-202-3p-mimics (NC-mimics). Finally, we detected both firefly and renilla bioluminescence using the DLR Gene Assay Kit . The firefly luciferase activity was then adjusted with the control renilla luciferase activity for a measurement of the constructed reporter luciferase activity.We inserted wild-type (WT) or mutant-type (Mut) SIT1-WT:TCTGTGCTGACGCCTCAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGSIT1-Mut:CTCACATCGGTATTCTAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGcircRNA_21767-WT:CCCAGAATCTTTTAGTCCTGCCCTACAGCTGCACCTCGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTAcircRNA_21767-Mut:TTTGGAATCTTTTAGTCCCATTCTACAGCCATGTTCTGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTABelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2"}
+{"text": "C. elegansvulva is a polarized epithelial tube that has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis. Here we used endogenous fusions to show that the spectrin cytoskeleton is polarized in this organ, with conventional beta-spectrin (UNC-70) found only at basolateral membranes and beta heavy spectrin (SMA-1) found only at apical membranes. The sole alpha-spectrin (SPC-1) is present at both locations but requiresSMA-1for its apical localization. Thus, beta spectrins are excellent markers for vulva cell membranes and polarity.The There, spectrins serve as linkers to connect plasma membranes and transmembrane proteins to the actin cytoskeleton . They play numerous roles in membrane and cytoskeletal organization and stability, tissue mechanics, and morphogenesis The spectrin cytoskeleton lines the cytoplasmic sides of cell plasma membranes . Vertebrates have multiple \u03b1-spectrin and \u03b2-spectrin genes. Invertebrates such asDrosophila melanogasterandCaenorhabditis elegans have just one \u03b1-spectrin and two \u03b2-spectrin genes, a conventional \u03b2-spectrin and a larger or \u201cheavy\u201d \u03b2-spectrin (\u03b2H).C. elegans\u03b1-spectrin is encoded byspc-1, and \u03b2-spectrin and \u03b2H-spectrin are encoded byunc-70andsma-1, respectively .SPC-1functions with eitherUNC-70orSMA-1to control different processes. In general,UNC-70is widely expressed and plays key roles in neuron and muscle structure , whileSMA-1is expressed more specifically in epithelial cells and affects tissue morphogenesis . We also discovered a role forSMA-1in blastomere cytokinesis .Spectrins exist as tetramers composed of two dimers, each with an alpha (\u03b1) and beta (\u03b2) subunit . InC. elegans, immunolocalization and/or transgenic reporter studies in the embryo detectedUNC-70/\u03b2-spectrin at many sites of cell-cell contact butSMA-1/\u03b2H-spectrin only at apical membranes , suggesting that apical vs. basal partitioning of different \u03b2-spectrins is conserved; however, there has been limited analysis of spectrin polarity in larval or adult epitheliaInC. elegansvulva. The vulva is a polarized epithelial tube used for egg-laying. It has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis . Vulva anatomy is well characterized and apical vs. basal cell surfaces can be distinguished easily using simple light microscopy of live animals.Here we visualized the spectrin cytoskeleton in theSPC-1/\u03b1-spectrin andUNC-70/\u03b2-spectrin have been reported previously . We used CRISPR/Cas9 to tag the endogenousSMA-1/\u03b2H-spectrin protein with GFP (Methods) and then examined the localization patterns of all three fusions in the developing vulva. At the mid-L4 larval stage, vulva cells are organized into a series of 7 stacked rings (named vulA-vulF), surrounding a central lumen cavity .SPC-1::GFP outlined all 7 rings   . Thus, .2020) '.SMA-1/SPC-1tetramer formation, we found thatSMA-1/\u03b2H-spectrin is needed to localizeSPC-1/\u03b1-spectrin to apical membranes. In allsma-1(ru18)null mutants examined,SPC-1::GFP failed to localize to apical membranes and was found only at basolateral membranes .The similarly polarized distributions of \u03b2-spectrin and \u03b2H-spectrin inet al.1999). These rings adopt specific shapes and undergo stereotypical movements during morphogenesis. The vulva also must connect appropriately with the uterus to allow the passage of eggs, and with the sex muscles and neurons that control egg-laying . Many of these cell behaviors depend on the actin cytoskeleton and/or on interactions with apical or basal extracellular matrices . We observed a variety of vulva shape abnormalities insma-1mutants  and the panels assembled with Adobe Illustrator.SMA-1with GFP was performed by Suny Biotech. The tag is inserted at theSMA-1C-terminus, immediately preceding the stop codon, as shown below. This endogenous fusion is functional based on normal body morphology, brood size and embryonic viability of the homozygotes.CRISPR/Cas9-mediated genome editing to tag endogenous1) Wild type sequence:TAGatacctcaccacacgctgatcttcataTTATTCAAGCGTGGATCCAAACATTCAAAG*sma-1; asterisk is where the GFP was insertedBold TAG is the stop codon ofsma-1(syb4954[SMA-1::GFP])V2) Precise sequence knock-in,CTGTTCAAGCGTGGATCCAAACATTCAAAGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGatacctcaccacacgctgatcttcataBold italics indicate silent mutations; underline indicates the GFP sequence.Strains used:GOU2043vab-10(cas602[VAB-10a::GFP])IGOU2936spc-1(cas815[SPC-1::GFP])Xunc-70(cas962[UNC-70::GFP)]VGOU3103sma-1(syb4954[SMA-1::GFP])VPHX4954sma-1(ru18) V;spc-1(cas815[SPC-1::GFP])XUP4241vab-10(cas602[VAB-10a::GFP]) I;sma-1(ru18)VUP4252"}
+{"text": "Insults to the developing cerebellum can cause motor, language, and social deficits. Here, we investigate whether developmental insults to different cerebellar neurons constrain the ability to acquire cerebellar-dependent behaviors. We perturb cerebellar cortical or nuclei neuron function by eliminating glutamatergic neurotransmission during development, and then we measure motor and social behaviors in early postnatal and adult mice. Altering cortical and nuclei neurons impacts postnatal motor control and social vocalizations. Normalizing neurotransmission in cortical neurons but not nuclei neurons restores social behaviors while the motor deficits remain impaired in adults. In contrast, manipulating only a subset of nuclei neurons leaves social behaviors intact but leads to early motor deficits that are restored by adulthood. Our data uncover that glutamatergic neurotransmission from cerebellar cortical and nuclei neurons differentially control the acquisition of motor and social behaviors, and that the brain can compensate for some but not all perturbations to the developing cerebellum. Cerebellar injury in early life can impair the development of motor and social behaviors. Here, the authors show that cerebellar cell types are differentially important for the acquisition of these behaviors in pups and adult mice. Preterm injuries affect the exponential phase of granule cell proliferation, although they often also alter the early development of all glutamatergic neurons in the cerebellum. The resulting defects lead to long-term changes in gray matter volume in the cerebellar cortex that are correlated to the severity of neural deficits in infants6. Cerebellar cortical injuries further impact the development and function of downstream cerebellar nuclei neurons, which serve as the predominant output from the cerebellum and link it to the rest of the motor network7. Accordingly, cerebellar defects can also impair the development and function of the cerebral cortex9. The combined injury to the cerebellum and neocortex may help explain the broad neural deficits observed in many infants that experience cerebellar injury during the perinatal period.Cerebellar injury in preterm infants is often associated with movement disorders, language impairments, and social deficits11. Intriguingly, over time, patients largely recover these impaired neural functions with only minor residual symptoms, most commonly persisting in their motor coordination13. Patients and apes with lesions localized to the cerebellar nuclei mainly demonstrate motor symptoms with limited recovery following the injury15. When the cerebellar cortex is the primary site of the lesion, deficits in social cognition, language, and motor performance arise, but they often persist following the initial injury with limited recovery, especially in the non-motor domains affecting cognition, sociability, language, and affect2. Similarly, studies in rodents have provided compelling evidence that disrupting cerebellar cortical function during development can lead to motor impairments, altered vocalizations, and social deficits21.Accumulating evidence suggests that the site of injury within the developing cerebellum may determine behavioral outcomes. When damage is confined to the cerebellar nuclei neuron axons that project and travel through the superior cerebellar peduncle, affected patients  can develop posterior fossa syndrome, which is hallmarked by ataxia, mutism, and changes in social interactionsThese clinical outcomes illustrate that while damage to the cerebellar cortex or to the downstream cerebellar nuclei neurons during infancy is sufficient to impair motor function, language, and social behavior, there are instances when the cerebellum can remarkably overcome perturbations and restore functions. Importantly, the degree of compensation may be linked to the cerebellar neurons that are primarily affected by the lesion, suggesting a unique role for each cerebellar neural subtype in the regulation of cerebellar-associated behaviors. These studies have inspired the need for a deeper examination of how the relatively few neuron types in the cerebellum contribute to a wide range of motor and non-motor functions. However, it remains largely unknown where in the circuit cerebellar-associated behaviors originate, whether the same neuronal subtypes contribute equally to the acquisition of these diverse behaviors, and whether the perturbation of all neuronal subtypes can be equally compensated for during development. Additionally, it remains specifically unexplored how the cerebellar nuclei contribute to the acquisition of different behaviors during postnatal life.22. The unique identities of the lineages can be used to manipulate GABAergic or glutamatergic cerebellar neurons. The Ptf1a lineage that originates in the ventricular zone gives rise to GABAergic neurons, including GABAergic cerebellar nuclei neurons and Purkinje cells23. In contrast, the Atoh1 lineage is derived from the rhombic lip and gives rise to glutamatergic neurons, including glutamatergic granule cells and cerebellar nuclei neurons  to eliminate the transport of glutamate into the synaptic vesicles of Atoh1 lineage neurons. This manipulation results in an effective silencing of fast neurotransmission in the genetically targeted neurons  from the Atoh1 lineage, which resulted in a lack of VGluT2 protein in pre-synaptic vesicles of the Atoh1 lineage, Vglut2-expressing neurons33. As a result, when an action potential arrives at the synapse, synaptic vesicles fuse to the pre-synaptic membrane but do not functionally affect the postsynaptic cells because no neurotransmitter is released into the synaptic cleft , and therefore the effects of our genetic manipulation do not actively and directly continue through adulthood. Unlike Atoh1 null and cerebellum-specific Atoh1 conditional knockout mice32, Atoh1Cre/+;Vglut2fl/fl mice were viable and survived into adulthood, likely because Atoh1Cre/+;Vglut2fl/fl mice had normal respiratory rhythms , but no Vglut2 expression was found in the granule cell layer   assays in adult animals showed that nearly all cerebellar nuclei neurons from the yer Fig.\u00a0. We vali01) Fig.\u00a0. Immunohtex Fig.\u00a0. We visuice Fig.\u00a0 since, aAtoh1Cre/+;Vglut2fl/fl conditional knockout mice have no major differences in zonal stripe patterning of VGluT2+ mossy fiber synapses in the anterior or posterior zones of the cerebellum, regions into which the spinocerebellar projections are targeted  and regularity (CV2). In line with our hypothesis, we found that loss of Vglut2 from the Atoh1 lineage resulted in a reduction of simple spike firing rate  to the prelimbic area in the medial prefrontal cortex36. Other studies have also suggested that these long-range projections from glutamatergic cerebellar nuclei neurons may mediate non-motor behaviors49. We therefore tested whether the Vglut2-expressing, Atoh1 lineage neurons make long-range projections to areas previously implicated in non-motor behavior. To investigate the complete population of neurons that were affected by our manipulation, we used an intersectional reporter allele (Rosafsf-lsl-tdTomato) that expresses tdTomato only after FlpO- (Atoh1FlpO/+) and Cre- (Vglut2IRES-Cre) mediated excision of two stop cassettes. In these mice, only the neurons with a history of Atoh1 and Vglut2 expression express tdTomato VPM Fig.\u00a0 and the VPM Fig.\u00a0. We alsoVPM Fig.\u00a0 and red VPM Fig.\u00a0. Furtherlei Fig.\u00a0. We alsoons Fig.\u00a0. We provAtoh1Cre/+;Vglut2fl/fl mice , nearly three-quarters of Vglut2+ neurons in the interposed nucleus were also YFP+ (73.7\u2009\u00b1\u20091.3%), and around a sixth of Vglut2+ neurons in the fastigial nucleus were also YFP+ (16.5\u2009\u00b1\u20091.6%). Overall, around half of all Vglut2+ cerebellar nuclei neurons were also YFP+ (52.5\u2009\u00b1\u20091.1%)  Fig.\u00a0.Ntsr1Cre-expressing nuclei neurons send long range projections to areas associated with motor and non-motor behaviors. To this end, we crossed the Ntsr1Cre mice to Atoh1FlpO/+;Rosafsf-lsl-tdTomato mice to obtain mice that expressed tdTomato solely in the Ntsr1Cre expressing cerebellar nuclei neurons , may affect adult behaviors. We found that compared to control mice, adult Ntsr1Cre/+;Vglut2fl/fl mice had no deficits in ambulatory activity  and specific (only the glutamatergic transporter Vglut2 is deleted). We have used the elimination of vesicular transporters to study the contribution of specific circuit components in several previous studies. In these studies, we did not detect changes in synaptic connectivity or developmental compensation that overcome the functional elimination of neurotransmission72. Therefore, we propose that our genetic methods allow for testing how synaptic VGluT2-dependent neurotransmission only in neurons within the intersectional domain influences circuit function and mouse behavior. Our study confirms that Atoh1 lineage cerebellar nuclei neurons are necessary for the refinement of motor behavior, and we also unveil that Atoh1 granule cells contribute to social vocalizations during postnatal development.Our genetic silencing approach provides a compelling in vivo demonstration of how the function of This study provides in vivo experimental evidence showing that glutamatergic neurons in the cerebellum are critical for the acquisition of motor function and social behaviors. Specifically, the glutamatergic neurons in the cerebellar cortex and nuclei are differentially required for the early postnatal development of motor behavior and social behaviors. These data also raise the possibility that parallel signaling from the GABAergic nuclei neurons may provide some compensation for functional lesions of the glutamatergic nuclei neurons during development, leading to improvements in early motor and social deficits. By leveraging the ability to restore cerebellar-dependent behaviors after normalizing cerebellar cortical function, tapping into cerebellar neurons that communicate social and motor signals to extra-cerebellar regions may provide an ideal therapeutic target to restore motor and non-motor functions after developmental\u00a0brain injury and disease.Ai14 ;73Ai32 ;74Ai65 ;74Atoh1Cre;75Atoh1FlpO (JAX: 036541);32Ntsr1Cre (MMRRC: 030648);57Vglut2IRES-Cre (JAX: 028863);76Vglut2fl (JAX: 012898)72. We used ear clippings from pre-weaned pups for genotyping and identification of transgenic alleles. Mice of both sexes were used in all experiments. We considered the day the pups were born as postnatal day 0 (P0). We did not observe gross differences in weight between control and Atoh1Cre;Vglut2fl/fl conditional knockout mice . The ages of the adult mice were between two and fourteen months old. All mice were kept under a 14\u2009h/10\u2009h light/dark cycle, a daily temperature between 68 and 72\u2009F, and humidity between 30 and 70%.All mice included in the experiments for this manuscript were housed in a Level 3, AALAS-certified facility. The Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine (BCM) reviewed and approved all studies that involved mice. We used the following mice for our experiments: 29. We anesthetized mice with Avertin and tested them for effective anesthesia by toe or tail pinch. Analgesia was also provided. We then accessed the chest cavity and penetrated the heart with a small butterfly needle for perfusion. We first perfused with 1\u2009M phosphate-buffered saline (PBS pH 7.4) to flush out blood until the liver turned clear. Next, we perfused the mouse body with 4% paraformaldehyde (PFA) to fix the tissue. After the tail and hind paws were stiff from fixation, we decapitated the mouse and dissected the brain from the skull or spinal cord from the spinal column. We post-fixed brain and spinal cord tissue in 4% PFA overnight or until the tissue was used for cryoprotection. We cryoprotected the tissue by serial sucrose gradients (15% \u2192 30% sucrose in PBS) at 4\u2009\u00b0C, each step until the tissue sinks. Finally, we froze the tissue in an optimal cutting temperature (OCT) solution and stored it at \u221280\u2009\u00b0C. Brain sections were cut into 40\u2009\u00b5m free-floating tissue sections, and spinal cord sections were cut into 25\u2009\u00b5m sections on the slide. Cut sections were stored at 4\u2009\u00b0C until it was used for immunohistochemistry (tissue was stored for a maximum of two months). Tissues from mice expressing the tdTomato allele were stored in aluminum foil at all steps to prevent photobleaching.We collected brain and spinal cord tissue for analyses as previously described29. We blocked tissue sections in 500\u2009\u03bcL 10% normal goat or normal donkey serum in 0.1% Triton-X in PBS (PBS-T) for 2\u2009h. We then incubated tissue sections overnight at room temperature in 500\u2009\u03bcL blocking solution with primary antibodies at desired concentrations. After washing the tissue sections three times in PBS-T, we incubated the tissue for two hours in 500\u2009\u03bcL PBS-T with secondary antibodies at desired concentrations. Subsequently, we incubated tissue sections with DAPI  in PBS for 10\u2009min. After a final two washes, we mounted the sections using VECTASHIELD Vibrance\u00ae mounting medium . All mounted slides were stored at 4\u2009\u00b0C until they were imaged. We used the following primary antibodies: guinea-pig (gp)-anti-VGluT2 , rabbit (rb)-anti-VGluT1 , rb-anti-Parvalbumin (PV) , rb-anti-Neurogranin (NG) , rb-anti-carbonic anhydrase 8 (Car8) , and sh-anti-tyrosine hydroxylase (TH) . We used the following secondary antibodies which were conjugated to an Alexa-488 fluorophore: goat-anti-gp , goat-anti-rb , or goat-anti-sh .We stained free-floating tissue sections as previously describedVglut2, Vglut1, YFP, and tdTomato expression in unfixed, fresh frozen tissue cut in 25\u2009\u03bcm-thick coronal brain sections. Digoxigenin (DIG)-labeled mRNA antisense probes against Vglut2, Vglut1, YFP, and tdTomato were generated using an RNA DIG-labeling kit from Roche. The specific sequences of the antisense probes that we used were as follows:ISH was performed by the RNA ISH Core at Baylor College of Medicine using an automated robotic platform. ISH was used to visualize Vglut2: GGTGCTGGAGAAGAAGCAGGACAACCGAGAGACCATCGAGCTGACAGAGGACGGTAAGCCCCTGGAGGTGCCTGAGAAGAAGGCTCCGCTATGCGACTGCACGTGCTTCGGCCTGCCGCGCCGCTACATCATAGCCATCATGAGCGGCCTCGGCTTCTGCATATCCTTCGGCATCCGCTGTAACCTGGGCGTGGCCATCGTGGACATGGTCAACAACAGCACTATCCACCGCGGAGGCAAAGTTATCAAGGAGVglut1: CAGAGCCGGAGGAGATGAGCGAGGAGAAGTGTGGCTTTGTTGGCCACGACCAGCTGGCTGGCAGTGACGAAAGTGAAATGGAGGACGAGGCTGAGCCCCCAGGGGCGCCCCCCGCGCCGCCTCCGTCCTACGGGGCCACACACAGCACAGTGCAGCCTCCGAGGCCCCCGCCCCCTGTCCGGGACTACTGACCACGGGCCTCCCACTGTGGGGCAGTTTCCAGGACTTCCACTCCATACACCTCTAGCCTGAGCGGCAGTGTCGAGGAACCCCACTCCTCCCCTGCCTCAGGCTTAAGATGCAAGTCCTCCCTTGTTCCCAGTGCTGTCCGACCAGCCCTCTTTCCCTCTCAACTGCCTCCTGCGGGGGGTGAAGCTGCACACTAGCAGTTTCAAGGATACCCAGACTCCCCTGAAAGTCGTTCTCCGCTTGTTTCTGCCTGTGTGGGCTCAAATCTCCCCTTTGAGGGCTTTATTTGGAGGGACAGTTCAACCTCTTCCTCTCTTGTGGTTTTGAGGTTTCACCCCTTCCCCCAAGACCCCAGGGATTCTCAGGCTACCCCGAGATTATTCAGGTGGTCCCCTACTCAGAAGACTTCATGGTCGTCCTCTATTAGTTTCAAGGCTCGCCTAACCAATTCTACATTTTTCCAAGCTGGTTTAACCTAACCACCAATGCCGCCGTTCCCAGGACTGATTCTCACCAGCGTTTCTGAGGGAYFP: AGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGtdTomato: ATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAANissl staining was performed with the tissue sections mounted and dried overnight on the slides. The next day, the mounted sections were immersed in 100% xylene two times for five minutes each time. Subsequently, they were placed through a rehydration series of 3 immersions in 100% ethanol, followed by 95% ethanol, 70% ethanol, and tap water, with each step lasting 2\u2009min. Afterward, the sections were immersed in cresyl violet solution for approximately two minutes. They were then dehydrated following a reversed order of the rehydration series and followed by a final immersion in xylene, with each step lasting up to one minute. Coverslips were mounted on the slides immediately after using Cytoseal XYL mounting media .We acquired photomicrographs of cerebellar sections using a Leica DM4000 B LED microscope with a DPX365FX camera or using a Zeiss Axio Imager.M2 microscope with an AxioCam MRc5 camera. We stitched the whole mount images for the intersectional lineage tracing together using the Adobe Photoshop Photomerge function. We adjusted color brightness and balance using ImageJ software, and we cropped the images to the desired size for figures using Adobe Illustrator.77. Prior to surgery, mice were anesthetized using a mixture of ketamine (80\u2009mg/kg) and dexmedetomidine (16\u2009mg/kg). During the surgery, the mouse was placed on a heated surgery pad and received additional isoflurane (1%) when necessary. The mouse\u2019s head was stabilized by ear bars in a stereotaxic surgery rig. When the mouse was fully anesthetized, we removed the hair from the head and made an incision in the skin over the anterior part of the skull. Next, we used a dental drill to make a large craniotomy over the cerebellar cortex. The craniotomy spanned from around bregma \u22125.60\u2009mm to \u22126.64\u2009mm, and from around 0.5\u20133\u2009mm lateral to bregma. Purkinje cells across several lobules were randomly sampled from this craniotomy, preventing any potential bias from local activity patterns  to acquire breath waveforms and used custom-written MATLAB (Mathworks) code to derive Tidal Volume, Respiratory Frequency, and Minute Volume parameters.We tested respiratory parameters in room air as described in our previous publication47. Mice were placed head down on a negative incline (\u221235\u00b0) that was covered with a sterile Poly-Lined drape. We measured the time until mice turned 90\u00b0 in either direction. Mice were tested three times at each time point. We suspended the test if mice did not turn within 60\u2009s or fell down the slope. We recorded the falls that occurred within 60\u2009s.We tested the negative geotaxis reflex at P7, P9, and P11 as previously described and shown47. Mice were placed in the supine position in an empty, clean cage. We measured the time until the mice turned onto their four paws. Mice were tested three times at each time point. We suspended the test if mice did not turn within 1\u2009min (60\u2009s).We tested the surface righting reflex at P7, P9, and P11, as previously described and shownAtoh1Cre/+;Vglut2fl/fl mice and littermate controls, vocalizations were recorded using a CM16 microphone (Avisoft Bioacoustics). Sound was amplified and digitized using UltraSoundGate 416H at a 250\u2009kHz sampling rate, and bit depth of 16 while Avisoft RECORDER software was used to collect the recordings. For Ntsr1Cre;Vglut2fl/fl mice and littermate controls, we measured vocalizations using a Noldus microphone and UltraVox XT software. Due to minimal congruency between the number of vocalizations recorded between these two recording systems79, we only compared mutants to control littermates whose vocalizations were measured using the same system.We measured pup ultrasonic vocalizations in a social isolation task at P7, P9, and P11. The ultrasonic vocalizations of each animal were monitored for 2\u2009min in a sound-attenuating chamber (Med Associates Inc.). For We measured open-field activity using automated Fusion software. Mice were placed in the center of an open field (40x40x30 cm chamber) that has photo beams for detecting horizontal and vertical activity. The chamber was placed in a room with the light set to 200\u2009lux and ambient white noise to 60\u2009dB. Each mouse was allowed to explore the chamber for 30\u2009min.81 to assess the sociability of adult mice. The apparatus consisted of three chambers\u2014a center chamber with doorways to two side chambers, all of equal dimensions . For three days prior to testing, the age- and sex-matched mice to be used as partners for Atoh1Cre/+;Vglut2fl/fl, Ntsr1Cre;Vglut2fl/fl, and their control littermates during the social interaction test were placed under a wire cup for 1\u2009h each day. The assay consisted of a 10-min habituation session followed by a 10-min test phase performed in dim lighting conditions (15\u2009lux). During the habituation session, the test subject was placed in the center chamber and allowed to freely explore for 10\u2009min. Once the subject returned to the center chamber, the doorways to the side chambers were blocked with plexiglass walls. Before the test phase starts, a novel partner mouse (pre-habituated to the apparatus 3 days prior) is placed under a wire cup in one side chamber, while a novel object (Lego block of similar size and color) is placed under a wire cup in the other side chamber. The plexiglass walls covering the doorways are then lifted, and the test session begins as the test subject is allowed to freely investigate all three chambers for 10\u2009min. The amount of time spent interacting with either the novel object or novel mouse during the two phases was scored manually by an experimenter blinded to genotype during the assay using ANY-Maze. Additional activity data that was acquired from each chamber during the two sessions was calculated automatically using ANY-Maze. The placement of the novel mouse and novel object within the side chambers was randomized to prevent chamber bias.We used the three-chamber test53. As is standard for this task, mice were placed on an accelerating rod on which they locomote according to the motion of the rod (4\u201340\u2009rpm over 5\u2009min) . Time was stopped and noted when one of three events occurred; the mouse fell off, the mouse made two consecutive rotations while hanging on the rod without walking, or the mouse successfully stayed on the rod for the total duration of the trial (5\u2009min). We recorded the trial duration for four trials per day for three consecutive days.We assessed rotarod performance using a previously established protocol51. The tremor monitor consists of a lightweight plastic container that is suspended in the air by eight elastic cords, one on each corner. The container is large enough for mice to move around and explore freely, providing us with readings related to movement. An accelerometer is mounted onto the underside of the container, where it functions to convert the detected movements into an electrical signal that is digitized using a Brownlee amplifier and records using Spike2 software. We also used the Spike2 software to detect the tremor power using a Fourier transform analysis based on two full minutes of mouse movements. For the recordings, mice were placed in the tremor monitor and were allowed to acclimate for three minutes. After this period, the recordings for analysis were initiated once the animals started actively exploring and utilizing the space.We recorded tremors using our custom-built tremor monitord for effect size (and reported these in the figure legends) by dividing the absolute difference in the parameter average of each group by the combined standard deviation.All statistical analyses were performed using MATLAB (Mathworks). When performing a two-way (sex*genotype) ANOVA, we did not observe an effect of sex or an interaction effect between sex and genotype on pup behavior. We, therefore, combined mice from both sexes in the experiments and statistical analyses. We performed a two-tailed T-test to assess differences between control and conditional knockout mice when only a one-time point was involved. For analyses of pup behavior and rotarod performance, we performed a repeated measures ANOVA analysis with a Tukey post hoc analysis to test for differences at each time point. For the analyses of electrophysiological recordings in Purkinje cells, we used a linear mixed model analysis with genotype as a fixed variable and mouse number as a random variable. We calculated Cohen\u2019s Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"}
+{"text": "RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current \u201cRNA-sequencing\u201d methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods. N6-methyladenosine (m6A), and others are complex multistep modifications, resulting in wybutosine  and a sugar-phosphate backbone. The bases and sugar of RNA undergo chemical alteration, leading to >150 modified nucleotides  methods do not sequence RNA, rather RNA is converted back to DNA and the resultant DNA is sequenced; in the reverse transcription, all modifications are lost  and the >150 modifications, it is necessary to train base-calling algorithms to establish the correspondence between the nucleotides and their corresponding current patterns. Many groups have developed methods to convert the current signals to sequence  was cloned from human fibroblast DNA into the pcDNA3.1-GFP(1\u201310) (Addgene cat# 70219) vector. The clone sequence was as follows:UUGAGCAUUUCAUCCGGAGUCUGGCCGCCCUGACCUUCCCCCAGCCGCCUGCAGGGGGCGCCAGAGGGCCGGAGCACGGAAAGCAGCGGAUCCUUGAUGCUGCCUUAAGUCCGGCUCAGAGGGGCGCAGCGUGGCCUGGGGUCGCUAUCUUCCCAUCCGGAACAUCUGCCCUGCUGGGGGACACUACGGGCCUUCCCUUGCCUGAGGGUAGGGUCUCAAGGUCACUUGCCCCCAGCUUGACCUGGCCGGAGUGGCUAUAGAGGACUUUGUCCCUGCAGACUGCAGCAGCAGAGAUGACACUGUCUCUGAGUGCAGAGAUGGGGGCAGGGAGCUGGGAGAGGGUUCAAGCUACUGGAACAGCUUCAGAACAACUAGGGUACUAGGAACUGCUGUGUCAGGGAGAAGGGGCUCAAGGACUCGCAGGCCUGGGAGGAGGGGCCUAGGCCAGCCAUGGGAGUUGGGUCACCUGUGUCUGAGGACUUGGUGCUGUCUGGAUUUUGCCAACCUAGGGCUGGGGUCAGCUGAUGCCCACCACGACUCCCGAGCCUCCAGGAACUGAAACCCUGUCUGCCCCCAGGGUCUGGGGAAGGAGGCUGCUGAGUAGAACCAACCCCAGGUUACCAACCCCACCUCAGCCACCCCUUGCCAGCCAAAGCAAACAGGCCCGGCCCGGCACUGGGGGUUCCUUCUCGAACCAGGAGUUCAGCCUCCCCUGACCCGCAGAAUCUUCUGAUCCCACCCGCUCCAGGAGCCAGGAAUGAGUCCCAGUCUCUCCCAGUUCUCACUGUGUGGUUUUGCCAUUCAUCUUGCUGCUGAACCACGGGUUUCUCCUCUGAAACAUCUGGGAUUUAUAACAGGGCUUAGGAAAGUGACAGCGUCUGAGCGUUCACUGUGGCCUGUCCAUUGCUAGCCCUAACAUAGGACCGCUGUGUGCCAGGGCUGUCCUCCAUGCUCAAUACACGUUAGCUUGUCACCAAACAUACCCGUGCCGCUGCUUUCCCAGUCUGAUGAGCAAAGGAACUUGAUGCUCAGAGAGGACAAGUCAUUUGCCCAAGGUCACACAGCUGGCAACUGGCAGAGCCAGGAUUCACGCCCUGGCAAUUUGACUCCAGAAUCCUAACCUUAACCCAGAAGCACGGCUUCAAGCCCCUGGAAACCACAAUACCUGUGGCAGCCAGGGGGAGGUGCUGGAAUCUCAUUUCACAUGUGGGGAGGGGGCUCCCCUGUGCUCAAGGUCACAACCAAAGAGGAAGCUGUGAUUAAAACCCAGGUCCCAUUUGCAAAGCCUCGACUUUUAGCAGGUGCAUCAUACUGUUCCCACCCCUCCCAUCCCACUUCUGUCCAGCCGCCUAGCCCCACUUUCUUUUUUUUCUUUUUUUGAGACAGUCUCCCUCUUGCUGAGGCUGGAGUGCAGUGGCGAGAUCUCGGCUCACUGUAACCUCCGCCUCCCGGGUUCAAGCGAUUCUCCUGCCUCAGCCUCCCAAGUAGCUAGGAUUACAGGCGCCCGCCACCACGCCUGGCUAACUUUUGUAUUUUUAGUAGAGAUGGGGUUUCACCAUGUUGGCCAGGCUGGUCUCAAACUCCUGACCUUAAGUGAUUCGCCCACUGUGGCCUCCCAAAGUGCUGGGAUUACAGGCGUGAGCUACCGCCCCCAGCCCCUCCCAUCCCACUUCUGUCCAGCCCCCUAGCCCUACUUUCUUUCUGGGAUCCAGGAGUCCAGAUCCCCAGCCCCCUCUCCAGAUUACAUUCAUCCAGGCACAGGAAAGGACAGGGUCAGGAAAGGAGGACUCUGGGCGGCAGCCUCCACAUUCCCCUUCCACGCUUGGCCCCCAGAAUGGAGGAGGGUGUCUGGAUUACUGGGCGAGGUGUCCUCCCUUCCUGGGGACUGUGGGGGGUGGUCAAAAGACC5)] of the 5-mers of the RNA synthesized with just the canonical nucleotides.The sequence includes 691 distinct 5-mers, about 70% 5-deoxyadenosine, 2.5 mM MgCl2, and 5 mM Tris-HCl buffer pH 8.0. The digestion mixture was incubated at 37\u00b0C for 6 h.RNA from each sample (2 \u00b5g) was hydrolyzed in a 50-\u00b5L digestion cocktail containing 8 U benzonase, 5 U calf intestinal alkaline phosphatase, 0.15 U phosphodiesterase I, 0.1 mM deferoxamine, 0.1 mM butylated hydroxytoluene, 5 ng coformycin, 50 nM internal standard [N1-methyladenosine (m1A), m/z 282 \u2192 150, 1.2 min; m6A, m/z 282 \u2192 150, 8.7 min; 5-hydroxymethylcytidine (hm5C), m/z 274.1 \u2192 142.1, 0.88 min; Y, m/z 245 \u2192 191, 0.88 min. Signal intensities for each ribonucleoside were normalized by dividing by the sum of the UV signal intensities of the 4 canonical ribonucleosides recorded with an in-line UV spectrophotometer at 260 nm.After digestion, all 9 samples were analyzed by chromatography-coupled triple-quadrupole mass spectrometry (LC-MS/MS). For each sample, 600 ng of hydrolysate was injected into each of 3 technical replicates. Using synthetic standards, HPLC retention times of RNA modifications were confirmed on a Waters Acuity BEH C18 column  coupled to an Agilent 1290 HPLC system and an Agilent 6495 triple-quadrupole mass spectrometer. The HPLC system was operated at 25\u00b0C and a flow rate of 0.3 mL/min in a gradient [Escherichia coli poly(A) polymerase, followed by Oxford Nanopore's protocol for direct RNA sequencing (SQK-RNA002). RNA samples were sequenced using an Oxford Nanopore MinION Mk1B device or a 5-cell GridION sequencer, with FLO-MIN106D flow cells . Reads were aligned to the clone sequence using minimap2 v2.17 , using tIn each sample, at each nucleotide position, we compared the RNA sequence to the corresponding sequence of the DNA sense strand, and then, we calculated the error rate as a ratio of mismatches to the total sequence reads at position (matched + mismatched). Those ratios are presented as percentages in the figures.For each sample, we also break down the errors by the 4 nucleotides in the DNA template. For each of the 4  nucleotides in the DNA, we looked for the number of mismatches in the RNA. In each sample, for each nucleotide, we determined the ratio of the number of mismatches to the total number of reads for the nucleotides and reported the ratios in percentages as A-to-X, C-to-X, G-to-X, and U-to-X where X is A, C, G, and U from Nanopore. The error rates calculated as implemented in our Python scripts are the same as those from EpiNano.To compute dwell times, reads were first resquiggled using f5c version 0.8  and principal component 1 (PC2) scores are plotted.Principal component analysis (PCA) was performed by using 7 variables  for the 45 samples . The data were normalized by using the Z-score. The PCA was calculated as the total variance explained.We also carried out a PCA with up to 5,000 randomly selected reads from each sample and the current and dwell times for the 5-mers that are most different among the modifications were used as variables. To select the variables for the PCA, for each 5-mer in our data, we performed ANOVA to identify the current signals that are most variable among the samples vs within, the top 5% of the most variable current signals were used as variables. The same analysis was used to identify the dwell times for the PCA. The variables were normalized by Z-score. The PCA was calculated as the total variance explained. For each sample, we then looked for the sites with the most modified nucleotides based on the PC1 and PC2 scores. We identified the outlier sites as those with PC scores furthest by Euclidean distance from the center of the plots; then at those locations, in each read, we looked for errors and longer dwell times to identify the most likely modified nucleotides. For the m6A, biotinylated-C, and Y samples, we only looked at the A, C, and U, respectively.Another PCA was performed for each sample with 7 variables  for each nucleotide. The data were also normalized by We synthesized a set of RNA transcripts with only canonical nucleotides and also with 10 different modified nucleotides. We then performed direct RNA sequencing using the Oxford Nanopore technology on those 2 kb RNA transcripts.O-methyladenosine (Am), 2\u2032-O-methylcytidine (Cm), 2\u2032-O-methylguanosine (Gm), and 2\u2032-O-methyluridine (Um). Each of the modified nucleotides was added with its corresponding canonical nucleotide in 1:100, 1:10, 1:5, and 1:2 proportions .Some of the IVT reactions included canonical nucleotides only, others included different proportions of a modified base or sugar nucleotide and the corresponding canonical nucleotide. The modified bases consist of m1A, m6A, biotinylated cytidine, hm5C, 5-methylcytidine (m5C), Y, and the modified sugars were 2\u2032-To verify that the modified nucleotides were incorporated into the RNA transcripts, the samples were checked by chemiluminescent assay and LC-MS/MS. The samples with biotinylated cytidine were checked by chemiluminescence using streptavidin-horseradish peroxidase. Additionally, to obtain a more accurate assessment of the modifications, 9 samples were analyzed by mass spectrometry. These include 1 sample synthesized with just canonical nucleotides and samples with m1A, m6A, hm5C, and Y at 2 concentrations . All the synthesized RNA transcripts were polyadenylated, and then sequenced using the Oxford Nanopore MinION. The base-calling was carried out using Guppy, alignment was performed with Minimap2 , and seqWe started by analyzing the 10 independently synthesized and sequenced sets of RNA with just canonical nucleotides. The samples were sequenced deeply with an average coverage of 5,563 reads  and is prone to slippage in particular when transcribing A/T homopolymers  and those from EpiNano  in the samples with and without modified bases. We found that all 6 nucleotides with modified bases have highly significantly different , the cytidines in the samples with modified cytidines  and the uridines in the Y samples. By motif analysis, we found that neighboring adenosines contributed to errors in the m6A samples but not in the m1A samples, this may allow for the 2 modifications to be distinguished. There is no such combination of features that allows us to tell apart the 3 modified cytidines. While all 6 modified bases led to significantly different dwell times in the pores, dwell time alone does not tell apart the different modifications.O-methylated sugars  at different proportions in the IVT reactions. The samples are deeply sequenced . We also carried out PCA with sequence reads to ask whether we can distinguish them. We used a similar concept in population genetic studies where individuals are grouped by populations based on their genotypes of genetic markers that have different allele frequencies across populations . We usedMaterials and Methods) and at those positions, we annotated the sequence reads with errors or longer dwell time as modified nucleotides  DNA template for IVT with 10 different modified nucleotides in a range of concentrations to generate RNA transcripts with modified bases or sugars. We also included RNA transcripts with only canonical nucleotides. The incorporation of the modified nucleotides was confirmed by chemiluminescence assay and mass spectrometry analyses. The RNAs were then sequenced on a Nanopore MinION with deep coverage, most samples having >5,000 reads. These are available for benchmarking algorithms for base-calling.We generated independently 10 sets of RNA with only canonical nucleotides for comparison with their counterparts with modified ribonucleotides. Direct RNA sequencing of the samples with only canonical nucleotides revealed that the error rate of Nanopore RNA sequencing is still >10%.Our analyses show that, while some modifications can be detected with existing algorithms such as Tombo and analysis of error patterns, we are far from sequencing RNA with all modifications. To obtain the complete sequences with all modifications, several technological advancements are needed. These include reducing the error rates to <0.1%, improving the resolution from 5-nucleotide to single-nucleotide, and developing base-callers that identify the canonical bases, known modifications, and yet-to-be discovered modifications. Given the large number of ribonucleotides per cell, even if we assume 10 pg of RNA per cell, there are >10 billion ribonucleotides in each cell, and thus, even a low error rate of 0.1% has many errors. Improvement in resolution to the single-nucleotide level will facilitate base-calling. Most probably, many modifications are transient and occur at a low level, so they are difficult to detect, and therefore, technical barriers such as poor resolution need to be improved. The biggest challenge of direct RNA sequencing is base-calling. Even if the instrument can capture current signals of a single nucleotide or uses methods such as exo-seq (RNA-sequencing technologies that provide the complete sequences with all the modifications, at affordable cost, will transform RNA biology and clinical medicine. The complete sequences will elucidate the basic aspects of RNA from splicing to stability. The sequences are crucial for the development of effective RNA-based therapeutics. The exact sequences are necessary for targeting specific RNA, replacing dysfunctional ones and in manufacturing vaccines. Development of sequencing technologies, coupled with analysis methods, is a necessary breakthrough to advance RNA biology and therapeutics.jkad200_Supplementary_DataClick here for additional data file."}
+{"text": "Peng D, Ando K, Hu\u00dfmann M, Gloger M, Skoczylas R, Mochizuki N, Betsholtz C, Fukuhara S, Schulte-Merker S, Lawson ND, Koltowska K. 2022. Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells. Published 22 March 2022This error occurred during the misaligned \u201ccopy and paste\u201d from the primer list while drafting the paper. It did not affect anyone qPCR experiments or the results. We regret any confusion or difficulties it may have caused.In the Materials and methods session - FACS and qPCR analysis, there is a mix-up for the dll4 primersCorrected sequence:GGACAAATGCACCAGTATGCGTTTGCGCAGTCGTTAATGTOriginal sequence:CGTTCCCAGGAGCCTTTTCTTTGGGATCAGGGATGGGGATThe article has been corrected accordingly."}
+{"text": "Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly. Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24\u2009h. Antibodies have also recently garnered attention as potential countermeasures for emerging pathogens, having been used as both prophylaxis and therapy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and coronavirus disease 2019 (COVID-19)6.Antibodies are widely used in diagnostics and as drugs. They are the critical component in immunoassays enabling rapid diagnostics8 sequences to a pool of 103 to 104 candidates targeting the desired antigen. However, once this pool of candidates is generated, state-of-the-art workflows rely on labor-intensive procedures , etc.) to evaluate and identify the best antibody candidates8. These procedures take weeks to months and represent a major speed and throughput bottleneck in antibody discovery.Contemporary workflows for antibody discovery commonly utilize synthetic selections or the isolation of single B cell clones from convalescent patients or animals to go from >1010 or non-overlapping epitopes12 to resist viral escape and increase the ability to neutralize viral variants13; both of which require intensive screening campaigns. A further challenge is that existing antibody discovery processes frequently have low efficiency, with few of the screened candidates having potent neutralizing activity, as has been the case for SARS-CoV-2 (Supplementary Table\u00a0The effort to identify antibodies against emerging threats like SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic has highlighted the importance of (i) rapid and high-throughput antibody discovery platforms and (ii) identifying high-affinity antibodies targeting conserved15, the manufacture of proteins without living cells using crude extracts or purified components, is an attractive tool to overcome these limitations. A variety of CFPS systems for antibody expression have been developed22; however, few of these studies have focused on the functional screening of antibodies, and most methods rely on techniques that are not suitable for high-throughput screening like the use of purified plasmids or labor-intensive ELISAs23.Cell-free protein synthesis (CFPS)24, and (iv) acoustic liquid handling that enables a highly parallel and miniaturized workflow. This integrated workflow enables a single researcher to express and profile the antigen-specific binding of hundreds of antibodies in less than 24\u2009h. As a model, we apply our workflow to profile a diverse set of 135 previously published antibodies targeting the SARS-CoV-2 spike glycoprotein, and show that our workflow identifies all 8 neutralizing antibodies that had been granted emergency use authorization (EUA) by the United States Food and Drug Administration for the treatment of COVID-19. In addition, we screen 119 antibodies derived from mice immunized against the SARS-CoV-2 spike glycoprotein and identify several candidate neutralizing antibodies.Here, we describe a CFPS-based integrated pipeline for antibody expression and evaluation to address screening limitations in current antibody discovery pipelines. The workflow leverages four key developments Fig.\u00a0: (i) DNA25. The method consists of a Gibson assembly step, followed by PCR amplification of the linear expression template (LET) using the unpurified Gibson assembly product as a template. The key idea was to create a versatile approach for construction of DNA templates without the requirement of cell culture, allowing for DNA assembly and amplification in less than 3\u2009h entirely in 384-well plates.We first implemented a method for cell-free DNA assembly and amplification by adapting and optimizing recently reported protocols for rapid construction of DNA templates for CFPS26. These sequences were subsequently amplified by PCR to generate LETs  strain, which contains mutations in the E. coli reductase genes trxB and gor to enable the formation of disulfide bonds in the cytoplasm27. By pretreating the extract with the reductase-inhibitor iodoacetamide (IAM) to further stabilize the redox environment30 and supplementing the reaction with purified E. coli disulfide bond isomerase DsbC and prolyl isomerase FkpA32, we successfully expressed and assembled full-length trastuzumab 31. Consistent assembly under the same experimental conditions is important for screening since individual conditions cannot be optimized for hundreds or thousands of antibodies. We thus opted to use the synthetically dimerized antigen-binding fragment  format and, like Ojima-Kato et al.18, found that the assembly of sdFabs were more uniform than their corresponding Fabs in CFPS for a panel of antibodies  for binding of the SARS-CoV-2 Receptor Binding Domain (RBD). Our determined rank order of IC50s Fig.\u00a0.Furthermore, we utilized AlphaLISA to develop a sdFab assembly screen to monitor antibody fragment expression and assembly in CFPS, a step that traditionally requires SDS-PAGE. The measurement immobilizes the heavy and light chains of the sdFab to the AlphaLISA beads, resulting in signal when the two chains are assembled 50. We used these assays as a rapid screen for S6P binding and ACE2 competition at a single unknown concentration of CFPS-derived antibody.Using the developed workflow, we next evaluated a set of 115 SARS-CoV-2 targeted antibodies that were selected based on the availability of sequence, structural, binding, and neutralization data, with 84 being drawn from Brouwer et al.FPS Fig.\u00a0. MeasureTo determine the robustness of the workflow, antibody fragments were expressed and evaluated in triplicate. We observed that independent replicates were consistent with one another and exhibited average coefficients of variation (standard deviation divided by the mean) in the range of 0.15\u20130.22 , r\u2009=\u20090.41 , and r\u2009=\u20090.40  for Figs.\u00a0While there is only a weak correlation between our AlphaLISA data and the corresponding Brouwer et al. S6P binding ELISA data, the RBD binding ELISA data, and the pseudovirus neutralization data 67. For all 8 EUA antibody fragments, we observed S6P binding, RBD binding, ACE2 competition, and assembly  by the US Food and Drug Administration for prophylaxis or treatment of COVID-19 67. Notably, we observed that S2H97 exhibited ACE2 competition, which like S309, is reported to bind an epitope adjacent to the receptor binding motif and not compete with ACE2 for binding10. The antibody 54042-4 exhibited weak binding to the RBD but showed low assembly signal, indicative of poor expression or assembly , we observed binding and competition for 10 of 11 antibody fragments tested, with the results largely consistent with published literatureata Fig.\u00a0. Based o68. For these antibodies, the normalized S6P binding AlphaLISA measurement correlates well with loss in neutralization potency ). Also consistent with literature, S309 exhibited binding to the SARS-CoV spike protein, whereas no other EUA Ab is reported to bind SARS-CoV59.We further profiled the binding of this set of high interest antibodies against SARS-CoV-2 variants of concern (VOC) including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron  as well as several other human coronaviruses including SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E Fig.\u00a0. Our dat64. S2K146 did not exhibit binding to the SARS-CoV spike, despite being reported to neutralize SARS-CoV65. S2P6 additionally exhibited binding to MERS-CoV and HCoV-OC43 consistent with the literature on this antibody61. For S2P6, we observed heterogeneity in binding signal to different S6P variants, possibly a result of the target epitope of S2P6 being near the C-terminus of S6P and near to the C-terminal avi tag (a site-specific biotinylation), which may impede immobilization on the AlphaLISA bead. Neutralization data against all VOC for the tested bnAbs are not available, but the binding profiles for those characterized are generally consistent with literature. Against Omicron BA.1, S2K146 exhibits strong binding whereas S2X259, S2H97, and SARS2-38 all exhibit reduced binding70. Similarly, S2K146 exhibits strong binding to all Omicron variants except BA.4/5, whereas S2X259 and S2H97 exhibit reduced binding to the other Omicron sub-lineages72. Taken together, our results indicate that the CFPS-derived antibody fragment binding patterns are consistent with those reported in literature.For the set of bnAbs, we observed cross-reactivity with the SARS-CoV spike protein for S2P6, S2X259, DH1047, C118, and S2H97 all of which are reported to bind to this antigen73 and isolated spike-positive activated B cells 10 days later using fluorescence-activated cell sorting. The pooled sorted B cells were sequenced to identify paired heavy and light chains, which were codon optimized and ordered commercially as synthetic DNA .No statistical method was used to predetermine sample size. Previous experience with the measurement techniques and their dynamic ranges was used to determine sample sizes. Large-scale experiments . The Benjamini and Hochberg False Discovery Rate procedure50 was used to correct for multiple testing. Statistical analyses were performed in python. Two-sided t-tests  were performed using the scipy package and the FDR procedure was performed using the statsmodels package with a family-wise error rate of 5%. For antibody screening, the following samples were used as a measurement of background, and the combined data were used in the t-test. Assembly: No DNA and beads only controls. S6P binding: No DNA and beads only controls. RBD binding: No DNA and beads only controls. ACE2 competition: No DNA and \u03b1HER2.Samples were considered different from background if they exhibited Pearson correlation coefficients were calculated using GraphPad Prism 9.Single-cell BCR sequences were analyzed with Cell Ranger v3.1.0. AlphaLISA data were analyzed with Prism 9.5.1. Images were processed using ImageJ2 v2.9.0/1.53t. Plate reader data were processed using Python 3.8.8. The Python code to analyze plate reader data was not central to the research and was not deposited.18. Example plasmid maps of the aHER2 heavy and light chain sdFabs can be found in the Source data. Antibody sequences were collected from literature and their light chains were classified as either kappa or lambda via the terminal residue of the J-segment in the VL domain. The VH and VL domains were subsequently fused to their corresponding human constant heavy (Uniprot P0DOX5) or human constant light (kappa CL Uniprot P01834 or lambda 1 CL Uniprot P0CG04) chains. At the N-terminus of the VH and VL domains, we chose to include a modified expression tag based on the first 5-residues of the E. coli chloramphenicol acetyltransferase gene followed by a Tobacco Etch Virus (TEV) protease cleavage site 79 as opposed to the previously published SKIK tag80. The heavy chain was fused to the LZA heterodimer subunit (AQLEKELQALEKENAQLEWELQALEKELAQK) and a strep II tag or super FLAG (sFLAG) tag81. The light chain was fused to the LZB heterodimer subunit (AQLKKKLQALKKKNAQLKWKLQALKKKLAQK). Antibodies in the screen in Fig.\u00a0sdFabs were assembled based on a modified version of previously published protocolssdFab heavy chain constant strepII tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[WSHPQFEK].sdFab heavy chain constant super FLAG (sFLAG) tagged:[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[DYKDEDLL].sdFab light chain kappa:[MEKKIENLYFQS][VL_Sequence][RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].sdFab light chain lambda 1:[MEKKIENLYFQS][VL_Sequence][GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].Proteins to be manufactured via CFPS were codon optimized using the IDT codon optimization tool and ordered as double-stranded linear DNA containing the desired Gibson assembly overhangs from IDT or GenScript. sfGFP was ordered containing the two pJL1 Gibson assembly overhangs. Antibody VH DNA was ordered with the pJL1 5\u2032 and the human IgG1 heavy chain constant 5\u2032 Gibson overhangs. Antibody VL DNA was ordered with the pJL1 5\u2032 and the human Ig light chain kappa or lambda 1 Gibson assembly overhangs. DNA was resuspended at a concentration of 50\u2009ng/\u03bcL and used without amplification.Additional linear DNA components for Gibson assembly  were ordered as gblocks from IDT. These components were amplified using PCR using Q5 Hot Start DNA polymerase  following manufacturer instructions. Amplified DNA was purified using the DNA Clean and Concentrate Kit  and diluted to a concentration of 50\u2009ng/\u03bcL. Sequences of the utilized components are listed below, with Gibson assembly sequences being denoted by underlined lowercase text and primers for a given amplicon being listed below the DNA sequence.Gibson assembly overhangs:tttgtttaactttaagaaggagatatacat.pJL1 5\u2032 Gibson: gtcgaccggctgctaacaaagcccgaaagg.pJL1 3\u2032 Gibson: gcgtcaacaaaaggtccttcagttttcccattagcccct.Human IgG1 heavy chain constant 5\u2032 Gibson: cgcacggtcgcggcgccgtctgtctttatttttcctcct.Human Ig light chain kappa 5\u2032 Gibson: ggccaacccaaagcaaacccaactgtcactttgttcccg.Human Ig light chain lambda 5\u2032 Gibson: Linear pJL1 plasmid backbone (Addgene plasmid # 69496):gtcgaccggctgctaacaaagcccgaaaggAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCTTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATtttgtttaactttaagaaggagatatacat.pJL1_F: gtcgaccggctgcta.pJL1_R: atgtatatctccttcttaaagttaaacaaaattatttcta.Linear sdFab heavy chain constant strepII tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTTCTAGCGCATGGTCACATCCCCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.Linear sdFab heavy chain constant super FLAG tagged:gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTGGAGCCAGTCCAGCAGCTCCTGCGCCTGGCGGGGACTACAAAGATGAAGACCTTCTTTAAgtcgaccggctgctaacaaagcccgaaagg.IgGC_F: GCGTCAACAAAAGGTCCTTCAGTTTTC.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain kappa constant:cgcacggtcgcggcgccgtctgtctttatttttcctcctTCTGATGAACAGCTTAAATCTGGGACAGCTTCTGTTGTATGTTTATTAAACAACTTTTACCCGCGTGAGGCAAAAGTTCAATGGAAGGTAGACAACGCACTGCAAAGCGGAAATTCGCAGGAGTCAGTTACCGAACAGGATTCCAAGGATAGTACCTACTCCTTAAGTTCAACATTAACCCTGTCAAAGGCGGACTATGAAAAACATAAGGTATATGCCTGCGAAGTAACTCATCAGGGCTTATCATCCCCAGTTACAAAATCTTTCAACCGTGGAGAATGCGGCGGCGGAGGTAGCGCGCAGCTTAAGAAAAAATTGCAAGCCCTTAAAAAAAAAAATGCCCAACTTAAATGGAAGCTGCAAGCCTTAAAAAAGAAATTGGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.kLC_F: TCGCGGCGCCGTCTG.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.Linear sdFab light chain lambda 1 constant:ggccaacccaaagcaaacccaactgtcactttgttcccgCCCTCAAGCGAGGAACTTCAGGCTAATAAGGCCACGCTTGTTTGCCTGATCTCAGACTTTTATCCCGGTGCCGTAACAGTGGCTTGGAAGGCAGATGGTTCGCCGGTCAAAGCGGGCGTGGAAACTACAAAGCCATCGAAACAGTCAAACAATAAATATGCGGCATCAAGTTACTTGAGCCTTACCCCAGAACAGTGGAAGTCACACCGCTCGTACAGTTGTCAAGTTACACACGAGGGAAGTACAGTTGAAAAGACCGTTGCCCCAACTGAATGTTCAGGCGGTGGTGGCTCAGCGCAGTTAAAGAAAAAACTGCAGGCTTTGAAGAAAAAGAATGCTCAATTAAAGTGGAAATTGCAGGCGTTGAAGAAGAAACTTGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.lLC_F: GGCCAACCCAAAGCAAACC.pJL1_3\u2032Gib_R: CCTTTCGGGCTTTGTTAGCAGC.82.Linear sfGFP (same DNA sequence as Addgene Plasmid #102634). Note that the sequence of sfGFP is heavily modified and contains mutations from Bundy et al.tttgtttaactttaagaaggagatatacatATGAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGTCACAAATTCAGCGTGCGTGGTGAAGGTGAAGGCGATGCCACGATTGGCAAACTGACGCTGAAATTTATCTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACGCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGTCGCTATCCGGATCACATGAAACGTCACGATTTCTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGTACGATTAGCTTTAAAGATGATGGCAAATATAAAACGCGCGCCGTTGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCACGGATTTTAAAGAAGATGGCAATATCCTGGGCCATAAACTGGAATACAACTTTAATAGCCATAATGTTTATATTACGGCGGATAAACAGAAAAATGGCATCAAAGCGAATTTTACCGTTCGCCATAACGTTGAAGATGGCAGTGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAATCATTATCTGAGCACGCAGACCGTTCTGTCTAAAGATCCGAACGAAAAAGGCACGCGGGACCACATGGTTCTGCACGAATATGTGAATGCGGCAGGTATTACGTGGAGCCATCCGCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.84. 20\u2009ng of purified, linear pJL1 backbone, 20\u2009ng of purified, linear sdFab VH or VL constant DNA, and 20\u2009ng of the protein open reading frame insert were combined in 2\u2009\u03bcL Gibson assembly reactions and incubated at 50\u2009\u00b0C for 30\u2009min. The unpurified assembly reactions were diluted in 40\u2009\u03bcL of nuclease-free water  and 1\u2009\u03bcL of the diluted reaction was used as the template for a PCR to generate linear expression templates (LETs) for CFPS. Linear expression templates were amplified via PCR using the pJL1_LET_F (ctgagatacctacagcgtgagc) and pJL1_LET_R (cgtcactcatggtgatttctcacttg) primers in a 50\u2009\u03bcL PCR reaction using the Q5 Hot Start DNA polymerase  following manufacturer instructions.Gibson assembly was used to assemble protein open reading frame DNA with the pJL1 backbone following the published protocol with the addition of 3.125\u2009\u03bcg/mL of ET SSB P. pyralis luciferase containing a c-terminal strepII tag  used as a negative control is below and was cloned into the pJL1 vector.The DNA sequence of the atggaagacgctaagaacattaagaagggacctgctccattctaccccctcgaagacggcactgcaggtgagcagcttcataaagcgatgaagcgttatgcgttagttcctggcacgatcgccttcactgacgcgcacatcgaagtcaatatcacctacgctgaatactttgagatgagtgtgcgtctggcggaagccatgaagcgttatggccttaacacgaaccaccgcatcgttgtttgtagcgagaattccttacaattcttcatgcccgtccttggcgcgctgtttattggtgtggccgttgcaccagccaatgacatctataatgagcgcgagttgttgaactccatgaacatttctcaaccaacagtggtgttcgtttcaaagaaaggcttacagaaaatcttaaacgttcaaaagaaactgccgattatccagaagatcatcattatggatagtaagactgactaccagggcttccagtcaatgtatacattcgtgacgagtcacctgcccccgggttttaacgagtacgactttgtcccagagagctttgatcgcgacaagaccatcgccctcattatgaatagcagtggttcgacgggtagcccaaagggagtggccctgccccatcgtaccgcgtgcgtccgtttctcccatgcccgcgacccaattttcggcaatcaaatcatccccgacacggcaatcttgtcggtcgtcccgtttcaccatggctttggaatgtttacgacactcggttacctcatctgcggtttccgcgtcgttctgatgtatcgcttcgaggaagagttgttcttacgttcgcttcaggactacaagattcaatccgcccttctggtccccactttgttcagtttctttgctaagagcaccttaattgataagtatgacctctccaacttacacgagattgcgagcggtggtgctcccctcagcaaagaggttggagaggcggttgctaagcgttttcatctgcccggtatccgtcaaggttacggcctcaccgaaaccacttctgccattcttatcactccggaaggtgacgataagcctggggcagtgggtaaagttgtacccttcttcgaggctaaggttgtggatttagatacggggaagaccttaggtgtgaaccagcgcggtgaactgtgcgttcgcggtccgatgattatgtcgggttatgttaatgaccccgaggctacgaacgcgcttatcgataaggacggttggcttcattccggcgacatcgcttactgggatgaggatgagcacttcttcatcgttgaccgtctgaagagtctcatcaagtataagggatgtcaagtcgctccggcagagttagagagcatcttactccagcaccctaatatcttcgatgctggggttgccgggctcccaggcgacgatgccggcgagctgccggcggcggtagttgttttagagcatggcaagaccatgaccgaaaaggagattgtagactacgtcgcgagtcaagtaaccacagcgaagaagctccgcggtggagtggtctttgttgacgaggtgcctaaaggcctgacgggcaaacttgacgcgcgtaagatccgtgagatcctcatcaaagcgaagaagggtgggaagagtaagctggggagttcaggttggtcccacccgcaatttgagaagtga.E. coli OrigamiTM B(DE3)  extracts were prepared using a modified version of established protocols86. Briefly, a 150\u2009mL OrigamiTM B(DE3) starter culture was inoculated in LB from a glycerol stock and cultured in a 250\u2009mL baffled flask at 37\u2009\u00b0C for 16\u2009h. The 2xYTP was prepared without glucose in 75% of the final volume and sterilized using an autoclave. A 4x glucose solution was prepared and autoclaved separately, then added to the medium immediately before use. The starter cultures were used to inoculate 1\u2009L of 2xYTPG media  in a 2.5\u2009L Full-Baffle Tunair shake flask at an initial OD600 of 0.08. Cells were cultured at 37\u2009\u00b0C at 220 RPM in a shaking incubator. Cultures were grown until OD600 0.4-0.6, at which point the expression of T7 RNA polymerase was induced by the addition of IPTG to a final concentration of 0.5\u2009mM. Cells were harvested at an OD600 of 2.5 via centrifugation at 12,000\u2009\u00d7\u2009g for 1\u2009min at 4\u2009\u00b0C. Cell pellets were washed three times with 25\u2009mL S30 buffer per 50\u2009mL culture . Pellets were resuspended in 1\u2009mL S30 buffer per gram of cell mass. Cell suspensions were lysed using a single pass on an Avestin EmulsiFlex-B15 Homogenizer at a lysis pressure of 24,000 PSI. Cell debris was separated via centrifugation at 18,000\u2009\u00d7\u2009g for 20\u2009min, and the clarified lysate was collected, flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C.E. coli MRE600 tRNA (Roche 10109541001), 100\u2009mM NAD, 50\u2009mM CoA, 4\u2009mM oxalic acid, 1\u2009mM putrescine, 1\u2009mM spermidine, 57\u2009mM HEPES pH 7.2, 2\u2009mM of each amino acid, 33.3\u2009mM PEP, 20% v/v E. coli extract, varying concentrations of DNA template, and the remainder water. The preparation of these reagents has been described in detail elsewhere87. For DNA templates, plasmids were used at a concentration of 8\u2009nM, and unpurified linear PCR products were used at 6.66% (v/v). For the expression of antibodies, each template was added to a final concentration of 6.66% (v/v). For antibody and sdFab expression 4\u2009mM oxidized glutathione, 1\u2009mM reduced glutathione, 14\u2009\u03bcM of purified DsbC, and 50\u2009\u03bcM FkpA were also supplemented to the reactions. In addition, for oxidizing CFPS reactions, cell-extracts were treated with 500\u2009\u03bcM iodoacetamide (IAM) at room temperature for 30\u2009min before use in CFPS88. All reaction components were assembled on ice and were either run as 12\u2009\u03bcL reactions in 1.5\u2009mL microtubes or 2\u2009\u03bcL reactions in 384-well plates . For 2\u2009\u03bcL reactions, components were transferred to the plate using an Echo 525 acoustic liquid handler. A mix containing all the CFPS components except for the DNA was dispensed from 384PP Plus plates  using the BP setting. The DNA (unpurified PCR products) was dispensed from a 384LDV Plus plate  using the GP setting. Reactions were allowed to proceed at 30\u2009\u00b0C for 20\u2009h.CFPS reactions were composed of the following reagents: 8\u2009mM magnesium glutamate, 10\u2009mM ammonium glutamate, 130\u2009mM potassium glutamate, 1.2\u2009mM ATP, 0.5\u2009mM of each CTP, GTP, and UTP. 0.03\u2009mg/mL folinic acid, 0.17\u2009mg/mL 86. Radioactive leucine was added to CFPS at a final concentration of 10\u2009\u03bcM of L-[14C(U)]-leucine , followed by precipitation of the expressed proteins and scintillation counting89. To quantify sfGFP fluorescence, 2\u2009\u03bcL of a CFPS reaction was diluted in 48\u2009\u03bcL of water in a Black Costar 96 Well Half Area Plate. Fluorescence was measured using a BioTek SynergyTM H1 plate reader with excitation and emission wavelengths of 485 and 528, respectively. Scintillation counts and fluorescence were fit to determine a standard curve for use with non-radioactive samples.To quantify sfGFP fluorescence, a standard curve was prepared using previously reported methodsTM . FluoroTectTM was included in the CFPS reaction at 3.33%v/v. After protein synthesis, RNAseA  was added to 0.1\u2009mg/mL and the sample was incubated at 37\u2009\u00b0C for 10\u2009min. 3\u2009\u03bcL of the CFPS and RNAseA mixture were combined with 4x loading buffer  and the samples were subsequently denatured at 70\u2009\u00b0C for 3\u2009min, then separated via SDS-PAGE and imaged using a LI-COR Odyssey Fc imager on the 600 channel. Densitometry was performed using the ImageJ software.To visualize antibody assembly, proteins were labeled during CFPS with FluoroTect77. DsbC  and FkpA  were ordered as gBlocks from IDT containing a c-terminal, TEV cleavable his tag (GSENLYFQSGSHHHHHHHHHH) and cloned into pET28a. Plasmid maps of both DsbC and FkpA are available in the Source Data. Plasmids were transformed into BL21 StarTM DE3, plated on LB agar, and cultured overnight at 37\u2009\u00b0C. 1\u2009L of Overnight Express TB  was inoculated by scraping all colonies on a transformation plate and cultured at 37\u2009\u00b0C in 2.5\u2009L tunair flasks  at 220\u2009rpm overnight. Cells were harvested, resuspended at a ratio of 1\u2009g cell mass to 4\u2009mL resuspension buffer , 1\u2009mg/mL lysozyme, 62.5\u2009U/mL cell suspension of benzonase ) and lysed using an Avestin B15 homogenizer at 24,000 PSI. The lysate was spun down 14,000\u2009\u00d7\u2009g for 10\u2009min and the clarified supernatant was incubated with Ni-NTA Agarose  for 60\u2009min on an end-over-end shaker. The resin was spun down 2500l\u2009\u00d7\u2009g for 2\u2009min, the supernatant removed, resuspended in wash buffer , loaded on a gravity flow column, and subsequently washed with 20X resin volumes of wash buffer. Protein was eluted using elution buffer  and exchanged into 50\u2009mM HEPES pH 7.4, 150\u2009mM NaCl using PD-10 desalting columns  according to manufacturer instructions.Protein expression, purification, and his tag removal were performed similarly to previously reportedHis tags were removed via cleavage by ProTEV Plus . Before cleavage, 10% v/v glycerol was added to the protein. ProTEV Plus was added to a concentration of 0.5 U/\u03bcg purified protein and DTT was added to a concentration of 1\u2009mM. Cleavage reactions were carried out at 30\u2009\u00b0C for 4\u2009h. Free His tag and ProTEV Plus were removed by incubating with Ni-NTA Agarose for 1\u2009h at 4\u2009\u00b0C and collecting the supernatant. Proteins were subsequently concentrated to >1\u2009mg/mL . His tag removal was validated via SDS-PAGE and the AlphaScreen Histidine (Nickel Chelate) Detection Kit .90.AlphaLISA reactions were carried out in 50\u2009mM HEPES pH 7.4, 150\u2009mM NaCl, 1\u2009mg/mL BSA, and 0.00015\u2009v/v TritonX-100 (hereafter referred to as Alpha buffer). All components were dispensed using an Echo 525 liquid handler from a 384-Well Polypropylene 2.0 Plus microplate  using the 384PP_Plus_GPSA fluid type. All components of the AlphaLISA reactions were prepared as 4x stocks and added as 0.5\u2009\u03bcL to the final 2\u2009\u03bcL reaction to achieve the desired concentration. All AlphaLISA reactions were performed with CFPS reactions diluted to a final concentration of 0.025\u2009v/v. AlphaLISA beads were combined to prepare a 4X stock in Alpha buffer immediately before use and added to the proteins to yield a concentration of 0.08\u2009mg/mL donor beads and 0.02\u2009mg/mL acceptor beads in the final reaction. All reactions were incubated with AlphaLISA beads for at least 1\u2009h before measurement. AlphaLISA measurements were taken on a Tecan Infinite M1000 Pro plate reader using the AlphaLISA filter with an excitation time of 100\u2009ms, an integration time of 300\u2009ms, and a settling time of 20\u2009ms. Before measurement, plates were allowed to equilibrate inside the instrument for 10\u2009min. For measurements involving sdFabs, protein A AlphaLISA beads were avoided due to the ability of protein A to bind human subgroup VH3 FabsThe impact of CFPS reagents on AlphaLISA was determined by serially diluting the specified reagents in Alpha buffer and combining them with the specified AlphaLISA conditions. The TrueHits kit  was used to assess the impact of the CFPS reagents on the Alpha detection chemistry. CFPS reagents were mixed with the donor and acceptor beads and incubated for 2\u2009h before measurement. His tagged RBD  and human FC tagged human ACE2  were used to evaluate the impact of CFPS reagents on capture chemistries. RBD and ACE2 were diluted in Alpha buffer, mixed at a final reaction concentration of 10\u2009nM each, combined with the CFPS reagents, and allowed to incubate for 1\u2009h. Donor and acceptor beads were subsequently added and allowed to incubate for a further hour before measurement. Protein A Alpha donor beads , Ni-Chelate AlphaLISA acceptor beads , and anti-6xhis AlphaLISA acceptor beads  were utilized for detection.50 values were recorded from the product page at the time of purchase and converted to \u03bcg/mL assuming a MW of 150,000\u2009Da if reported in M. Antibodies were serially diluted in Alpha buffer and mixed with SARS-CoV-2 RBD  at a concentration of 10\u2009nM in the final reaction and incubated for 1\u2009h. Mouse FC tagged human ACE2  was subsequently added and incubated for 1\u2009h, followed by simultaneous addition of the acceptor and donor beads. AlphaLISA detection was performed using Anti-Mouse IgG Alpha Donor beads  and Strep-Tactin AlphaLISA Acceptor beads . IC50 values were calculated using Prism 9 by fitting the normalized data to [Inhibitor] vs. response\u2013Variable slope (four parameters) fit with the max constrained to a value of 1.The commercial neutralizing antibody ACE2 competition experiment was performed with the following antibodies: nAb1 , nAb2 , nAb3 , nAb4 . ELISA ICFor all antibody screening experiments, the different reagents and AlphaLISA conditions used are described in Supplementary Table\u00a0Assembly AlphaLISA reactions consisted of sdFab expressing CFPS and either Rabbit Anti-Human kappa light chain antibody  or Rabbit Anti-Human lambda light chain . CFPS reaction containing the expressed sdFab of interest was mixed with the appropriate anti-light chain antibody and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 S6P binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 S6P CFPS reaction containing the expressed sdFab of interest was mixed with the S6P and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.SARS-CoV-2 RBD binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 RBD. CFPS reaction containing the expressed sdFab of interest was mixed with the RBD and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.ACE2 and RBD competition AlphaLISA reactions consisted of sdFab expressing CFPS, human ACE2, and SARS-CoV-2 S6P. CFPS reaction containing the expressed sdFab of interest was first mixed with S6P and allowed to incubate for 1\u2009h. Subsequently, ACE2 was added and allowed to equilibrate for a further 1\u2009h before the simultaneous addition of the acceptor and donor beads.77), SARS-CoV-2 S6P Beta/B.1.351 , SARS-CoV-2 S6P Gamma/P.1 , SARS-CoV-2 S6P Delta/B.1.617.2 , SARS-CoV-2 S6P Omicron/BA.1 , SARS-CoV-2 S6P Omicron/BA.2 , SARS-CoV-2 S6P Omicron/BA.2.12.1 , SARS-CoV-2 S6P Omicron/BA.4/5 , SARS-CoV S2P , MERS-CoV S2P , HCoV-HKU1 S , HCoV-OC43 S , HCoV-NL63 S , and HCoV-229E S .For SARS-CoV-2 variant and other non-SARS-CoV-2 coronavirus binding experiments, AlphaLISA measurements were carried out in the same manner as described for SARS-CoV-2 S6P. The following Hisx6-tagged proteins were used. SARS-CoV-2 S6P , SARS-CoV-2 S6P Alpha/ B.1.1.7  of human ACE2. All three components were incubated for an additional hour prior to simultaneous addition of AlphaLISA beads. Reactions were incubated for 2\u2009h prior to measurement.For RBD and ACE2 bridging experiments SARS-CoV-2 RBD, human ACE2, and the specified dilution of CFPS  were incubated for 1\u2009h prior to the simultaneous addition of the AlphaLISA beads. Reactions were incubated for 2\u2009h prior to measurement.10 viral particles (vp) of ChAd-SARS-CoV-2-S73 in 50\u2009\u00b5l of PBS via intramuscular injection in the hind leg. Draining inguinal lymph nodes were collected 10 days later and processed into a single-cell suspension. Cells were stained with biotinylated recombinant SARS-CoV-2 spike (S2P) for 30\u2009min at 4\u2009\u00b0C then washed twice with FACS buffer followed by staining with anti-CD19 BV421 (BioLegend # 115537), anti-CD4 FITC (BioLegend # 100405), anti-IgD-PE-Cy7 (BioLegend # 405719), Streptavidin APC (BioLegend # 405207), aqua cell viability dye (Invitrogen L34957), and anti-mouse CD16/CD32 Fc block (BioLegend # 156607). Spike-positive activated B cells (live singlet CD4- CD19+ IgDlo Streptavidin+) were bulk sorted on BD FACSAriaII sorter.Female C57BL/6 (Strain: 000664) were purchased from The Jackson Laboratory. Six-week-old animals were immunized with 10The following 10x Genomics kits were used for libraries preparation: Chromium Single Cell 5\u2032 Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Mouse B cell (96rxns) (PN-1000072), and Single Index Kit T (PN-1000213). The GEM generation and barcoding was followed by cDNA preparation then GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 10\u201314 cycles then cleaned up using SPRIselect beads. cDNA concentration was determined by running samples on a Bioanalyzer. BCR target enrichments were done on the full-length cDNA followed by BCR libraries preparation as recommended by 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1 Chemistry) user guide. The cDNA Libraries were sequenced on Novaseq S4 (Illumina), targeting a median sequencing depth of 5000 read pairs per cell.Cell Ranger v3.1.0 for alignment against the GRCm38 mouse reference v3.1.0 , generating 3760 assembled high-confidence BCR sequences for 4420 cells. Sequences for screening were selected randomly from the top clonal groups with >10 members in the clonal group. Cellranger vdj output was then parsed using Change-O v0.4.6 within the immcantation suite. Additional quality control included examining sequences to be productively rearranged and have valid V and J gene annotations. Furthermore, only cells with exactly one heavy chain sequence paired with at least one light chain sequence were kept.Demultiplexed pair-end FASTQ reads from 10x Genomics single-cell V(D)J profiling were preprocessed using the \u201ccellranger vdj\u201d command from Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary"}
+{"text": "Shigella flexneri to selectively internalize into glioblastoma (GBM) brain tumor cells as an initial step to generating a bacterial-based drug delivery system.The use of microorganisms as drug delivery systems to treat cancer has expanded recently, including FDA approval of certain viruses as oncolytics. Microorganisms have several unique benefits compared to traditional pharmacologic agents including dose independence, the ability to produce therapeutic proteins locally within the tumor, and simplicity of administration. However, current microbial delivery systems such as AAV9 and herpes virus have limited cassette sizes, minimal cancer cell selectivity, and low innate cytotoxicity. To address these issues, we sought to generate a strain of S. flexneri that selectively internalize into GBM cells using iterative co-cultured assays.We generated After 50 rounds of co-culture, the new strain infected 95 percent of GBM cells in 2 hours. GBM-infecting Shigella demonstrate a 124-fold preference for internalizing in nine different GBM cell lines compared to Normal Astrocytes (NA) controls. Additionally, we developed an in-cell western to identify GBM-infecting Shigella clones that preferentially internalize in patient samples without iterative co-culture. Finally, we demonstrate internalization into GBM cells is mediated via a factor modified by myristoylation.In conclusion, here we present a novel bacterial platform that preferentially internalizes in brain tumor cells. This system provides numerous potential benefits over current interventions and other microbial strategies for treating brain tumors. Microorganism-based drug delivery systems are emerging as a promising approach to treating solid tumors \u20134. Most Glioblastoma (GBM) is the most common malignant, primary brain tumor observed in adults, and patients diagnosed with GBM demonstrate a median survival of only 15 months . The staShigella flexneri, a gram-negative intracellular bacterium similar to the viruses discussed above. Importantly, Shigella has a type 3 secretion system capable of administering toxic proteins into the host cell cytosol  (30 g/L) + agar (15 g/L), and Congo Red (100 mg/L) sterile plates and incubated overnight at 30\u00b0C or 37\u00b0C. Individual colonies were expanded in sterile Tryptic Soy Broth (30 g/L) using a shaking incubator (250 rpm) at 30\u00b0C.Shigella was streaked out from the frozen stock onto TSB (30 g/L) + Congo Red (0.01% w/v) plates and incubated overnight at 37\u00b0C. Colonies were picked and grown for approximately 2 hours (OD650\u00a0=\u00a00.6) (\u00ae EHEC (Cat#: 751630). Band intensity was measured using ImageJ.At 24 hours prior to testing, 0\u00a0=\u00a00.6) . SamplesShigella colony was picked using a pipet tip and resuspended in 20 \u03bcL nuclease-free water. A colony was incubated for 10\u00a0min at 95\u00b0C and then centrifuged at 1,000 rcf for 1\u00a0min to remove membrane components.A single Supernatant at a volume of 5 \u03bcL was used as the template for a PCR assay to quantify the presence of DNA encoding for Shiga toxin(s). Potential DNA regions were amplified with the Phusion taq polymerase kit  using previously described primers that are specific for Shiga toxin 1 (Stx1 \u2013 primer pair: ATGTCAGAGGGATAGATCCA and TATAGCTACTGTCACCAGACAAT), Shiga toxin 2 (Stx2 \u2013 primer pair: AGTTCTGCGTTTTGTCACTGTC and CGGAAGCACATTGCTGATT), or positive control virulence factor F (virF \u2013 primer pair: AGCTCAGGCAATGAAACTTTGAC and TGGGCTTGATATTCCGATAAGTC) . Bands wS. flexneri was streaked onto TSB agar plates with 0.01% Congo Red and grown at 37\u00b0C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip. Each colony was added into 400 \u03bcL of TSB in a deep-well block plate and incubated at 37\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). Shigella suspensions were combined and pelleted into a 50-mL conical tube. After washing three times with 25 mL of 10% glycerol, Shigella was resuspended in 5 mL of 10% glycerol. Shigella was electroporated with 200 ng of pRedTKI plasmid DNA (Addgene Plasmid #51628) and recovered in TSB for 1 hour at 30\u00b0C, followed by plating on TSB + 50 \u03bcg/mL Kan agar plates with 0.01% Congo Red overnight. The pRedTKI plasmid introduces the Lambda red genes necessary for homologous recombination along with conferring Kan resistance.Genes were deleted as previously described using standard Lambda red recombineering methods with minor modifications , 27. S. Shigella + pRedTKI colonies were picked and incubated at 30\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 3 hours); during the last 30\u00a0min of incubation, l-arabinose was added at 10 mmol/L to induce the Lambda red recombination genes along with 50 \u03bcg/mL kanamycin to maintain transmission of the pRedTKI plasmid. Bacteria were pelleted in a 50-mL conical tube by centrifugation at 4,000 \u00d7 g, transferred to a 1.5-mL Eppendorf tube, washed three times with 500 \u03bcL of 10% glycerol, and resuspended in 50 \u03bcL of 10% glycerol. Using an electroporation cuvette with a 2\u00a0mm gap, Shigella were transfected via electroporation with 200 ng of I-SceI-flanking resistance cassette with 70-bp homology adjacent to the gene to be deleted. The cassette for MsbB1 was as follows: TGGTGCGGGGCAAGTTGTGCCGCTACACTATCACCAGATTGATTTTTGCCTTATCCGAAACTGGAAAAGCTAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTATAAAAGCCTCTCGCGAGGAGAGGCCTTCGCCTGATGATAAGTTCAAGTTTGCTTCAGAATATTCGAAATCT.The cassette for MsbB2 was as follows: AATTAAGGTTAGATGTATTCTCTGAATAAAATATTAATGATGATTATGGTAGGGGCATTCGCACTAAATATAGGGATAACAGGGTAATATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTACGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATAGGGATAACAGGGTAATAATTATAAAGTACAGGTATTTCCACTAGTTGTTTCTTACAGGTTACCAATCGAAACACATCCCCCTTCCG.Shigella was allowed to recover in Super Optimal broth with Catabolite repression (SOC) for 3 hours at 30\u00b0C and plated on TSB + 50 \u03bcg/mL Kan + 12.5 \u03bcg/mL chloramphenicol (Cam) agar plates with 0.01% Congo Red overnight.The primer pair for identifying MsbB1 was TTGAACTTATCATCAGGCGAAGGCCTC and CGGCTTTTTTTATTTGGTGCGGGG, and that for MsbB2 was CTGCTATCCGCTCTTTGGATGCA and CTACACAGTCCTCCGTGCCAA. Electroporated Shigella + pRedTKI + Cam resistance cassette was incubated at 30\u00b0C, 250 rpm, until an OD650 of 0.6 (approximately 3 hours) in 3 mL TSB + 50 \u03bcg/mL Kan + 20 mmol/L isopropyl \u03b2-d-1-thiogalactopyranoside (IPTG) to induce I-SceI expression to excise the Cam resistance gene. A sample of liquid culture was seeded onto TSB + 50 \u03bcg/mL Kan + agar plates with 0.01% Congo Red and incubated overnight. Modifications were verified by PCR.To complete the gene deletion protocol, a single colony of l-cysteine, NaOH, CaCl2, DNAse I, EDTA, and papain for 35\u00a0min, and then inactivated in serum media. Cells then underwent trituration, were strained through a 100-\u03bcm cell strainer, and were centrifuged for 5\u00a0min at 200 \u00d7 g, 4\u00b0C. The supernatant was aspirated, and cells were counted and replated at 750,000 cells/mL. Neurobasal media at a volume of 2 mL with B-27 supplement, GlutaMAX, and 1.25% FBS was added to a culture plate. Thirty percent of the media was replaced twice per week. After 3 days of culture, 5-fluoro-2\u2032-deoxyuridine (FUDR) was added to remove dividing cells. All mammalian cells were cultured at 37\u00b0C, 5% CO2.GBM cell lines were obtained from Dr. John Kuo  or ATCC . Normal astrocytes were purchased from Lonza (Cat#: CC-2565) and grown according to the manufacturer\u2019s protocol without antibiotics. Mammalian cells were grown in tissue culture-treated T25 or T75 flasks in low-glucose Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with 10% fetal bovine serum (FBS), GlutaMAX, and 1% sodium pyruvate (without pen/strep) \u201330. CellMammalian cells were maintained in culture as described above. At 24 hours prior to co-culture assays, the media was changed without removing cells from the tissue culture flask. Cells grown to 80% confluency in a T25 flask were used for each round of co-culture.S. flexneri was streaked on TSB agar plates with 0.01% Congo Red and grown at 37\u00b0C overnight. The next day, 96 red colonies of Shigella were picked using a filtered p200 pipet tip and added to individual wells of a deep-well block plate that contained 400 \u03bcL of TSB. The deep-well block plate was incubated at 37\u00b0C, 250 rpm, until an OD650 of 0.6 was reached (approximately 2.5 to 3 hours). A control well was used to estimate the Shigella growth rate. After reaching the mid-log phase (0.6 OD650), individual wells were pooled, and the Shigella was concentrated to 2 \u00d7 108 cfu/mL (OD650 of 1.0\u00a0=\u00a08.0 \u00d7 108). Next, 250 \u03bcL of Shigella concentrate was added to ~5 mL of GBM, normal astrocyte (NA), or rNeuron media. Shigella-containing media at a volume of 5 mL was added to the T25 cell culture flask containing mammalian cells. The T25 culture flask was centrifuged at 200 \u00d7 g for 10\u00a0min and then incubated at 37\u00b0C, 5% CO2, for 30\u00a0min. After 30\u00a0min, culture media was removed by aspiration. The T25 flask was washed four times using 5 mL of phosphate-buffered saline (PBS) for 1\u00a0min with gentle agitation. Next, 5 mL of mammalian culture media containing 20 \u03bcg/mL of gentamicin was added to the T25 flask. The T25 flask was incubated at 37\u00b0C, 5% CO2, for an additional 90\u00a0min. After 90\u00a0min, the media was aspirated, and cells were washed four times with PBS, 1\u00a0min per wash, with gentle agitation.2, until GBM, NA, or rNeuron cells detached from the plate. Mammalian cell membranes were mechanically disrupted by pulling the cell suspension through a 27Ga needle 10 times. The mammalian cell lysate was centrifuged at 1,000 \u00d7 g for 2\u00a0min to pellet any membrane-associated  Shigella. Mammalian cell lysate at a volume of 20 \u03bcL was plated onto a TSB + 0.01% Congo Red agar plate and grown at 37\u00b0C overnight. Individual colonies were expanded and frozen in 25% glycerol or used directly for the next round of co-culture assays.After the final wash, 1 mL of Accutase  was added to the T25 flask. The T25 flask was incubated at 37\u00b0C, 5% COd-Lysine, No. 1.5 glass-bottom dishes .At 24 hours prior to internalization, GBM cells were grown to ~80% confluence using the protocol described above in the mammalian cell culture section on Poly-Shigella colony was picked, grown to the mid-log phase, and co-cultured with mammalian cells in the microscopy dish using the protocol described above in the co-culture assay section. After the final wash, cells were fixed with 4% paraformaldehyde (PFA)  for 10\u00a0min and then washed three times with PBS.An individual PBS staining solution at a volume of 1 mL containing wheat germ agglutinin-fluorescein  and Hoechst 33342  was added to the fixed cells and incubated for 15\u00a0min. Cells were washed three times with PBS. After the final wash, 1 mL of PBS was added to the dish so cells did not dry out during imaging. Cells were imaged using a 60\u00d7 objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Shigella antibody  in 1 mL PBS + 30 mg/mL BSA + 0.1% Triton X-100 for 90\u00a0min at room temperature. After incubation, cells were washed three times with PBS. Next, cells were incubated with anti-rabbit Alexa Fluor 488 secondary  in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100  for 60\u00a0min. Cells were washed three times with PBS. Next, cells were incubated with Phalloidin Dye 594  and Hoechst 33342  in PBS  for 15\u00a0min. Cells were imaged using a 60\u00d7 objective on a Nikon Eclipse Ti2 confocal microscope. Images were processed and analyzed using the publicly available ImageJ software package.Cells were permeabilized by adding 1 mL of 0.1% Triton X-100 in PBS for 5\u00a0min. Blocking buffer at a volume of 1 mL  + 5% donkey serum + 0.1% Triton X-100) was added to each dish, and the cells were incubated at 4\u00b0C overnight. The next day, cells were washed three times with PBS. Next, cells were incubated with an anti-At 24 hours prior to internalization, GBM or NA cells were grown to 80% confluence as described in the mammalian tissue culture section above on a tissue culture-treated, black well, clear bottom, 96-well tissue culture plate.Shigella colonies were picked and expanded as described above in a deep block 96-well plate. Shigella culture at a volume of 40 \u03bcL from a single well was added to 200 \u03bcL of GBM or NA media previously plated into each well of the clear-bottom, black, 96-well plate. The 96-well plate was centrifuged at 200 \u00d7 g for 10\u00a0min and then incubated at 37\u00b0C, 5% CO2, for 30\u00a0min. Culture media was removed after 30\u00a0min by dumping the supernatant into a sterile glass dish containing bleach. The plate was washed four times with PBS using a similar method to remove the supernatant. Next, 200 \u03bcL of mammalian cell media containing 20 \u03bcg/mL gentamicin was added to each well. The plate was incubated at 37\u00b0C, 5% CO2, for 90\u00a0min. After incubation, the plate was washed four times with PBS. Cells were fixed by adding 200 \u03bcL of 4% PFA to each well and incubating for 10\u00a0min. The plate was washed three times with PBS. Cells were permeabilized by adding 200 \u03bcL of PBS containing 0.1% Triton X-100 to each well for 5\u00a0min. Blocking buffer containing 1% BSA and 1% donkey serum was added to each well, and the plate was incubated at 4\u00b0C overnight. The next day, the plate was washed three times with PBS. The plate was incubated with anti-Shigella antibody  in PBS + 30 mg/mL BSA + 0.1% Triton X-100 (100 \u03bcL per well) for 90\u00a0min. The plate was washed with PBS three times. Finally, each well was incubated with IR800CW anti-rabbit secondary  in PBS + 30 mg/mL BSA + 5% donkey serum + 0.1% Triton X-100 for 60\u00a0min. The plate was washed three times with PBS and imaged on a LI-COR Fc scanner using a 2-min medium resolution scan. A standard curve of Shigella was used on each plate to convert the IR800 signal to the number of bacteria per well. It was important to ensure that the moles of anti-Shigella antibody greatly exceeded the moles of Shigella to ensure the saturated binding assumption was valid in order to quantify the number of bacteria per well.Individual Shigella internalization in cell lines were determined using ANOVA with a 1% false discovery rate used as the threshold for significance. For microscopy assays, ANOVA was used to determine differences between groups with a 5% false discovery rate used as the threshold for significance.Data are generally presented as means with standard deviation. All validation experiments were conducted with a minimum of two independent replicates. For microscopy experiments, a minimum of three fields were quantified from at least two independent experiments. Significant differences between Shigella clones that preferentially internalize into brain tumors using iterative co-culture assays. Briefly, S. flexneri, an intracellular bacterium, was streaked onto agar plates. Clones containing the virulence factor needed to survive inside mammalian cells were expanded and incubated with a brain tumor cell line, U-251 GBM. Bacteria that internalized into GBM cells were harvested and used for additional rounds of co-culture.As described in Shigella strain that is safe to use as a therapeutic platform, we identified a strain that did not contain Shiga toxins. We probed S. flexneri strain 2475 serotype 2a for DNA regions encoding Shiga toxins using previously described primers. As expected, we did not observe a band at the expected size for the PCR product  when stained with a DNA intercalating dye Hoechst 33342 (rendered blue). The number of GBM cells demonstrating an internalized bacterium increased after each round of Shigella co-culture, starting with 5% \u00b1 2.5% in round 10 and enriching to 95% \u00b1 1% by round 50 of co-culture. The percentage of GBM cells with an internalized bacterium was calculated by taking three randomly selected fields and dividing the total number of infected cells by the total number of U-251 cells present , phalloidin-AF594, and Hoechst 33342 then imaged via confocal microscopy. The DNA rod-like structures observed inside GBM cells throughout this study co-localized with an anti-Shigella antibody  lines were incubated with GBM-infecting Shigella. Internalized Shigella was harvested and plated to count the number of internalized bacteria per group. GSC 115 showed the highest internalization at 123,800 bacteria/5 \u00d7 105 GBM cells. To demonstrate the specificity of the platform, GBM-infecting Shigella were incubated with normal astrocytes and internalized bacteria counted. Approximately 383 bacteria internalized/~300 NA cells (two separate patient cell lines) were observed. Next, GBM-infecting Shigella and compared the data to those of the parental strain. We identified 177 mutations in 46 genes . Removal of both MsbB1 and MsbB2 completely abrogated internalization to an average of <1 bacterium recovered. Confocal imaging of GBM cells treated with either R50, MsbB1 KO, or MsbB1 and MsbB2 KO demonstrate a significant decrease in cells internalizing Shigella (Shigella-positive cells dropped from 95% to 25% (p < 0.05 via ANOVA). Quantifying the fluorescent Shigella signal indicated that MsbB1 removal reduced the mean signal to 0.425 rfu compared to 6.16 rfu for R50 . Removal of both MsbB1 and MsbB2 resulted in 0% of cells with a Shigella internalized and a fluorescent signal of 0.009 rfu (~684-fold decrease). GBM cells that did demonstrate Shigella internalization after removal of myristoylation factor MsbB1 had similar numbers of internalized Shigella compared to R50. The growth curves of R50, MsbB1 KO, and MsbB1 and MsbB2 KO Shigella are presented in We removed MsbB1 and MsbB2 myristoylation factors from the round 50 GBM-infecting ineering \u201337, remoShigella Figure\u00a05S. flexneri clones that selectively internalize into GBM cell lines. We first selected a strain of Shigella that does not express Shiga toxins to improve the safety profile of the platform  , doxycycline, or polymyxins, can serve as master suicide switch(es) to immediately halt GBM-infecting Shigella activity in the case of adverse events such as fever or general infection  , 41. A rblastoma . Additioe genome , 27. Thun system , 20. In n system , 24, 27.nfection . It is iShigella to internalize into GBM cells as a foundation for a drug delivery system. We envision this platform serving as a tool for neurosurgeons to clean the margins of a GBM post-resection. GBM-infecting Shigella could be administered into the surgical cavity before closing to infect and eradicate cancerous cells in the invasive margin that cause disease recurrence. This approach bypasses the need for systemic administration and reduces the potential for internalization into off-target cells in the body. However, many steps are required before GBM-infecting Shigella are suitable for a drug delivery platform including stabilizing genetic material, controlling the bacterial population, ensuring intracellular replication, modulating immunogenicity, and weaponizing the system with cytotoxic proteins such as ribosome toxins (gelonin), cytokines (IL-2 and TNF), and autophagy-inducing proteins (caspases)  \u201346. AddiShigella into GBM cells. Removal of MsbB1 and MsbB2 myristoylation enzymes completely abrogates the internalization of GBM-infecting Shigella in GBM cells. Previous reports indicate that removing MsbB genes does not impair the internalization of Shigella in intestinal cells, the typical target of wild-type Shigella or Salmonella Typhimurium invasion into tumor cells  is essential for GBM-infecting Shigella to internalize into GBM cells. However, our data do not indicate what that factor is. Whole genome sequencing did not identify obvious mutations in MsbB genes or a known myristoylated protein; however, future RNA-sequencing, epigenetic, and/or proteomic studies of Shigella membrane-bound proteins could identify factors with differential copy number, alterations in promoters, and/or de-novo localization to bacterial membranes that are driving the observed changes in Shigella internalization in GBM cells. These studies can be narrowed bioinformatically by eliminating proteins of interest that do not contain an N-terminal glycine  that is required for modification with myristic acid  can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.AS designed assays and wrote the manuscript, GF designed assays and edited the manuscript, BD conceptualized the study and edited the manuscript, and BU conceptualized the study, designed assays, and wrote the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "Chlamydomonas reinhardtii delta tubulin (uni3) and epsilon tubulin (bld2) genes through an unknown mechanism. Our studies revealed that intact PCR products for both these genes could be obtained upon PCR amplification from plasmid templates carrying these genes. However, interestingly, purification of these PCR products led to their cleavage through an unidentified mechanism. This cleavage persisted despite using different PCR purification kits. Deleting a synthetic intron within the delta tubulin gene also did not have any effect on this cleavage.In this study, we report an unusual phenomenon of the self-cleavage of purified PCR products of codon-optimized Centrosomes consist of orthogonally-paired cylindrical centrioles enveloped by the protein-dense pericentriolar material .Chlamydomonas reinhardtii delta and epsilon tubulin proteins that are coded by theuni3 andbld2 genes respectively, play a crucial role in maintaining the proper triplet microtubule architecture of centrioles . To obtain further insights into the functions of these genes in regulating centriole architecture, we wanted to generate purified PCR products ofChlamydomonas reinhardtiidelta and epsilon tubulin cDNAs carrying synthetic introns that were codon-optimized for expression inC. elegans. These purified PCR products would enable us to perform downstream functional assays to further characterize the functions of these genes. Although we were successful in obtaining full length PCR products of codon-optimizedChlamydomonas reinhardtiidelta and epsilon tubulin genes, these PCR products exhibited an unexpected cleavage upon their purification using either the Invitrogen Purelink\u2122 Quick PCR purification kit or the Qiaquick\u00ae PCR purification kit.As shown in, both the delta and epsilon tubulin PCR products but not a non-relevant control PCR product (dyn-1) exhibited cleavage after their purification using the Invitrogen Purelink\u2122 Quick PCR purification kit or the Qiaquick\u00ae PCR purification kit. Purification of the delta and epsilon tubulin PCR products using a non-kit-based isopropanol precipitation method as well as upon their gel extraction using a Qiaquick Gel Extraction kit , both yielded this similar pattern of cleavage. To confirm that the cleaved fragments indeed correspond to the PCR products of the delta and epsilon tubulin genes, DNA sequencing of these lower sized fragments was performed after excising these separated bands from the agarose gel. Our sequencing results confirmed that the lower sized fragments that were observed between 650 bp and 1000 bp upon agarose gel electrophoresis of the purified delta and epsilon tubulin PCR productscontained DNA sequences from these respective genes. Our sequencing analysis identified a synthetic intron close to the genomic region where the delta and epsilon tubulin PCR products were being cleaved. Since the sequence of this synthetic intron was identical between the delta and epsilon tubulin genes, we questioned whether the presence of this synthetic intron was responsible for the cleavage of the purified PCR products of these genes. To address this, site-directed mutagenesis was performed to remove this synthetic intron from the sequence of the delta tubulin gene and the PCR purification procedure was repeated. As shown indeletion of the synthetic intron close to the cleavage region within the delta tubulin gene did not eliminate cleavage of the purified PCR product. These data indicate that this synthetic intron does not contribute to this observed cleavage phenomenon. In the future, studies should be directed to further characterize the molecular mechanisms mediating the cleavage of theChlamydomonas reinhardtii delta and epsilon tubulin PCR products upon purification. Since we have invested a considerable amount of time and resources on this project, we would like to share these findings with the scientific community and with other researchers working on these genes to prevent a further loss of their valuable time and resources.Centrosomes are the major microtubule organizing centers of most animal cells. They contribute to a variety of cellular functions including spindle assembly, cell polarity, cell shape and cilia assembly (Reviewed in NiggI) PCR conditions, analysis, and purificationPCR was performed using a C1000\u2122 Thermal Cycler . 35 cycles of amplification were used for 50 \u00b5L PCR reaction volume consisting of:uni3,bld2,dyn-1), 1 \u00b5L - 10 mM dNTPs, 2 \u00b5L- 50 mM MgSO4, 5 \u00b5L - 10X Hi-Fi Buffer, 0.2 \u00b5L - Platinum\u00aeTaqDNA Polymerase Hi -Fi enzyme, and molecular biology water was used to bring the final volume to 50 \u00b5L.1 \u00b5L- 10 \u00b5M forward primer, 1 \u00b5L- 10 \u00b5M reverse primer, 35 ng- plasmid template  was obtained from a synthesized plasmid construct that contains the coding sequence for blue fluorescent protein with homology arms to theC. elegans dyn-1 gene.The control PCR product (dyn-1) PCR:PCR conditions for the control  Site-directed mutagenesisuni3 gene except that the Phusion\u00ae High-Fidelity DNA Polymerase was used for PCR amplification.For performing the site-directed mutagenesis, the PCR reaction mixture comprised of all the same components used in PCR amplification of thePCR conditions for delta tubulin intron deletion using site-directed mutagenesis:Initial denaturation: 98\u00b0C - 30 sDenaturation: 98\u00b0C - 10 sAnnealing: 68.6\u00b0C - 30 sExtension: 72\u00b0C - 6 minFinal Extension: 72\u00b0C - 10 minSite-directed mutagenesis was followed by digestion with 5 units of Dpn1 for 2 hours, followed by bacterial transformation and plasmid purification using the QIAprep\u00ae Spin Miniprep kit. The isolated plasmid samples were sent for whole plasmid sequencing . Upon analyzing the sequencing results, the plasmid with successful intron deletion was selected and used for further analysis.Primers and sequences used for studyI) Primers for PCR amplification of Epsilon tubulinForward primer: 5\u2019- ATT CGA ATA TAT ATT GTC AGT TG -3\u2019Reverse primer: 5\u2019- ATA CGA GGA TTA TGG TAC AAG -3\u2019II) Primers for PCR amplification of Delta tubulinForward primer: 5\u2019- TTG TTT CTT TCT TTT AAT GTT AAA TAT TTC CAG AAC TAT GCC ATG -3\u2019Reverse primer: 5\u2019- GAT AAA ATA ATT ATT CGG GCA GTA ATA AAA CAG GGA TCT ATC ACT TC -3\u2019III) Primers for site-directed mutagenesis for Delta tubulin with one intron deletedForward primer: 5\u2019- TCG AGG AGG CCG GAC TCA AGG GAC AAT CCT CCG GAC CAG G -3\u2019Reverse primer: 5\u2019- CCT GGT CCG GAG GAT TGT CCC TTG AGT CCG GCC TCC TCG A -3\u2019(dyn-1):IV) Primers for amplification of controlForward primer: 5\u2019- AAA ATC GAT TTT CAG GTA GTT CAG C -3\u2019Reverse primer: 5\u2019- TTG ATC ACA GGG ATC AAC GCC -3\u2019Chlamydomonas reinhardtii delta tubulin (uni3) cDNA codon optimized for expression inC. elegans (contains synthetic introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lowercase)):V) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGTCGTCGCCGGAGCCCGTTCCGCCGCCGCCGCCTCCGGATCCTGGTGGCGTTACCCATCCTCCGGATACCTCGTCATGCAATCCGGATCCGGAAACAACTGGGCCCAAGGATTCCACGGATACGGACCACAAGTCCACGAGGACGCCCTCGACCTCGTCCGTAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGAGGTCGAGCACGCCGACTCCCTCACCGGATTCCTCCTCCTCCAATCCATGGCCGGAGGAACCGGAGCCGGACTCGGAACCTACGTCGCCGAGGCCCTCCGTGACGAGTACCACTCCGCCTTCGTCGCCAACTGCTGCGTCTGGCCATACGAGTCCGGAGAGGTCATCGTCCAACCATACAACACCCTCCTCACCCTCTCCCACCTCGCCGACGTCTCCGACGGACTCGTCCTCCTCGAGAACGAGGCCCTCCACCGTACCGCCGCCAAGCTCTACGGAATCGCCCGTCCATCCTTCGGAGTCCGTGGACGTGTCCTCGGACGTGCCGGAGAGTCCCGTGTCGAGGAGGCCGGACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGACAATCCTCCGGACCAGGAGGATGGGGAGTCTGCACCGCCCCACTCGCCGAGCTCGTCACCCGTCTCTGCGGACACCCAGCCTACCGTCTCCTCACCCTCCGTTCCGTCCCACAACTCCCACCAGCCAACATCGACTTCACCACCTTCACCTGGCCAGCCCTCACCAAGCGTCTCCGTCAAATGCTCGTCACCGGATCCGTCCTCGAGGAGGGACTCGACTGGTCCATCACCCCACAATCCCCAGGAGCCGCCGCCGCCCTCGGAGCCGGACTCGCCGGACCAACCGTCAACCGTGCCCTCGCCTCCTGGCTCATCCTCCGTGGACAAGGAGCCGCCGAGGCCGACGTCGGAGAGTTCGCCGACCCAGCCCTCTCCGCCGCCTGGTCCCCAGAGCCACTCTCCGTCTCCTACTCCACCGGACGTTTCGGACGTTGCGCCATGTCCGCCTGCCTCCTCTCCAACGACCGTCACTGCGTCGGACCAATCCAACGTATGCAAGAGCACGCCTACGGAATGCTCGAGTCCCGTGCCTTCGTCCACCAATACGAGAAGTACGGACTCTCCGTCGCCGAGTTCCAAGACTGCTTCGCCCGTATCGAGGACATCGCCCAACGTTACGCCCGTCTCGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGATTACAAGGATGACGATGACAAGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgatagatccctgttttattactgcccgaataattattttatc-3\u20195\u2019-ttgtttctttcttttaatgttaaatatttccagaactATGCCATGCATCACCCTCCAACTCGGACAATGCGGAAACCAACTCGGATGCTCCCTCTTCAACACCCTCGCCACCGAGTTCTCCTCCCACGACTACGGAACCGACGCCGTCCACGAGTACTTCCGTCCATCCGCCGACCCAAACCTCTACACCGCCCGTTCCGTCCTCATCGACATGGAGCCAAAGChlamydomonas reinhardtii epsilon tubulin (bld2) cDNA codon optimized for expression inC. elegans (contains introns (lowercase + underlined) and homology arms for CRISPR genome editing (only lower case)):VI) Sequence ofgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagATCCGTCTCACCGCCGAGGACTGCGACTCCCTCCAATCCTTCATGGTCCTCCACTCCCTCGGAGGAGGAACCGGATCCGGAGTCGGAACCTACATCGTCCGTATGCTCGCCGACGAGTTCCCAGGAGTCTTCCGTTTCACCGGATCCGTCTTCCCATCCGAGGACGACGACGTCGTCACCTCCCCATACAACGCCATGCTCGCCCTCGGACAACTCGTCGAGCACGCCGACTGCGTCCTCCCAATCGAGAACCAAGCCCTCATCGACATCGTCAACCGTACCGAGGCCGCCCGTGACCGTGCCGCCGCCGCCGACGCCGCCGCCTCCGCCGTCTCCGGACTCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGATCCGGAGGAGGATCCAAGCCATTCGACTCCATGAACGGAGTCGCCGCCTCCCTCCTCCTCCACCTCACCGCCTCCGTCCGTTTCGAGGGACCACTCAACGTCGACCTCAACGACATCACCATGAACCTCGTCCCATACCCACGTATGCACTTCCTCCTCTCCTCCATGTCCCCACTCCAACCACCACCAAAGGACAAGGACCCACGTACCCTCGACCAAGTCCGTGTCTTCGGAGACGTCTTCTCCCGTGAGCACCAACTCATCCGTGCCGACCCACGTGCCGCCACCTACCTCGCCTGCGGACTCATCGCCCGTGGACCAACCGCCACCATGGCCGACATCAACCGTAACGTCGCCCGTCTCCGTCCACAACTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagATGGTCCACTGGAACTCCGAGGGATTCAAGCTCGGAATCTGCTCCACCCCACCAGTCGGATGCCCATTCGGACTCCTCTGCCTCGCCAACAACACCGCCATCGCCCACACCTTCACCACCATGCGTGAGCGTTTCGACAAGCTCTACAAGCGTCGTTTCTACACCCACCACTACGAGCAATACATGGACCCAGGAGGATTCACCTCCGCCATGGAGGTCGTCGGAGACCTCACCGCCCAATACCGTGCCCTCGAGGGAGCCACCCAAGCCCCACCACTCACCCGTCTCCGTCCACGTGGACTCTCCTTCCTCCCAGGAGGTTCCGGTGGTTCTGGTGGATCCGGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGtgataatattcatttaatccaacttgtaccataatcctcgtat-3\u20195\u2019- attcgaatatatattgtcagttgttctgtttgtcgtcgtgATGCCACGTGAGCTCGTCACCATCCAAGTCGGACAATGCGGAAACCAAGTCGGATGCCGTTTCTGGGAGCTCGCCCTCCGTGAGCACGCCGCCTACAACACCAAGGGAGTCTACGACGAGGCCCTCTCCTCCTTCTTCCGTAACGTCGACACCCGTGTCGAGCCACCACGTAACCTCCCAGTCGGAGAGGGACGTGGAGCCATCCGTACCCTCAAGGCCCGTTCCGTCATCGTCGACATGGAGTGCGGAGTCATCAACGAGATGCTCAAGGGACCACTCGGAGAGGTCCTCGACACCCGTCAACTCGTCTCCGACGTCTCCGGAGCCGGAAACAACTGGGCCCACGGACACCACGAGTACGGACCACGTTACCACGACGCCATCCTCGACAAG(dyn-1):VI) Sequence of the positive control used in this study5\u2019- AAAATCGATTTTCAGGTAGTTCAGCGTATAACCACCAGGATCAGCGATGGTTTCGGAATTGATTAAAGAAAATATGCACATGAAGCTCTACATGGAGGGAACCGTCGACAACCACCACTTTAAATGTACCTCCGAGGGAGAGGGAAAGCCATACGAGGGAACCCAAACCATGCGTATCAAGGTCGTCGAGGGTGGTCCGCTCCCATTCGCCTTTGATATCCTCGCCACCTCCTTCCTCTATGGTTCCAAGGTAAGTTTAAACATATATATACTAACTAACCCTGATTATTTAAATTTTCAGACCTTCATCAACCACACCCAAGGAATCCCAGACTTTTTTAAACAATCCTTCCCAGAGGGATTCACCTGGGAGCGTGTCACCACCTACGAGGACGGAGGAGTCCTCACCGCCACCCAAGACACCTCCCTCCAAGACGGATGCCTCATCTACAACGTCAAGGTAAGTTTAAACAGTTCGGTACTAACTAACCATACATATTTAAATTTTCAGATCCGTGGAGTCAACTTCACCTCCAACGGACCAGTCATGCAAAAGAAGACCCTCGGATGGGAGGCCTTCACCGAGACCCTCTACCCAGCCGACGGAGGACTCGAGGGACGTAACGACATGGCCCTCAAGCTCGTCGGAGGATCCCACCTCATCGCCAACGCCAAGGTAAGTTTAAACATGATTTTACTAACTAACTAATCTGATTTAAATTTTCAGACCACCTACCGTTCCAAGAAGCCAGCCAAGAACCTCAAGATGCCAGGAGTCTACTACGTCGACTACCGTCTCGAGCGTATCAAGGAGGCCAACAACGAGACCTACGTCGAGCAACACGAGGTCGCCGTCGCCCGTTACTGCGACCTCCCATCCAAGCTCGGACACAAGCTCAACTACCCATATGATGTTCCAGATTACGCTGGAGGATCTGGAGGCGGTTCTGGCGGAGGTTCTGGTATGTCGTGGCAAAACCAGGGAATGCAGGCGTTGATCCCTGTGATCAA \u2013 3\u2019"}
+{"text": "Blumeria graminis forma specialis tritici (B.g. tritici) is the airborne fungal pathogen that causes powdery mildew disease on hexaploid bread wheat. Calmodulin-binding transcription activators (CAMTAs) regulate plant responses to environments, but their potential functions in the regulation of wheat\u2013B.g. tritici interaction remain unknown. In this study, the wheat CAMTA transcription factors TaCAMTA2 and TaCAMTA3 were identified as suppressors of wheat post-penetration resistance against powdery mildew. Transient overexpression of TaCAMTA2 and TaCAMTA3 enhanced the post-penetration susceptibility of wheat to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing compromised wheat post-penetration susceptibility to B.g. tritici. In addition, TaSARD1 and TaEDS1 were characterized as positive regulators of wheat post-penetration resistance against powdery mildew. Overexpressing TaSARD1 and TaEDS1 confers wheat post-penetration resistance against B.g. tritici, while silencing TaSARD1 and TaEDS1 enhances wheat post-penetration susceptibility to B.g. tritici. Importantly, we showed that expressions of TaSARD1 and TaEDS1 were potentiated by silencing of TaCAMTA2 and TaCAMTA3. Collectively, these results implicated that the Susceptibility genes TaCAMTA2 and TaCAMTA3 contribute to the wheat\u2013B.g. tritici compatibility might via negative regulation of TaSARD1 and TaEDS1 expression. Triticum aestivum L.) has served as a major staple food for thousands of years and provided about 20% of the calories consumed by humans [Blumeria graminis forma specialis tritici (B.g. tritici), leading to 5\u201350% yield losses [B.g. tritici-resistant wheat cultivars [B.g. tritici and identify key regulators of wheat resistance against powdery mildew disease.As one of the most widely grown small-grain cereal crops, bread wheat  transcription factors, calmodulin-binding transcription activators (CAMTAs) play important roles in regulating plant growth, development, and responses to environmental stresses [CAMTA genes differentially respond to environmental cues like drought, salinity, and extreme temperatures in the model plant Arabidopsis thaliana [Arabidopsis mutant camta1 exhibited hypersensitivity to cold and drought stress, and AtCAMTA1 was shown to regulate the expression of cold and drought-responsive genes like AtRD26, AtERD7, AtCBF2, and AtRAB18 [Arabidopsis mutant camta6 exhibited hypersensitivity to NaCl treatment, and AtCAMTA6 was demonstrated to regulate expression of salt resilience-related genes, including + TRANSPORTER1HIGH-AFFINITY K, SALT OVERLY SENSITIVE1, and +/H+ ANTIPORTERNa [Arabidopsis AtCAMTA3 was shown to function in concert with AtCAMTA1 and AtCAMTA2 in suppressing plant defense responses [B.g. tritici remains largely unknown.As Castresses ,20,21. Fthaliana ,24,25,26 AtRAB18 ,24,25,26TIPORTER . In addiesponses ,30,31,32TaCAMTA2 and TaCAMTA3, were characterized as Susceptibility (S) genes contributing to wheat\u2013B.g. tritici compatibility. Transient overexpression of TaCAMTA2 and TaCAMTA3 resulted in enhanced wheat post-penetration susceptibility to B.g. tritici, while transient silencing of TaCAMTA2 and TaCAMTA3 led to attenuated wheat post-penetration susceptibility to B.g. tritici. Furthermore, overexpressing TaSARD1 and TaEDS1 could confer wheat post-penetration resistance against powdery mildew, while silencing TaSARD1 and TaEDS1 enhanced wheat post-penetration susceptibility to B.g. tritici. Moreover, TaCAMTA2 and TaCAMTA3 were demonstrated to negatively regulate the expression of the defense genes TaSARD1 and TaEDS1. These results strongly support that S genes TaCAMTA2 and TaCAMTA3 partially redundantly suppress wheat post-penetration resistance against B.g. tritici presumably via the negative regulation of expressions of defense genes TaSARD1 and TaEDS1.In this research, two CAMTA transcription factor genes, Arabidopsis CAMTA transcription factor AtCAMTA3 plays a vital role in the regulation of plant immunity [B.g. tritici interaction. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtCAMTA3 (At2g22300) as a query and obtained TaCAMAT2 and TaCAMTA3, the most closely related homologs of AtCAMTA3, in bread wheat. Three highly homologous sequences of TaCAMAT2 genes separately located on chromosomes 4A, 4B, and 4D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA2-4A (TraesCS4A02G407100), TaCAMTA2-4B (TraesCS4B02G306300), and TaCAMTA2-4D (TraesCS4D02G304500). Similarly, three highly homologous sequences of TaCAMAT3 genes separately located on chromosomes 2A, 2B, and 2D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA3-2A (TraesCS2A02G163000), TaCAMTA3-2B (TraesCS2B02G188800), and TaCAMTA3-2D (TraesCS2D02G169900).Previous studies revealed that the immunity ,30,31,32Arabidopsis AtCAMTA3. In addition, TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, and TaCAMTA3-2D proteins all contain a conserved CG-1 DNA-binding domain at their N-terminal parts, a transcription factor immunoglobulin-like (TIG) DNA-binding domain, several ankyrin repeats (ANK) in the middle parts, as well as two IQ CaMB motifs (IQXXXRGXXXR) at their C-termini  increased from 56% for the empty vector (OE-EV) control to above 70% on wheat cells overexpressing TaCAMTA2 or TaCAMTA3 genes. These results suggested that overexpression of TaCAMAT2 and TaCAMTA3 could significantly enhance wheat post-penetration susceptibility to B.g. tritici.To study the function of TaCAMAT2 and TaCAMTA3 in the regulation of wheat\u2013B.g. tritici interaction, we employed transiently induced gene silencing (TIGS) assays to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. After inoculation of conidia from the virulent B.g. tritici isolate E09, the frequency of fungal haustorium formation in the transformed plant cells was scored. As shown in TaCAMAT2 or TaCAMTA3 genes resulted in a marked HI% decrease to about 27%, compared to 33% for empty vector (EV) controls. Significantly, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further decrease in HI% to approximately 13%, suggesting that TaCAMTA2 and TaCAMTA3 might partially redundantly suppress post-penetration resistance of wheat to B.g. tritici.To further verify the function of TaCAMAT2 or TaCAMTA3 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. qRT-PCR showed that the endogenous transcript level of TaCAMAT2 or TaCAMTA3 was substantially reduced in the indicated VIGS plants  declined to approximate 40% on BSMV-TaCAMTA2as plants and 47% on BSMV-TaCAMTA3as plants, compared with 55% for the BSMV-\u03b3 plants -induced gene silencing (BSMV-VIGS) to silence all endogenous S plants C. Therea\u03b3 plants D. NotablSYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (AtSARD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (AtEDS1) in A. thaliana [Arabidopsis AtSARD1 (At1g73805) and AtEDS1 (At3g48090) as a query and obtained TaSARD1 and TaEDS1, the most closely related homologs of AtSARD1 and AtEDS1, in bread wheat. Five highly homologous sequences of TaSARD1 genes separately located on chromosomes 6A, 6B, and 6D were obtained from the genome sequence of the hexaploid wheat and designated as TaSARD1.1-6A (TraesCS6A02G091700), TaSARD1.1-6B (TraesCS6B02G119900), TaSARD1.1-6D (TraesCS6D02G080500), TaSARD1.2-6A (TraesCS6A02G296600), and TaSARD1.2-6D (TraesCS6D02G276800). Similarly, three highly homologous sequences of TaEDS1 genes separately located on chromosomes 5A, 5B, and 5D were obtained from the genome sequence of the hexaploid wheat and designated as TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D [Previous studies revealed that AtCAMTA3 could regulate the expression of defense genes thaliana ,30,31,32aEDS1-5D .Arabidopsis AtSARD1. In addition, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, and TaSARD1.2-6D proteins all contain a CBP60-conserved domain  resistance genes and quantitative trait loci (QTL) contributed to wheat resistance to B.g. tritici and have been employed in wheat breeding for powdery mildew resistance [B.g. tritici underlies wheat\u2019s susceptibility to powdery mildew. A plethora of wheat S genes have been identified to facilitate compatibility by inducing B.g. tritici (pre)penetration, suppressing wheat immunity, and supporting the sustenance of B.g. tritici [S genes TaWIN1, TaKCS6, and TaECR were revealed to facilitate the conidial germination of B.g. tritici by promoting the biosynthesis of wheat cuticular wax, whereas wheat S gene TaSTP13 encodes a sugar transporter facilitating wheat hexose accumulation for B.g. tritici acquisition [TaMLO, TaEDR1, and TaPOD70 genes contribute to wheat susceptibility to powdery mildew by suppressing plant defense responses [B.g. tritici by suppressing defense-related transcriptional reprogramming in bread wheat [Powdery mildew, caused by the adapted fungal pathogen oduction ,4. To imeraction ,4. Powdesistance ,4. Compa tritici ,35. For uisition ,39,40,41esponses ,45,46,47ad wheat ,51,52,53TaCAMAT2 and TaCAMTA3 were identified as the most closely related homologs of AtCAMTA3, which is consistent with the reported phylogenetic analysis of the CAMTA homologs in different species [TaCAMAT2 and TaCAMTA3 are characterized as wheat S genes contributing to the wheat post-penetration susceptibility to B.g. tritici in this study. Overexpression of TaCAMTA2 and TaCAMTA3 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat susceptibility to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing resulted in compromised wheat post-penetration susceptibility to B.g. tritici. Interestingly, a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes the Arabidopsis homologs of wheat TaCAMTA2 and TaCAMTA3, could suppress the edr2-associated powdery mildew resistance [sr1-4D single mutant is more susceptible to Arabidopsis powdery mildew (Golovinomyces cichoracearum), whereas the sr1-1 null mutant plants displayed enhanced post-penetration resistance against G. cichoracearum [Arabidopsis AtCAMTA1 was revealed to function partially redundantly with AtCAMTA2 and AtCAMTA3 in suppressing plant immunity [TaCAMAT2 and TaCAMTA3 could lead to a further decrease in the HI% and MI% compared with single silencing of TaCAMAT2 or TaCAMTA3, supporting the fact that TaCAMTA3 functions partially redundantly with TaCAMAT2 in suppressing wheat post-penetration resistance against B.g. tritici. In Arabidopsis, CAMTA transcription factors AtCAMTA1, AtCAMTA2, and AtCAMTA3 partially redundantly suppress the biosynthesis of salicylic acid (SA) and N-hydroxypipecolic acid (NHP), a metabolite duo essential for systemic acquired resistance (SAR) [S genes TaCAMAT2 and TaCAMTA3 in the regulation of SA and NHP biosynthesis, as well as SAR establishment, in bread wheat in future research.Through homology-based searching,  species . TaCAMATsistance . The sr1racearum . In addiimmunity ,31,32. Ice (SAR) ,31,32. TTaSARD1 and TaEDS1 are identified as positive regulators of wheat resistance against B.g. tritici in this study. Overexpression of TaSARD1 or TaEDS1 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat post-penetration resistance to B.g. tritici, while knockdown of TaSARD1 or TaEDS1 expression using transient- or virus-induced gene silencing resulted in increased wheat post-penetration susceptibility to B.g. tritici. In Arabidopsis, transcription factor AtSARD1 functions in concert with AtCBP60g to activate the expression of SID2 (SA INDUCTION DEFICIENT 2), which encodes isochorismate synthase 1 (ICS1), essential for pathogen-induced SA biosynthesis [Arabidopsis AtEDS1 was shown to heterodimerize with its partners, phytoalexin deficient 4 (PAD4) or senescence-associated gene 101 (SAG101), to play signaling roles in ETI as well as SA-dependent and SA-independent PTI pathways [TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were attenuated by silencing of TaSARD1 or TaEDS1, suggesting that the SARD1-EDS1-SA defense axis might be partially conserved between model plant Arabidopsis and crop plant bread wheat. Therefore, it is intriguing to examine the potential regulation of wheat SA biosynthesis and signaling by TaSARD1 and TaEDS1 in future research.ynthesis ,55,56. Apathways ,62,63,64TaSARD1 and TaEDS1 were significantly enhanced by silencing TaCAMTA2 and TaCAMTA3. Notably, simultaneous silencing TaCAMAT2 and TaCAMTA3 could lead to a further increase in the expression levels of TaSARD1 and TaEDS1 compared with single silencing TaCAMAT2 or TaCAMTA3, indicating that TaCAMTA2 and TaCAMTA3 partially redundantly suppress expressions of TaSARD1 and TaEDS1. In Arabidopsis, AtCAMTA3 could bind to the promoter region of AtEDS1 by recognizing the CGCG box, thereby directly repressing the expression of AtEDS1 [AtSARD1 was demonstrated to be negatively regulated by partially redundant AtCAMTA1, AtCAMTA2, and AtCAMTA3, presumably via an indirect effect [SARD1 and EDS1 by partially redundant CAMTA3 and its homologs might be partly conserved between the model plant Arabidopsis and the important crop bread wheat. Indeed, the expressions of SA defense marker genes TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were found to be potentiated by silencing TaCAMAT2 or TaCAMTA3 in this study. However, binding sites for TaCAMAT2 and TaCAMTA3 in the promoter regions of TaSARD1 and TaEDS1 genes remain to be identified.In this study, expression levels of f AtEDS1 ,29,30,31t effect ,29,30,31TaCAMAT2 and TaCAMTA3 are identified as wheat S genes partially redundantly suppressing post-penetration resistance against powdery mildew, presumably via negative regulation of the expressions of defense genes TaSARD1 and TaEDS1. Genetic manipulation of S genes TaMLO and TaEDR1 via targeting induced local lesions in genomes (TILLING) and genome editing techniques like transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) 9 systems compromised wheat compatibility with B.g. tritici and conferred wheat resistance against powdery mildew [TaCAMAT2 and TaCAMTA3 in wheat breeding for powdery mildew resistance in future research.Herein, y mildew ,71,72,73B.g. tritici strain E09 was maintained on the leaves of Jing411 plants. Conidia of B.g. tritici strain E09 were used for the inoculation of Jing411 leaves in the study of wheat\u2013powdery mildew interaction. Arabidopsis thaliana used in this study was grown in the greenhouse under a 16 h/8 h light period at 23 \u00b1 1 \u00b0C with 70% relative humidity.The seedlings of bread wheat cultivar Yannong999 used in this study were grown in a growth chamber under a 16-h/8-h, 20 \u00b0C/18 \u00b0C day/night cycle with 70% relative humidity. The GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (TaGAPDH) was set as the internal control and expressions of TaGAPDH, TaCAMTA2, TaCAMTA3, TaSARD1, TaEDS1, TaPR1, TaPR2 and TaPR5 were analyzed using the primers 5\u2032-TTAGACTTGCGAAGCCAGCA-3\u2032/5\u2032-AAATGCCCTTGAGGTTTCCC-3\u2032, 5\u2032-TACAGAAGTTGCAACAG-3\u2032/5\u2032-ATCTCCGTCGACTCCTCA-3\u2032, 5\u2032-CCTGACAAACAACTTGA-3\u2032/5\u2032-CGCCAGCTGCA TCGCTT-3\u2032, 5\u2032-GCGAGTAATGAAAGCAT-3\u2032/5\u2032-TTAATCAACTTGATCCC-3\u2032, 5\u2032-TGAAAGACAGGGTGGGT-3\u2032/5\u2032-CGAAGGCACAAGTCTCG-3\u2032, 5\u2032-GAGAATGCAGACGCCCAAGC-3\u2032/5\u2032-CTGGAGCTTGCAGTCGTTGATC-3\u2032, 5\u2032-AGGATGTTGCTTCCATGTTTGCCG-3\u2032/5\u2032-AAGTAGATGCGCATGCCGTTGATG-3\u2032, and 5\u2032-CTTCTACATCAAGA ACAACTG-3\u2032/5\u2032-CAGTCGCCGGTCTGGCAG-3\u2032.Total RNA was extracted from the wheat leaves using the EasyPure Plant RNA kit  and 2 \u03bcg of RNA was used to synthesize the cDNA template using the TransScript one-step gDNA removal and cDNA synthesis supermix  according to the manufacturer\u2019s instructions. The real-time PCR assay was performed using the ABI real-time PCR system with the qPCR Master Mix . The expression of traditional housekeeping gene TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 was cloned into the pCa-\u03b3bLIC vector to create the BSMV-TaCAMTA2as, BSMV-TaCAMTA3as, BSMV-TaSARD1as, and BSMV-TaEDS1as constructs using the primer pair 5\u2032-AAGGAAGTTTATACCATCATTAGCACTTGG-3\u2032/5\u2032-AACCACCACCACCGTCACTTTTGGAATTACATTC-3\u2032, 5\u2032-AAGGAAGTTTACATTATGCACCTGCGAGGA-3\u2032/5\u2032-AACCACCACCACCGTTCAGTGCACTTTGGTGAGC-3\u2032, 5\u2032-AAGGAAGTTTATGGTTCTAGTATCTATAAG-3\u2032/5\u2032-AACCACCACCACCGTGTTTGGAACCAGTTATTCG-3\u2032, and 5\u2032-AAGGAAGTTTAAGCGAATTCCCAACAGGTG-3\u2032/5\u2032-AACCACCACCACCGTAGACGGGGAAGTGTCAATC-3\u2032. The BSMV-mediated gene silencing in wheat leaves was performed as described by Zhi et al. (2020) [B.g. tritici strain E09 conidia. About 72 h post-B.g. tritici inoculation, leaf segments were fixed with ethanol: acetic acid solution  and kept in the destaining solution . Before mounting for microscopy, B.g. tritici-infected leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure, as reported previously [The antisense fragment of . (2020) . About 1eviously .TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 were, respectively, amplified using the primers 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCATCATTAGCACTTGG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCACTTTTGGAATTACATTC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCATTATGCACCTGCGAGGA-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGCACTTTGGTGAGC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGGTTCTAGTATCTATAAG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGAACCAGTTATTCG-3\u2032, and 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGCGAATTCCCAACAGGTG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACGGGGAAGTGTCAATC-3\u2032, and cloned into the pIPKb007 vector using a Gateway cloning system to create the TIGS-TaCAMTA2, TIGS-TaCAMTA3, TIGS-TaSARD1, and TIGS-TaEDS1 constructs. The coding regions of TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, TaCAMTA3-2D, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, TaSARD1.2-6B, TaSARD1.2-6D, TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D were, respectively, amplified using the primers 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCT GGGTCCTAGAAATAGCCAGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATATTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAACAGTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATAGTGGACATCG-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCCCG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCACG-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, and 5\u2032-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3\u2032/5\u2032-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3\u2032, and cloned into the pIPKb001 vector. The single-cell transient gene silencing and expression were conducted essentially as described  [B.g. tritici strain E09 conidia, the leaf segments were stained for GUS activity 48 h post-B.g. tritici inoculation. Before mounting for microscopic analysis, the leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure.Antisense fragments of ., 2020) . Briefly"}
+{"text": "Tmarus Simon, 1875 is a relatively large spider genus, currently includes 227 species distributed worldwide. Fanjing Mountain Nature Reserve is one of China\u2019s most biodiverse regions. However, Tmarus can be regarded as being poorly represented in Fanjing Mountain, with only one species having been recorded so far: T.fanjing Yang & Yu, 2022.Tmarus species were brought to light by those expeditions: T.fanjing Yang & Yu, 2022 and T.circinalis Song & Chai, 1990. T.fanjing is redescribed, based on new material and the female is described and illustrated for the first time. The supplementary micrographs of T.circinalis are given for the first time. The DNA barcodes and a distribution map of both species are provided for future use.Recently, various expeditions to Fanjing Mountain Nature Reserve were carried out by the authors. In this paper, two  Tmarus Simon, 1875 is the second most speciose genus of Thomisidae Sundevall, 1833, with 227 valid species distributed worldwide so far, after Xysticus C. L. Koch, 1835 (293 species), 27 species of which are recorded from China  ; for manThomisidae represents a substantial fraction of southwest China foliage-dwelling spiders , Lysitelesfanjingensis Wang, Gan & Mi, 2020, L.inflatus Song & Chai, 1990, Phartatangi Wang, Mi & Peng, 2016, Phrynarachnemammillata Song, 1990, Strigoplusguizhouensis Song, 1990, Tmarusfanjing Yang & Yu, 2022, Thomisuslabefactus Karsch, 1881 and Xysticuskurilensis Strand, 1907  to redescribe the male and report the female of T.fanjing for the first time; 2) to re-illustrate T.circinalis, based on new material from Mt. Fanjing and give supplementary micrographs; 3) to provide the DNA barcodes and a distribution map of T.fanjing and T.circinalis for future use.Recently various short, but intensive field collections in Fanjing Mountain have been conducted by staff of the Guizhou Normal University and Guizhou Education University. This paper reports our findings on the study of recently-available samples from the area, which revealed a new record species of Fanjing Mountain, Specimens in this study were collected by beating vegetation. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope; details were studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3. software package .T.circinalis in Hubei Province and Chongqing City were originated from ArcGIS . Due to lack of locality coordinates in previous publications, locality coordinates for cGIS see .Tmarusfanjing: YHTHO014, \u2642, GenBank ON796486; YHTHO013, \u2640, GenBank ON796487. Tmaruscircinalis: YHTHO015, \u2642, GenBank OR075896; YHTHO016, \u2640, GenBank OR075897.A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for one male and one female specimen, respectively, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Yang & Yu, 202242208F9D-B77B-5D32-BABA-3628DD1444C6Type status:Holotype. Occurrence: recordedBy: Da Wang; Jaiyuan Xin; individualID: YHTHO001; individualCount: 1; sex: 1 male; lifeStage: 1 adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON392063GenBank: ; occurrenceID: 7FC42291-EC27-5A7D-9D64-6E9F98746510; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 755 m; decimalLatitude: 27.87; decimalLongitude: 108.80; Identification: identifiedBy: Jianshuang Zhang; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 4; day: 18; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Haonan Zhang; individualCount: 2; sex: 1 male, 1 female; lifeStage: 2 adults; behavior: foraging; preparations: whole animal (ETOH); occurrenceID: A38DE598-7685-5DA5-AB10-C63D1031394B; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1060 m; decimalLatitude: 27.90; decimalLongitude: 108.58; Identification: identifiedBy: Hao Yu; dateIdentified: 12-12-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2022; month: 7; day: 19; Record Level: basisOfRecord: PreservedSpecimenType status:Other material. Occurrence: recordedBy: Cheng Wang; Xiaoqi Mi; individualID: YHTHO013, YHTHO014; individualCount: 4; sex: 2 males, 2 females; lifeStage: 4 adults; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: ON796487; ON796486; occurrenceID: EE48AA39-2206-5DA9-8F4B-82BB2DA9B1D3; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: fanjing; taxonRank: species; scientificNameAuthorship: Yang & Yu; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Shiqian; locality: Fodingshan Nature Reserve; verbatimElevation: 858 m; decimalLatitude: 27.36; decimalLongitude: 108.0; Identification: identifiedBy: Cheng Wang; dateIdentified: 12-05-2022; identificationReferences: Yang et al. 2022; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2017; month: 4; day: 28; Record Level: basisOfRecord: PreservedSpecimenFemale . Overall body colour is dull brown in ethanol. Total length 6.34; carapace 2.33 long, 2.25 wide; abdomen 4.01 long, 2.23 wide.Chelicerae coloured as ocular area, both margins without teeth. Labium and endites uniformly light brown, endites depressed posteriorly, slightly convergent anteriorly, with dense scopulae on anterior margin; labium nearly diamond-shaped, anterior margin with sparse setae. Sternum yellowish-white, more or less cordiform or shield-shaped, 1.20 long, 0.93 wide.Carapace Fig. A dull brAbdomen Fig. A\u2013C elongLegs uniformly yellowish-white Fig. A and B. Epigyne Fig. C\u2013F. EpigMale .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGAGCGTGATCGGCTATAGTAGGAACTGCTATAAGAGTATTGATTCGAATAGAATTAGGTAATTCAGGAAGACTTTTTGGAAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTGATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGATTTGGTAATTGATTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTAGAAATAGGAGTAGGAGCTGGATGAACTGTATATCCACCTTTGGCTTCTAGTTTAGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGAAGAGTAGGAATGACTATAGAAAAGGTGCCTTTATTTGTCTGATCGGTGTTAATTACTGCTATTTTACTTTTATTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACTTCGTTTTTTGACCCTGCTGGTGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3'  . However, T.fanjing can be distinguished from T.piger by the following characters: for the males, embolus apex blunt in T.fanjing ; associatedSequences: GenBank: prepare to upload; occurrenceID: 9E9A0A7F-27B9-5732-81EE-36AD91E7A4E2; Taxon: order: Araneae; family: Thomisidae; genus: Tmarus; specificEpithet: Tmaruscircinalis; scientificNameAuthorship: Song & Chai; taxonomicStatus: accepted; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; municipality: Jiangkou; locality: Fanjingshan Nature Reserve; verbatimElevation: 1025 m; decimalLatitude: 27.98; decimalLongitude: 108.69; Identification: identifiedBy: Hao Yu; dateIdentified: 15-01-2023; identificationReferences: Song and Zhu 1997; Event: samplingProtocol: Beating; samplingEffort: 10 km by foot; year: 2021; month: 7; day: 20; Record Level: basisOfRecord: PreservedSpecimenSee OR075896).5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTAGGAGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTATATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' .5'TATTTGGGGCGTGGTCAGCTATAGTAGGAACTGCTATAAGAGTATTAATTCGAATAGAATTGGGTAATTCAGGAAGACTTCTTGGTAATGATCATTTATATAATGTAATTGTGACTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATGCCTATTTTAATTGGAGGTTTTGGTAATTGATTAGTACCTTTGATATTGGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTATTTTTATTATTTATATCTTCTATAGTGGAGATAGGAGTAGGGGCTGGGTGAACTGTGTATCCACCTTTAGCTTCTAGTTTGGGTCATATAGGGAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTGGCTGGGGCTTCTTCAATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGTAGAGTAGGAATAACTATAGAAAAAGTGCCTTTATTTGTTTGATCAGTGTTAATTACTGCTATTTTACTTTTACTATCATTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACAGATCGAAATTTTAATACATCGTTTTTTGACCCTGCTGGAGGGGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTT3' , Chongqing City (Xiushan County) and Guizhou Province (Mt. Fanjing), China Fig. ."}
+{"text": "In contrast, mutation of theC. elegansBRCA1 ortholog,brc-1,or itsbinding partner,brd-1, lead to only mild embryonic lethality. We show that inC. elegans,brc-1andbrd-1embryonic lethality is enhanced when53bp1 ortholog,hsr-9,is also mutated. This is not a consequence of activatingpolq-1-dependent microhomology-mediated end joining, aspolq-1mutation does not suppress embryonic lethality ofhsr-9;brc-1mutants. Together, these results suggest thatBRC-1-BRD-1andHSR-9function in parallel pathways and do not act antagonistically as in mammals.In mice, mutation of In contrast to what has been reported in mice, we observed elevated embryonic lethality inbrc-1andbrd-1null alleles . We found that thehsr-9;brc-1polq-1triple mutant had levels of embryonic lethality similar tobrc-1polq-1but higher thanhsr-9;brc-1, suggesting that the elevated embryonic lethality ofhsr-9;brc-1is not a consequence of activation of MMEJ . On thethality . To detof MMEJ . TherefCRISPR-mediated genome editing:hsr-9(xoe17)andpolq-1(xoe51)alleles were engineered by incorporating the stop-in cassettehsr-9repair template (gattttgcctcttaaataaaatttcagCAAAAAACCGAGGGGAGACTTGCAATAGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCTCTCGGATCATCTTGCAAACATGCTTATTGCTGgtaggtattgcaacc) and guide RNA (AGGGGAGACTTGCAATATCT) were injection intoN2and the resulting progeny were analyzed by PCR using TGAAATTAAGGTGGTCACTCGAAG and GTTGTTGTGGGGAGGCTGAA. Thepolq-1repair template (AGAGAATTCTCTGAAGATCCATTAATATTGCTTACCGAAGGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCAGAGTTTTCGCCGCAATTCTCAGACTTTGGTAATGATTTC) and guide RNA (ATTGCGGCGAAAACTCTCTT) were injected intoN2and the resulting progeny were analyzed by PCR using ATAGGCAAATGGCTGGACGG and TCAAAGCAGTCTTCTCGGCA. Worms were outcrossed a minimum of three times.Embryonic lethality:L4 hermaphrodites of indicated genotypes were picked onto individual plates and transferred to new plates every 24hr for 3 days. Embryonic lethality was determined by counting eggs and hatched larvae 24hr after removing the hermaphrodite and calculating percent as eggs/(eggs + larvae).Strains:"}
+{"text": "Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein . Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro\u2013transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation.Key featuresThis protocol is optimized for cap-dependent in vitro translation in the dynamic linear range.Details for generating capped reporter mRNA in one day are provided.Requires as little as one day to complete if starting with in vitro\u2013transcribed mRNA.This protocol requires access to an ultracentrifuge and a real-time PCR system. Graphical overviewCaenorhabditis elegans) (Caenorhabditis elegans)  . Elongatelegans) , which uelegans) . In yeaselegans) . Subsequelegans) . Upon laelegans) .Ribosome footprint profiling and classic polysome profiling can be used to detect stalled ribosomes; however, these approaches can be technically challenging, laborious, and rather low throughput. Additionally, ribosome profiling is not cost-effective when testing multiple specific mutations within reporter mRNAs or effector proteins. Here, we present a validated protocol that can be used to assess ribosome stalling in vitro that is medium-to-high throughput and can be performed in as little as one day if starting with in vitro\u2013transcribed mRNA.We take advantage of the selective nature of puromycin, an amino-acyl transfer RNA analog to incorporate into nascent polypeptides of only actively elongating ribosomes . PuromycReagents2-Propanol (isopropanol) 3\u2032-O-Me-m7G(5\u2032)ppp(5\u2032)G RNA Cap Structure Analog  Agarose LE, quick dissolve Bromophenol blue Chloroform, ethanol stabilized 10\u00d7 CutSmart buffer Cycloheximide (CHX) Dimethyl sulfoxide (DMSO) Dithiothreitol (DTT) DNA Clean & Concentrator-25 kit DNase I (Rnase-free) E. coli Poly(A) polymerase 500 mM EDTA, pH 8.0 ULTROL grade Ethanol, 200 proof 10 mg/mL ethidium bromide Flexi Rabbit Reticulocyte Lysate System 37% (w/v) formaldehyde Glycerol, biotechnology grade Glycogen, molecular biology grade Hi-Di Formamide HiScribe T7 High Yield RNA Synthesis kit iScript Reverse Transcription Supermix iTaq Universal SYBR Green Supermix, 5 mL 2 M KCl, pH 7.4 2 1 M MgClMillennium RNA size marker 10\u00d7 MOPS buffer Nuclease-free water Puromycin dihydrochloride 6\u00d7 purple gel loading dye Quick-Load Purple 1 kb Plus DNA ladder RNA Clean & Concentrator-25 kit Rnase inhibitor, murine Sucrose, Ultra-pure, Rnase- & Dnase-free 10\u00d7 TBE buffer 1 M Tris-HCl, pH 7.4 TRIzol reagent Xylene cyanol FF pcDNA3.1(+)/nLuc-3XFLAG Dual promoter plasmid pCR II pGL4.13 Solutions100 mg/mL cycloheximide (CHX) (see Recipes)10 mg/mL CHX (see Recipes)5 mg/mL CHX (see Recipes)1 M DTT, Cleland\u2019s Reagent (see Recipes)70% (v/v) ethanol (see Recipes)1 mg/mL ethidium bromide (see Recipes)10 mM GTP (see Recipes)1\u00d7 MOPS buffer (see Recipes)10 mg/mL (~18 mM) puromycin (see Recipes)0.6 mM puromycin (see Recipes)2\u00d7 ribosome dilution buffer (see Recipes)RNA loading dye (see Recipes)RNA sample buffer (see Recipes)60% (w/v) sucrose (see Recipes)35% (w/v) sucrose, buffered (see Recipes)1\u00d7 TBE buffer (see Recipes)Recipes100 mg/mL cycloheximide (CHX) (store at -20 \u00b0C)10 mg/mL CHX 5 mg/mL CHX 1 M DTT, Cleland\u2019s Reagent (store at -20 \u00b0C)70% (v/v) ethanol1 mg/mL ethidium bromide10 mM GTP (store at -20 \u00b0C)1\u00d7 MOPS buffer10 mg/mL (~18 mM) puromycin (store at -20 \u00b0C)0.6 mM puromycin (store at -20 \u00b0C)2\u00d7 ribosome dilution buffer (store at 4 \u00b0C)RNA loading dyeRNA sample buffer (make fresh day of use)60% (w/v) sucrose35% (w/v) sucrose, buffered (store at 4 \u00b0C)1\u00d7 TBE bufferLaboratory supplies0.2 mL, open-top thick wall polycarbonate tube, 7 mm \u00d7 20 mm 1.7 mL microcentrifuge tube, clear 500 mL Erlenmeyer flask8-strip PCR tubes Hard-shell PCR plates, 96-well, thin-well IceIce bucketsLight-dry tissue wipes Microseal \u2018B\u2019 seals Nitrile glovesP10, P20, P200, and P1000 calibrated pipettesP10, P20, P200, and P1000 pipette tips ParafilmPlastic wrapTube racks-80 \u00b0C freezer-20 \u00b0C freezer4 \u00b0C refrigeratorAspiratorCFX Connect Real-Time System Eppendorf centrifuge 5430 Fume hoodGelDoc Go Gel imaging system with Image Lab Touch software MicrowaveMilliporeSigma Synergy ultrapure water purification system NanoDrop One Microvolume UV-Vis spectrophotometer OWL EasyCast B1 Mini Gel electrophoresis system OWL EasyCast B1A Mini Gel electrophoresis system Paper towelsPlate centrifuge, PerfectSpin P Pointed tweezersS100-AT3 fixed angle rotor Sorvall Discovery M120 SE micro-ultracentrifuge (Hitachi)T100 thermal cycler Vortex mixer Bio-Rad CFX MaestroMicrosoft Excelhttps://www.graphpad.com/quickcalcs/molarityform/)Prism GraphPad Molarity Calculator Oligo Calc: Oligonucleotide Properties Calculator Transcription and Translation Tool  subcloned from pGL4.13 into pCR II, which is linearized with HindIII. For the experimental reporter, we typically use nanoLuciferase (nLuc) subcloned from pNL1.1 into pcDNA3.1(+), which is linearized with either XbaI or PspOMI. Both plasmids are available upon request from the corresponding author; pcDNA3.1(+)/nLuc-3XFLAG is also available from Addgene.ii. Choose a restriction endonuclease that cleaves downstream of the reporter coding sequence and either produces blunt ends or 5 overhangs. Restriction endonucleases that produce 3\u2032 overhangs should be avoided as they can elicit aberrant and antisense transcription . Choose In a microcentrifuge tube, combine 100 \u03bcL of plasmid DNA (300 ng/\u03bcL), 20 \u03bcL of 10\u00d7 CutSmart buffer, 20 \u03bcL of restriction enzyme , and 60 \u03bcL of Milli-Q water. Gently mix by inversion 20 times and collect contents by a short centrifugation spin .Pause point: Store completed digest at -20 \u00b0C.Digest the plasmid for 4 h at 37 \u00b0C and then hold at 4 \u00b0C. Note: Despite the excess of restriction enzyme in the reaction, we suggest a 4 h incubation for complete linearization due to the relatively large massof plasmid DNA.During the restriction digest, cast a 0.8% (w/v) agarose gel in an OWL EasyCast B1A Mini Gel electrophoresis system with a 1.5 mm 10-well comb. Add 100 mL of 1\u00d7 TBE buffer with 0.8 g of agarose in a 500 mL Erlenmeyer flask and then loosely plug the flask with a folded-up paper towel. Heat in the microwave for 1.5 min or until the agarose is completely dissolved. Allow to cool for 10 min on the benchtop and then add 5 \u03bcL of 10 mg/mL ethidium bromide. Gently swirl to mix and let the flask cool on the benchtop until it can be held comfortably for 10 s (or place in a 65 \u00b0C water or bead bath for 30 min). Pour ~70 mL into the casting tray and let the gel solidify at room temperature for ~1 h. Remove combs by gently pulling them up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1\u00d7 TBE buffer .Purify linearized plasmids using the DNA Clean and Concentrator-25 kit and the supplied solutions.g for 30 s, unless specified. To the 200 \u03bcL digest, add 800 \u03bcL of DNA binding buffer and mix by gentle inversion 20 times. Transfer mixture to the Zymo-spin column in a collection tube. Centrifuge and discard flowthrough. Wash the column with 200 \u03bcL of DNA wash buffer by centrifugation. Discard flowthrough and repeat wash step. Transfer column into a fresh and pre-labeled 1.7 mL microcentrifuge tube. Add 30 \u03bcL of nuclease-free water directly to the column (the white resin at the bottom of the column) and incubate at room temperature for 1 min. Elute by centrifugation . Store on ice for immediate use or at -20 \u00b0C for long-term storage.Perform all steps at room temperature and centrifugation at 16,000\u00d7 Determine DNA concentration and purity by UV spectrophotometry .Note: The Zymo-spin IICR column has a reported capacity of 25 \u03bcg of DNA. This protocol calls for 30 \u03bcg of DNA, which allows us to max out the column, resulting in most reporter plasmids eluting at ~1,000 ng/\u03bcL.Confirm linear plasmid integrity by 0.8% (w/v) agarose gel electrophoresis. Add 5 \u03bcL of Quick-Load Purple 1 kb Plus DNA ladder to the first well. On a piece of parafilm or in a microcentrifuge tube, gently mix 1 \u03bcL of purified linear plasmid DNA (250\u2013500 ng/\u03bcL), 1 \u03bcL of purple gel loading dye (6\u00d7), and 4 \u03bcL of Milli-Q water, and then load the entire sample into a single well. Run the agarose gel at 120 V (constant) for 30\u201345 min or until the desired resolution. Image gel via UV transillumination to confirm a single band that is running at the expected molecular weight.Note: If using two combs per casting tray, use the bottom half of the agarose gel before using the top half, as ethidium bromide in the gel runs toward the cathode. Unused parts of the gel may be stored for up to one week in plastic wrap at 4 \u00b0C.Synthesize in vitro\u2013transcribed reporter mRNA using the HiScribe T7 High Yield RNA Synthesis kit.To resuspend the ARCA cap analog to 40 mM, first quickly collect the contents (1 \u03bcmol) by a short centrifugation and then add 25 \u03bcL of nuclease-free water to the pellet. Gently mix by flicking the tube and collect the contents by brief centrifugation. Repeat the gently mixing procedure for a total of five times.It is critical to use 1 \u03bcL of 10 mM GTP and not 1 \u03bcL of 100 mM GTP. The 8:1 ARCA cap analog to GTP ratio ensures 90% co-transcriptional capping efficiency , 0.5 \u03bcL of RNase inhibitor, 1 \u03bcL of 10\u00d7 T7 reaction buffer, 1 \u03bcL of 100 mM ATP, 1 \u03bcL of 100 mM UTP, 1 \u03bcL of 100 mM CTP, 1 \u03bcL of 10 mM GTP, 2 \u03bcL of 40 mM ARCA cap analog, 1 \u03bcL of T7 RNA polymerase mix, and 0.5 \u03bcL of nuclease-free water in a PCR tube or microcentrifuge tube. ficiency .Notes:i. If transcribing multiple mRNAs at one time, assemble a master mix of all components except linear plasmid templates. Use 9 \u03bcL of master mix with 1 \u03bcL of linear plasmid template (~500 ng/\u03bcL). Reactions can also be scaled up linearly to 20 \u03bcL but beware of exceeding the binding capacity of the RNA clean up columns (see below).ii. Most T7 RNA polymerase\u2013mediated mRNA synthesis kits with a cap analog pre-mixed with the NTPs use a 4:1 ARCA cap to GTP ratio, which only provides ~80% co-transcriptional capping efficiency. These cap analog pre-mixed kits also do not provide the ability to generate A-capped or non-methylated G-capped mRNAs to test cap-dependency.Perform in vitro transcription for 2 h at 30 \u00b0C using a thermal cycler (for PCR tube) or heat block (for microcentrifuge tube).Note: In our hands, transcription at 30 \u00b0C yielded purer mRNA than transcription at 37 \u00b0C.To remove the DNA template, add 1 \u03bcL of DNase I (RNase-free) to each 10 \u03bcL reaction and gently mix by inversion. Incubate at 37 \u00b0C for 15 min.E. coli Poly(A) polymerase, and 28 \u03bcL of nuclease-free water, followed by incubation at 37 \u00b0C for 1 h.If polyadenylation of the RNA is desired, combine and gently mix on ice the 11 \u03bcL DNase-treated capped and transcribed mRNA reaction with 5 \u03bcL of 10\u00d7 Poly(A) polymerase buffer, 5 \u03bcL of 10 mM ATP, 1 \u03bcL of Note: This protocol uses thenuclease-treatedFlexi rabbit reticulocyte lysate (RRL) system from Promega. It is not entirely necessary to polyadenylate in vitro\u2013transcribed mRNA for efficient translation usingnuclease-treatedRRL. Polyadenylation provides a less than two-fold enhancement of reporter mRNA translation innuclease-treatedRRL Soto . If polyIf using a PCR tube, transfer the entire contents to a microcentrifuge tube.Purify mRNA using the RNA Clean and Concentrator-25 kit and the supplied solutions.g for 1 min, unless specified. To each 50 \u03bcL of mRNA sample, add 100 \u03bcL of RNA binding buffer and gently mix by inversion 20 times. Add 150 \u03bcL of 100% ethanol to each reaction and gently mix by inversion 20 times. Transfer the entire sample to the Zymo-Spin IICR Column in a collection tube and centrifuge. Discard the flowthrough. Add 400 \u03bcL of RNA prep buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 700 \u03bcL of RNA wash buffer to the column, centrifuge, discard flowthrough, and place column back into the collection tube. Add 400 \u03bcL of RNA wash buffer to the column, centrifuge for 4 min, discard flowthrough, and place column into a new RNase-free 1.7 mL microcentrifuge tube. Add 75 \u03bcL of nuclease-free water directly to the column matrix and allow to incubate at room temperature for 1 min. Elute by centrifugation. Store mRNA on ice moving forward.Perform all steps at room temperature and centrifugation at 16,000\u00d7 Determine RNA concentration and purity by UV spectrophotometry .Pause point.Aliquot mRNA in 3 \u03bcL volumes in PCR strips and store at -80 \u00b0C. Confirm in vitro\u2013transcribed mRNA quality and purity by denaturing agarose gel electrophoresis.Cast a denaturing 0.8%\u20131% (w/v) agarose formaldehyde gel in OWL EasyCast B1 Mini Gel electrophoresis system with a 1.5 mm 10-well comb placed in the top comb slot. Add 0.8\u20131 g of agarose to 80 mL of Milli-Q water in a 500 mL Erlenmeyer flask loosely plugged with a folded-up paper towel. Microwave for 1.5 min or until the agarose is dissolved and gently swirl to mix. Cool on countertop for 2 min.In a fume hood, add 10 mL of 10\u00d7 MOPS buffer and 10 mL of 37% formaldehyde to the flask containing dissolved agarose. Gently swirl to mix and allow to cool for 5\u201310 min.Pour 100 mL into the casting tray and let the gel solidify at room temperature for ~1 h in the dark by loosely covering with aluminum foil.Prepare mRNA samples by mixing 500 ng of in vitro\u2013transcribed mRNA, 1 \u03bcL of 1 mg/mL ethidium bromide, 1 \u03bcL of RNA loading dye (see Recipes), 5 \u03bcL of freshly-prepared RNA sample buffer (see Recipes), and nuclease-free water to 15 \u03bcL total volume.Prepare the RNA ladder by mixing 1 \u03bcL of Millennium RNA size marker (1 \u03bcg/\u03bcL), 1 \u03bcL of 1 mg/mL ethidium bromide, 1 \u03bcL of RNA loading dye (see Recipes), 3 \u03bcL of RNA sample buffer (see Recipes), and 4 \u03bcL of nuclease-free water.Heat ladder and samples at 70 \u00b0C for 5 min; then, place the ladder and samples on ice for 2 min.Remove combs by gently pulling up vertically. Remove and rotate the casting tray so that the wells are near the cathode. Fill the gel tank and completely cover the agarose gel with 1\u00d7 MOPS buffer .Load ladder and samples on the gel.Run at 60 V (constant) (or 5\u201310 V/cm gel width) in the dark  for 3\u20134 h (when the dye front reaches 2 cm from the bottom of the gel) or until the desired resolution.Image gel via UV transillumination to confirm a single band is running at the expected molecular weight (polyadenylating mRNA will add to the expected molecular weight).Note: Non-polyadenylated in vitro\u2013transcribed mRNA will run as a single, crisp band at the expected molecular weight on a denaturing agarose gel. The same in vitro\u2013transcribed mRNA that is polyadenylated will appear ~100\u2013200 nt heavier as a slightly broader band. An additional control reaction lacking the Poly(A) polymerase can be included to better define the poly(A) tail length. Large smears spanning far beyond the expected molecular weight indicate RNA degradation, rolling circle transcription due to the presence of non-linear template DNA, nondenatured RNA secondary structure, or errors during RNA clean up steps. We have also found that making fresh, day-of-use RNA sample buffer is critical.In vitro translationPre-cool the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor to 4 \u00b0C prior to setting up in vitro translation reactions. The S100-AT3 rotor can hold up to 20 samples.Note: For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA-containing reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1).In vitro translation of normalizing control mRNA:be sure to use the ssRNA setting; see Software). Using its molecular weight from above and the online Prism GraphPad Molarity Calculator (see Software), calculate the mass required for 3 nM in 10 \u03bcL. This is the mass of mRNA required in the complete 10 \u03bcL in vitro translation reaction for 3 nM mRNA . Dilute the purified mRNA in nuclease-free water such that 2 \u03bcL of RNA contains the mass calculated above, resulting in a final concentration of 15 fmol/\u03bcL. Other similar online tools are available and would suffice.Dilute normalizing mRNA to 15 fmol/\u03bcL in nuclease-free water and keep on ice. To do so, use the plasmid DNA sequence from the first transcribed nucleotide of the T7 RNA polymerase promoter to the restriction endonuclease cut site and determine the corresponding RNA sequence using the online Transcription and Translation Tool (see Software). Then, calculate the molecular weight of the control mRNA reporter with the online Oligo Calc: Oligonucleotide Properties Calculator , 0.1 \u03bcL of 1 mM amino acid mixture minus leucine, 0.1 \u03bcL of amino acid mixture minus methionine, 0.2 \u03bcL of 25 mM Mg(OAc), 0.4 \u03bcL of 2.5 M KCl, 0.2 \u03bcL of RNase inhibitor, and 4 \u03bcL of nuclease-free water. Perform in vitro translation for 15 min at 30 \u00b0C in a thermal cycler.Immediately place samples on ice.To each sample, add 4 \u03bcL of 5 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Samples should now be 14 \u03bcL. Keep samples on ice until step C1.Note: Cycloheximide is added in step B2 to robustly inhibit elongation and preserve ribosome-bound mRNAs when added to experimental samples that contain puromycin in step B3.In vitro translation of experimental mRNA:Dilute experimental mRNA(s) to 15 fmol/\u03bcL (as in step B2a) in nuclease-free water and keep on ice.On ice in a PCR tube, set up the following 10 \u03bcL translation reaction: mix 2 \u03bcL of 15 fmol/\u03bcL in vitro transcribed mRNA, 3 \u03bcL of Flexi RRL (nuclease-treated), 0.1 \u03bcL of 1 mM amino acid mixture minus leucine, 0.1 \u03bcL of amino acid mixture minus methionine, 0.2 \u03bcL of 25 mM Mg(OAc), 0.4 \u03bcL of 2.5 M KCl, 0.2 \u03bcL of RNase inhibitor, and 4 \u03bcL of nuclease-free water. If translating multiple experimental reporters or many biological replicates, prepare a master mix with all components except for the mRNA; add 8 \u03bcL of the master mix to 2 \u03bcL of 15 fmol/\u03bcL in vitro\u2013transcribed mRNA.Perform in vitro translation for 15 min at 30 \u00b0C in a thermal cycler.Immediately place samples on ice.To each sample, add 2 \u03bcL of nuclease-free water or 0.6 mM puromycin (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin.Place puromycin-containing samples on a thermal cycler and incubate at 30 \u00b0C for 30 min. Keep samples without puromycin  on ice.Place all samples on ice and immediately add 2 \u03bcL of 10 mg/mL CHX (see Recipes) . Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.All translation reactions should now be 14 \u03bcL.Ribosomal pelleting through low-speed sucrose cushionPrepare samples for low-speed ribosomal pelleting through a sucrose cushion.Mix a 14 \u03bcL normalizing control mRNA-containing reaction tube and a 14 \u03bcL experimental mRNA-containing reaction tube for a total sample volume of 28 \u03bcL. Keep samples on ice.Add 28 \u03bcL of ice-cold 2\u00d7 ribosome dilution buffer . Each sample should now be 56 \u03bcL.Gently mix by inversion 20 times and collect contents by a short centrifugation spin. Keep samples on ice.Prepare sucrose cushions and overlay samples.Label 7 mm \u00d7 20 mm polycarbonate thick-walled tubes. Mark a single spot on the rim of each tube . Place tAdd 130 \u03bcL of ice-cold 35% buffered sucrose  to the bottom of each tube.Very carefully, overlay all 56 \u03bcL of the diluted sample from step C1 on top of the sucrose cushion.Low-speed centrifugationWithout disturbing the sample\u2013sucrose interface, place the tubes into the pre-chilled S100-AT3 rotor. Be sure to position the marked spot facing outward and toward the back of the rotor. This spot will indicate the side of the tube where the ribosome pellet will be located after centrifugation . Using pCarefully place the rotor into the pre-cooled Sorvall Discovery M120 SE micro-ultracentrifuge.g for 1 h at 4 \u00b0C. Use an acceleration setting of 9 (fastest setting) and a deceleration setting of 5 (middle setting).Centrifuge samples at 50,000\u00d7 Carefully, remove the tubes from the rotor and place them on ice using the homemade ice bucket for the 7 mm \u00d7 20 mm tubes. Using pointed tweezers for this step is helpful.Using a pipettor, remove and discard the supernatant without disturbing the pellet. The glossy clear pellet should be at the bottom edge of the tube below the mark that was facing outward and toward the back of the rotor during centrifugation.Resuspend the pellet in TRIzol.i. First, add 200 \u03bcL of TRIzol to a new, labeled, nuclease-free microcentrifuge tube.ii. Add 100 \u03bcL of TRIzol to the ribosome pellet in the 7 mm \u00d7 20 mm tube and mix 20 times by gently pipetting up and down. The pellet will dissociate from the tube wall when TRIzol is first added and will float in solution until it ultimately dissolves.iii. Transfer this 100 \u03bcL to the microcentrifuge tube (now containing 300 \u03bcL).iv. Add another 200 \u03bcL of TRIzol to the 7 mm \u00d7 20 mm tube to wash off any remaining ribosomes by gently pipetting up and down 10 times.v. Transfer the 200 \u03bcL sample to the labeled microcentrifuge tube Pause point: Store samples at -80 \u00b0C.Mix samples end-over-end at room temperature for 15 min at 15 rpm. Collect contents by a short centrifugation spin. RNA extractions, cDNA synthesis, and RT-qPCRRNA extractionsThaw samples at room temperature if necessary. Add 100 \u03bcL of chloroform to each sample.Mix vigorously by hand for 1 min (do not vortex).g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Without disturbing or touching the protein interface, carefully remove the top 200 \u03bcL clear aqueous layer and transfer it to a new, labeled, nuclease-free microcentrifuge tube.Add 1.5 \u03bcL glycogen (20 mg/mL) to each sample. Upon dispensing, wash the tip in the aqueous phase by gently pipetting up and down three times.To each sample, add 500 \u03bcL of 100% isopropanol. Gently mix by inversion 20 times.g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Note: When pelleting RNA in microcentrifuge tubes, place the hinge upright facing the outside of the rotor. This will allow you to predict the location of the pellet at the bottom of the tube on the same side of the hinge.Aspirate off the isopropanol until ~100 \u03bcL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 \u03bcL with a P200 pipette. The pellet should be white but very small.Add 600 \u03bcL of ice-cold 70% ethanol. Vortex each sample for 1 s.g at 4 \u00b0C.Centrifuge for 15 min at 12,000\u00d7 Aspirate off the ethanol until ~100 \u03bcL remains in the tube, leaving the RNA pellet untouched. Remove the final ~100 \u03bcL with a P200 pipette and finally a P10 pipette. Aspirate any remaining ethanol off the tube walls. Allow to air dry with the top open for 2\u20133 min at room temperature on the bench.Place tubes on ice, add 30 \u03bcL of nuclease-free water to the pellet, and let stand on ice for 2 min. Gently resuspend the pellet by pipetting up and down 20 times. Be sure to wash down the side of the tube (same side as the hinge) to ensure complete resuspension of the RNA pellet.cDNA synthesisOn ice, combine 16 \u03bcL of RNA and 4 \u03bcL of 5\u00d7 iScript Reverse Transcription Supermix. Mix by gentle inversion 20 times and collect the contents by a short centrifugation spin. Store the remaining 14 \u03bcL of RNA at -80 \u00b0C.Using a thermal cycler, reverse transcribe using:i. 25 \u00b0C for 5 min (priming)ii. 46 \u00b0C for 20 min (reverse transcription)iii. 95 \u00b0C for 1 min (reverse transcriptase inactivation and RNA cleavage)iv. Hold at 4 \u00b0CPause point: Store samples at -20 \u00b0C.To each 20 \u03bcL cDNA sample, add 180 \u03bcL of nuclease-free water for a 1:10 dilution. Gently mix by inversion 20 times and collect contents by a short centrifugation spin. RT-qPCRDesign a 96-well plate layout scheme for all samples and negative controls. Each cDNA sample will be amplified with two primer sets: one targeting the normalizing control mRNA and the other targeting the experimental mRNA. A no-template negative control (where water is added instead of cDNA) should also be included for each primer set. Perform at least technical duplicates for each sample and primer set.Dilute qPCR primers by mixing 510 \u03bcL of nuclease-free water, 45 \u03bcL of 10 \u03bcM forward primer, and 45 \u03bcL of 10 \u03bcM reverse primer. See General notes below for the primer sequences we used for FFLuc and nLuc.Our typical RT-qPCR reaction is 15 \u03bcL. Each well will contain 7.5 \u03bcL of iTaq Universal SYBR Green Supermix (2\u00d7), 1.5 \u03bcL of the 1:10 diluted cDNA, and 6 \u03bcL of diluted primers. For both the normalizing control and experimental primer sets, create a master mix of iTaq Universal SYBR Green Supermix and diluted primers. Add 13.5 \u03bcL of this master mix to the appropriate wells. Then, carefully add 1.5 \u03bcL of the appropriate diluted cDNA samples. The no-template negative control contains 13.5 \u03bcL of appropriate master mix and 1.5 \u03bcL of nuclease-free water.Seal the plate with a microseal \u2018B\u2019 seal. Be sure not to touch the top of the plastic directly, but rather apply pressure to the seal by using a clean tissue wipe (or Kimwipe). Remove and discard the perforated edges.Centrifuge the plate in a plate centrifuge for ~30 s to pull the samples to the bottom of the wells.Place the plate into the Bio-Rad CFX Connect Real-Time System.Run the following RT-qPCR program:i. One cycle of 95 \u00b0C for 3 min.ii. Forty cycles of 95 \u00b0C for 10 s, 60 \u00b0C for 30 s, followed by a Plate Read.iii. Melt curve from 65 \u00b0C to 95 \u00b0C with an increment of 0.5 \u00b0C for 5 s and a Plate Read.Note: If using new primer sets, peel back the plastic film after a run and take out 10 \u03bcL to confirm the expected size amplicon on a 2% (w/v) agarose gel.For each experimental mRNA, at least six samples should be prepared and translated: three replicates that are translated and not puromycin-treated and three replicates that are translated and puromycin-treated (step B3). Each of the six samples is mixed with a translated normalizing control mRNA reaction (step B2), then diluted and layered on top of individual sucrose cushions (step C1). During RT-qPCR, each cDNA should be assayed in at least technical duplicates. Primer sets for both the normalizing control mRNA and experimental mRNA should be used in separate wells on the same plate.t-test with Welch\u2019s correction. For nLuc as the experimental mRNA reporter, we typically observe a relative ~60% reduction in signal with puromycin treatment  to the signal of the normalizing control mRNA  to account for any error during RNA extraction and/or cDNA synthesis. Once the gene expression has been calculated by the CFX Maestro software, export the data and perform the remaining analysis in Excel. For each experimental reporter, group the without puromycin-treatment replicates and set to 100%. Then, determine the relative signal for each replicate with puromycin-treatment. Statistical comparisons can be made using an unpaired reatment .Two additional controls should be incorporated in step B3:1) A no-template negative control, where water is added instead of either reporter mRNA, is critical to ensure specificity of RT-qPCR primers and determine background levels of detection.2) To confirm that the low-speed sucrose cushions are working as expected to selectively pellet ribosome-bound mRNA, set up translation reactions with the experimental mRNA without puromycin, incubate for 15 min on ice instead of 30 \u00b0C (step B3c), and proceed as directed above. The ice-incubated sample should have very few, if any, ribosomes loaded on the experimental mRNA and should be minorly detected by RT-qPCR when compared to the 30 \u00b0C\u2013incubated sample.Journal of Biological Chemistry, DOI: 10.1016/j.jbc.2022.102660. See Figure 6E and 6F.This protocol was validated in Scarpitti et al. (2022) These in vitro translation reaction conditions have been optimized to be in the dynamic linear range for time and mRNA input for a range of reporter mRNAs . These sg sucrose cushion in the Sorvall Discovery M120 SE micro-ultracentrifuge and S100-AT3 rotor. Increased centrifugal force resulted in loss of specificity for pelleting ribosome-bound mRNAs in a translation-dependent manner. Both variables may have to be re-optimized if using different translation extracts, if making substantial alterations to the translation reaction conditions, and/or if using a different rotor.We empirically optimized the 0.1 mM puromycin  for use with the in vitro translation reaction conditions described in this protocol. Increasing puromycin beyond 0.1 mM did not further reduce the amount of mRNA co-pelleted in a translation-dependent manner. We also optimized the 50,000\u00d7 If testing ribosome stalling by an mRNA-binding protein , we recoLastly, while we have always used FFLuc as the normalizing control mRNA and nLuc as the experimental mRNA, we believe other reporters  would function just as well for either the normalizing control or experimental mRNA. We have previously cloned FFLuc from pGL4.13 into the dual promoter plasmid pCR II. The use of pCR II is not required, but pCR II/FFLuc under control of the T7 RNA polymerase promoter is available upon request. pcDNA3.1(+)/nLuc-3XFLAG, which contains a T7 RNA polymerase promoter upstream of nLuc, is available upon request and from Addgene.See below for reporter coding sequences and qPCR primer sequences.FireFly Luciferase ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAAnanoLuciferase ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAART-qPCR primersF_pGL4.13 RT-qPCR: GCAGTACCGGATTGCCCAAGR_pGL4.13 RT-qPCR: GTCGGGGATGATCTGGTTGCF_nLuc (pNL1.1) RT-qPCR: CAGCGGGCTACAACCTGGACR_nLuc (pNL1.1) RT-qPCR: AGCCCATTTTCACCGCTCAG"}
+{"text": "Bacillus cereus strain PC2 (GenBank accession No. MZ314010) and Streptomyces praecox strain SP1 (GenBank accession No. MZ314009). In-site bacterial and fungal isolates defined in the current study were proficient in cleaning wastewater of chlorpyrifos pesticide residues.Pollutants cause a huge problem for humans, animals, plants, and various ecosystems, especially water resources. Agricultural, domestic, and industrial waste effluents change the water quality and affect living microorganisms. Therefore, the current study aimed to identify possible microorganisms in wastewater as potential bioremediation agents of pesticide residues. Wastewater samples were collected from El-Khairy agricultural drainage, which receives agricultural and domestic wastes. Bacteria and fungi species were isolated as clean cultures. Wastewater samples were analyzed for pesticide residues via gas chromatography-mass spectroscopy (GC\u2013MS) system. Results uncovered the presence of ten pesticides ranging from 0.0817 to 28.162\u00a0\u00b5g/l, and the predominant pesticide was chlorpyrifos. Along with that, about nine species  were relatively efficient in the removal of chlorpyrifos residues up to 2000\u00a0\u00b5g/l with removal percentages ranging from 24.16 to 80.93% under laboratory conditions. Two bacterial isolates proficiently degraded significant amounts of chlorpyrifos:  Therefore, the current study aimed to isolate indigenous bacteria and fungi and screen their potential as bioremediation agents of wastewater of pesticide residues.Acetonitrile HPLC-grade and culture media were purchased from local chemical providers. DNA purification kit (Germany) and PCR clean-up kit were from Maxim Biotech Inc. (USA). The internal standard (TPP) and extraction (Cat#5982\u20130650) and dispersive SPE clean-up (Cat#5982\u20135056) kits were purchased from Technoscient for Lab & Optical Product, Cairo, Egypt. Certified reference standard materials of pesticides were obtained from ULTRA Scientific Analytical Solutions  Table .Table 1L3/s  were collected by water sampler into cleaned and sterilized one-liter Pyrex borosilicate dark glass bottles  and plate count agar for isolation of the bacteria colonies  at 25\u00a0\u00b0C. Nine samples were collected; 3 from each collection point as replicates and 3 plates per replicate were planted from each sample under sterilized conditions and incubated at 37\u00a0\u00b0C. The complete growth of the microbe was reached after about 7\u00a0days of incubation. Each microorganism was transferred into a new Petri dish. The subculture of each microbe was repeated several times until a visually clean culture was obtained.4 (anhy) and NaCl, respectively, were thoroughly mixed for 1\u00a0min. Then the internal standard triphenyl phosphate (TPP) solution was added to tubes and shaken for 30\u00a0s. Then tubes were centrifuged at 1350\u2009\u00d7\u2009g for 10\u00a0min . About 1\u00a0ml of supernatant (acetonitrile) was mixed by hand for 5\u00a0min with 25\u00a0mg PSA sorbent and 150\u00a0mg MgSO4 (anhy) and centrifuged for 5\u00a0min at 1350\u2009\u00d7\u2009g. About 500\u00a0\u00b5l of each tube was filtered through 0.22-\u03bcm PTFE filters  into HPLC vials for GC\u2013MS analysis.Pesticide residues in wastewater were extracted and cleaned up using a modified method of Anastassiades . Separation conditions were as reported by AOAC AOAC, , where tThe intra-day assay (repeatability) and inter-day assay (intermediate precision) of the used analytical technique were calculated according to Ermer . Also, tThe radial growth of separated and identified fungi and bacteria on media mixed with the prevalent insecticide in wastewater, chlorpyrifos (CPF), was examined. Five concentrations of CPF  were prepared in the growing media. Five plates of each microorganism were used as replicates per each concentration. Microorganisms were incubated with the CPF at 37\u00a0\u00b0C for 1\u00a0week, and then their radial growth was photographed and recorded. Then competence of growth fungi and bacteria on such media with the insecticide was calculated compared to control plates. This experiment was repeated six times. Then the performance of the potential bioremediation activity of organisms was examined using 500, 1000, and 2000\u00a0\u00b5g/l. The residues of CPF in media after the incubation time were measured using the GC\u2013MS as described previously.The fungal isolates were cultured onto clean growth media until pure cultures were obtained and used for various evaluations and identification  following the manufacturer\u2019s guidelines.E. coli, forward, 5\u02b9 AGG ACG TGC TCC AAC CGC A \u02b93, and reverse, 5\u02b9 AAC TGG AGG AAG GTG GGG AT \u02b93  was performed by Macrogen Company . The 16S rRNA gene nucleotide sequences of isolated bacterial strains were submitted to the GenBank database under accession numbers MZ314009 and MZ314010.http://www.ebi.ac.uk/clustalw)  . The pesticide residues and microbial isolates\u2019 performance and growth were expressed as mean\u2009\u00b1\u2009SD. Significant means were contrasted using Tukey\u2019s honest significant difference test (HSD) (05) SAS, .The analytical method used in the analysis of pesticides was accurate and suitable based on obtained values of repeatability and intermediate precision Ermer, . ResultsAnalysis of pesticides residues revealed the presence of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, and permethrin at 0.721, 28.16, 4.14, 0.052, 7.881, and 0.208\u00a0\u00b5g/l, respectively, in samples collected from site A  were presented in Table Verification of the performance of isolated bacteria  and fungi  was examined by challenging their growth on media with 500, 1000, and 2000\u00a0\u00b5g/l of CPF Fig.\u00a0. ResultsAspergillus terreus, Aspergillus foetidus var. pallidus, Aspergillus fumigatus var. ellipticus, and Aspergillus fumigates according to the database identification program of RCMB For Aspergilli  and Bacillus cereus (I). For further differentiation between the two bacterial strains, similar morphology, physiological, and biochemical traits were reported, except for starch hydrolysis and lactose production that were negative in B. cereus and positive in S. praecox. Also, B. cereus produced acid from sucrose, but S. praecox did not.The characteristics of the most efficient bacterial isolates (F and I) in degrading CPF were listed in Table Bacillus cereus PC2 (GenBank Acc# MZ314010) and Streptomyces praecox SP1 (GenBank Acc# MZ314009) were constructed via molecular identification. Results of DNA sequences of the 16S gene of F and I bacterial isolates were as the following for F, identified as Streptomyces praecox, with a sequence of the following:GACGGCCTTCGGGTTGTAAACCTCGGGCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGCCCCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAAGGGGGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGCCCAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGTTGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTGGGGGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATThe 16S rRNA gene sequences of Bacillus cereus with a DNA sequence of the following:CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTAThe second bacteria (I) was identified as S. praecox strain SP1 was compared with the other 16 species of Streptomyces in GenBank. The neighbor-joining phylogenetic tree revealed high homology between S. praecox strain SP1 and S. praecox strain 7445   within 24\u00a0h  as the main metabolite with greater water solubility compared to TCP. Streptomyces sp. AC5 and AC7 species, effectively biodegraded CPF insecticide. The AC5 removed over 90% of CPF (50\u00a0mg/l) after 72\u00a0h. of incubation, while the AC7 strain was less effective  , I (Bacillus cereus), and A (Aspergillus terreus) removed about 80.82, 80.93, and 75.59% of the 2000\u00a0\u00b5g/l of CPF in 1\u00a0week. Following the molecular identification of these species, they were registered in the GenBank as Streptomyces praecox strain SP1 and Bacillus cereus strain PC2 with accession Nos. MZ314009 and MZ314010, respectively.Pollution of water resources with pesticides might cause serious problems. Indigenous microorganisms help lessen the adverse effects of pesticide residues through degradation. Agricultural wastewater samples were analyzed for pesticide residues, and results showed the detection of lenacil, chlorpyrifos, cypermethrin, bifenthrin, carbofuran, tolfenpyrad, oxamyl, dicloran, simetryn, sulfotep, ethofumesate, and permethrin. Chlorpyrifos insecticide was the dominant compound in wastewater samples. Indigenous bacterial and fungal species were isolated, and their ability to degrade chlorpyrifos insecticide was examined. After 1\u00a0week of incubation of isolated bacteria  and fungi  with CPF, results revealed efficiency % of removal ranging from 31.41 to 90.82%. Specifically, F ("}
+{"text": "Salmonella entericaserovar Typhimurium. Eight unique sRNAs were selected for deletion primarily based on their genomic location and/or putative targets. Quantitative and qualitative analyses confirm one of these, sRNA1186573, is required for efficient biofilm formation inS. entericafurther highlighting the significance of sRNAs duringSalmonellastress response.Small RNAs (sRNAs) are short noncoding RNAs of ~50-200 nucleotides believed to primarily function in regulating crucial activities in bacteria during periods of cellular stress. This study examined the relevance of specific sRNAs on biofilm formation in nutrient starved Salmonella entericais rod-shaped, motile, non-spore-forming gram-negative bacilli responsible for causing gastrointestinal disease and thousands of deaths across the world every year. Salmonella, like many other bacteria, forms biofilm when subjected to stressAcinetobacter baumanii, which is a classic biofilm formerE.coliandS. enterica:bssRandcsgD Small RNA (sRNA) are non-coding RNA molecules that are less than 200 nucleotides in length and have been found to control gene expression in many regulatory circuits in bacteria, such asSalmonella entericaserovar Typhimurium, we selected eight unique sRNAs for deletion primarily based on their genomic location and/or putative targets then performed qualitative and quantitative assays of biofilm formation on each mutant and controls . After 24-h of incubation at room temperature in a nutrient deficit condition (as nutrient starvation is an inducer of biofilm formation). In agreement with Congo Red assay results, standard microplate biofilm UV absorbance assays confirmed that the ability of the sRNA1186573 deletion mutant to form biofilms during nutrient deprivation was almost entirely lost, whereas the ability of the other sRNA deletion mutants to form biofilms did not significantly differ from wild type .To examine the relevance of specific sRNAs on biofilm formation inS. enterica, further highlighting the significance of sRNAs during Salmonella stress response. Finally of note, although RT-qPCR analyses confirm that sRNA1186573 deletion does not disruptcsgDgene expression, it is tempting to speculate that this sRNA may be involved with the post-transcriptional regulation ofcsgD, as csgD protein directly contributes to curli expression inSalmonella entericaIn conclusion, our results confirm that sRNA1186573 is required for efficient biofilm formation inPre-selection of sRNAs and mutant generationSalmonella entericaserovar TyphimuriumA total of 8 sRNAs  inThe primers used for generating mutants are mentioned below:sRNA924744 F: CACATTCACCGCTTACACAGGTCTGAACAAGGGGAGGCGAGTGTAGGCTGGAGCTGsRNA924744 R: AAGGCTCCAGTATATTTTTAAAGGATTTTTGGCATAATGAACATATGAATATCCTCCTsRNA1170414 F: GTAGTAATAGCGGTAGTTCCCCGGCAGTGATGGTCACTCAGTGTAGGCTGGAGCTGCsRNA1170414 R: AATGATGAGAGCTTTTAAGATGACAAGACCACCACCGGCGACATATGAATATCCTCCsRNA1186573 F: GTAATGGCTAGATTGAAAACAGTTAGTGTAGGCTGGAGCTGCTTCsRNA1186573 R: CCCCATAAAATAAAGGCACCAGAAGTACTGACAGATGTTGCATATGAATATCCTCCTTsRNA176086 F: TGAATTTGACACTGCGCACAGGGCGAsRNA176086 R: ACGACCTGCTTCTGAGGCTTTCTCTTTsRNA2594511 F: AAATAAGATCCCGGCCAGCCTGATACsRNA2594511 R: CGTGAACTGGGGAACTGGAAAGATTTsRNA3551252 F: TTTTAATATCATTAAAATCAAAAGTATAGACATTCATAGCGTGTAGGCTGGAGCTGCsRNA3551252 R: TGACTATACTTATTTGAGATACAAAAACAGCGCAAGAGTGCATATGAATATCCTCCTsRNA4130247 F: ATCTTGTGCTATTGGCAAAACCTATGGTAACTCTTTAGGTGTGTAGGCTGGAGCTGCsRNA4130247 R: TCGTCCAAGTGCAGCCCCGCACGGTGGGATAATAATCACCACATATGAATATCCTCCTsRNA4720054 F: CACAAAACTTATGGATTTATGCGTATAATCCGCGGCGCAAGTGTAGGCTGGAGCTGCsRNA4720054 R: CGTTATTGTGTCACTGTCTTACACACCGGTAAGACAGCAGACATATGAATATCCTCCThe primers used for confirming the mutants are here below:sRNA924744 F: GAATCCCCAGCAAACCAAG; sRNA924744 R: GCAGGCATAGTGATGATTTCCsRNA1170414 F: CTATGGAGATCGCGAATGGT; sRNA1170414 R: GAATGTCCGTACAGGGTGTTGsRNA1186573 F: AGGCACCAGAAGTACTGACAGA; sRNA1186573 R: ACGGCTATTTCAACCCACAGsRNA176086 F: GACATATCATATTTAAAACGCAACA; sRNA176086 R: CGCGATGTTCTGCCATAATsRNA2594511 F: TCTTCGTTGAGTCGCCTTT; sRNA2594511 R: CGTAAATAAATGCCTGGAAGGsRNA3551252 F: ACCATCCCGACAGACAA; sRNA3551252 R: TTGGAAGTGAAACCTCTGCATsRNA4130247 F: AGCCAAGATGCAAGAATAGACA; sRNA4130247 R: CCACGCTAATCACGACCAsRNA4720054 F: TTACTTACCGGAGGCGACAT; sRNA4720054 R: GAAAATTCTCCATCGCGGcsgDgene expression are as follows:The primers used to confirm that sRNA1186573 deletion does not disruptCsgD_F_qPCR: GGTCAGCGGATTACAGGGTA; CsgD_R_qPCR: TCGCGATGAGTGAGTAATGCBiofilm formation assaySalmonella Typhimurium: (i) rdar , (ii) pdar , (iii) bar , and (iv) saw et al.Both qualitative (Congo-Red agar test"}
+{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50\u2013200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors  suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target . The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space. Designing an antibody against the SARS-CoV-2 spike protein S is challenging due to its nonspecific nature, mainly conferred by each spike protein monomer . The spiRecent research and protein sequences designed to fit spike protein S failed to bind. A lack of specificity is a result of high mobility and flexibility combined with additional maximum solvent exposure of the monomer domains . A mass effect should virtually resolve this lack of specificity; in other words, by increasing the mass of the desired paratope, that will interact with the epitope (S protein), an alternative unavailable from the receptor\u2013ligand interaction point of view . Judging from the antigen\u2013antibody point of view, epitope amino acid (Aa) sequences located on the antigen must fit complementarily with the Aa chain  of the paratope (antigen variable (VL) and constant regions (CR)) .f) of one argument from a given domain is a polynomial function if a polynomial exists. Like any other functions, polynomial functions can be represented by a graph  in H2n. Stated precisely, this is a bilinear form. This is a symmetric form for n even , in which case the signature of M is defined to be the form\u2019s signature, and an alternating form for n odd . These can be referred to uniformly as \u03b5-symmetric forms, where \u03b5 = (\u22121)n = \u00b11, respectively, for symmetric and skew-symmetric forms . These f\u03b8 is the determinant, and cos\u03b8 represents the bound angle value in \u00c5, and if cos\u03b811, cos\u03b822, cos\u03b833, and cos\u03b844 are considered zero, the determinant matrix can be written as follows  \u00d7 [\u2212165 \u2212 (\u2212130)] = (\u22120.35) \u00d7 (\u221235) = 1225. The convergence of the two functions is determined via the following relationship:On the function interval \u22120.5 \u2264 x \u2264 \u22120.15, function f1 = \u221214x6) and (f1(x))/(\u2212x6) is computed: (f1(x))/\u3016\u2212x\u30176 =10 + 8/x + 6/x2 \u2212 (0.0002)/x3 \u2212 (0.0556)/x4 \u2212 (0.146)/x5 + (110.3)/x6 also (f2(x))/(\u2212x6) = 14 + 10/x \u2212 7/x2 +5/x3 + (0.0265)/x4 \u2212 (3.0432)/x5 + (165.5)/x6. Because A is a constant, the limits of the two functions are 10 and 14, respectively. While the limits have positive values , both of the functions have the following convergence domain: \u2212\u221e < x < +\u221e.The Riemann theorem shows that via permutation of a series of terms, the sum can be made equal to any number given before. In the case of functions f1(x) and f2(x), these numbers are 10 and 14. So, the functions are convergent.y-axis is described by = (x(t), \u03b8, y(t)). In Cartesian coordinates, these becomes (y(t)cos(\u03b8), y(t)sin(\u03b8), z(t) and (x(t)cos(\u03b8), x(t)sin (\u03b8), y(t)) [The surface of revolution in Euclidean space is a surface created by a rotating curve around an axis of rotation . Here, t), y(t)) ,46. ProtRamachandran plots are a tool for visualizing energetically allowed domains for backbone dihedral angles \u03c8 against \u03d5 of amino acid residues in proteins. Dihedral angle values are annular (0 degrees is the same as 360 degrees). Ramachandran plots warp from right to left and bottom to top . The \u03c9 aThe property space of both spike proteins demonstrates similar characteristics of both molecules. Solvent exposure of both molecules shares common characteristics. Spike protein SARS and SARS-CoV-2 solvent exposure are similar .Binding site identification of potential therapeutic targets is crucial in every drug design process as the expected spike protein has various binding sites. Also, many binding sites in various spike protein regions are described in the literature. An efficient vaccine can be designed by identifying the energetically and structurally favorable binding site. In the present study, the ligand\u2019s interaction with its receptor (spike protein) is a general process of applying various binding sites.Furthermore, the mirror symmetry of SARS-CoV-2 compared with SARS can potentially suggest potential ligands that can be used as vaccines. Symmetric molecules of SARS-proven ligands can be considered in such a way. Also, as seen in The design of an appropriate paratope that can lead to a COVID-19 antibody in in silico methods was used together with secondary data derived from the literature. In order to find a suitable paratope, a set of very low (VL) random sequences were used to assess the chemical space of epitope\u2013paratope interactions see also .Firstly, a target for a potential antibody was established. Spike protein S was chosen as a target due to its volume, shape, and frequency at the external membrane of COVID-19 compared to proteins E, M, and N, respectively. Computational studies and published data show that spike S protein is a valid target ,50,51,52Also, an Aa sequence was chosen as an epitope. The Aa sequence was chosen from the literature  ,54.A set of 47 PDB VL chains was chosen from the literature to explore the paratope space by evaluating the epitope\u2013paratope interactions using distinct computational techniques.https://www-cohsoftware.ch.cam.ac.uk, accessed on 1 July 2023). For example, the following sequence was obtained for 1A8J using Paratome: SYEGSDF. After checking that the sequence was of the outer external region and could contact the antigen, the probabilistic contribution of each Aa was computed using Parapred, in this case yielding the following values: S 0.1649, Y 0.486472, E 0.713605, G 0.32135, S 0.469778, and F 0.0840889. In order to take into account the epitopes\u2019 first light chains, interactions with the light chains were studied. The PSOPIA online server was used [Firstly, a light chain of 47PDB structures was used. Paratopes were detected for each structure using the Paratome online engine . Resultswas used . PSOPIA was used .iFrag, a protein\u2013protein interaction prediction server, was used to compute and quantify the interaction between epitopes and paratopes . First, Spike protein monomer:AYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVNCTFEYVSFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKGNYNYLYRKPFERDIYFPLQSYGFQPTNVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTSQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSACEII monomerSTTEELAKTFLETFNYEAQELSYQSSVASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQTYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEIn order to characterize the VL sequences, a cluster analysis was performed using molecular descriptors. The properties, after the cluster was computed, are the following: sum of atomic polarizabilities, number of hydrogen bond acceptors, number of acidic atoms, number of aromatic atoms, number of H donor atoms, the sum of the number of H bond acceptors and donors, number of heavy atoms, information content, medium information content, number of carbon atoms, number of hydrogen atoms, number of nitrogen atoms, number of oxygen atoms, Balaban index, difference in bonded atom polarizabilities, number of rotable single bonds, number of aromatic bonds, number of bounds, number of heavy bounds, number of single bounds, atomic connectivity index, atomic valence connectivity index, number of chiral centes, number of unconstrained chiral centers, density, diameter (most considerable vertex eccentricity in a graph), SlogP, molar refractivity, topological polar surface area, vertex adjency information, volume surface area, van der Waals acceptor surface area, molecular weight, winner path, Weiner polarizability, Zagreb index, surface rugosity, total energy, angular energy, electronic energy, non-bonded energy, solvation energy, strain energy, van der Waals energy, and globularity. As seen in The following PDB models were used: 6VXXfor SARS-CoV-2spike protein, 6CVR for SARS spike protein, 5X29for SARS-CoV-2 envelope protein, and3I6Gfor SARS-CoV-2 spike protein. Furthermore, the PDB models were energetically minimized, charges corrected, and protonated at pH 7.4, a salt concentration of 0.9 nmol/L, and at 315 K. The model preparations were performed using Schrodinger2009 and MOE 2009software packages ,58. ShapIn order to reduce the amount of data and to simplify the results, monomers for each protein were prepared computationally, as stated before. The trimers and pentamer PDB structures were energetically minimized, and charges were corrected using the AMBER force field available in MOE 2009 software package. For each monomer structure, the energy was minimized, charges corrected, and structures protonated at a physiological pH. Dihedral (torsion angles) for each monomer were computed using the Chemoffice 2008 software package.In probing the chemical space, some structural information, from simple dihedral angles to complex tertiary and quaternary organizations, was obtained computationally using Schrodinger 2009 software packages. Dihedral angles were represented using a scatter plot for each monomer . While an abrupt cut-off was observed in data, and an abrupt variation in data was noted, a logarithmic scale was used. Also, as discussed in the introductory part, by normalizing the data, clusters are more easily observed . A logarhttps://www.wolframalpha.com/, accessed on 1 July 2023) interface was used to perform a cluster analysis of all dihedral angles. To compare their function domains, the function table dn/dxn (f(x)) for n = 1 \u2026 4 was calculated for all four monomers. Surface plots were computed using the radius of gyration of each logarithmic trend line. Here, r2, while exploring a trend and not a correlation between two phenomena, is not significant. A histogram was computed using dihedral angle values on 300 intervals for all three monomers. Using logarithmic trendline equations, a derivate equation was computed for all three protein monomers to represent the dynamic of dihedral angles as a function graphically.In order to expand data dimensionality, a Ramachandran plot diagram was computed for all four monomers in order to characterize the dihedral angles of the structures using the Schrodinger 2009 software package. The Wolfram Alpha software (n/dxn (\u22120.059 log(x) + 6.1779) for n = 1 \u2026 4; for M (COVID-19) protein monomer table: dn/dxn(\u22121.51011 log(x) + 6.1779) for n = 1 \u2026 4; for E (COVID-19), protein monomer table: dn/dxn(\u22120.842 log(x) + 62.544) for n = 1 \u2026 4; and for S (COVID-19) protein monomer table: dn/dxn(\u22120.443 log(x) + 9.7581) for n = 1 \u2026 4.Radar plots were used to emphasize the dihedral angles of protein monomers. Furthermore, the surface of revolution for protein S for SARS and for M, E, and S for SARS-COV-2 were computed to retrieve the 3D dimensionality of monomers\u2019 dihedral angles. Surfaces were generated using the following formulas: for S (SARS) protein monomer table: dThe dimensionality of the COVID-19 interaction space is defined by its interaction with its core receptor. The interaction space has a finite dimensionality defined quantitatively by the polynomial discriminant. Judging by its 2D dimensionality, the interaction space has a negative and positive domain through which epitope\u2013paratope interactions are defined.SARS and SARS-CoV-2 spike proteins have similar solvent exposure and mirror symmetry regarding their dihedral angle chemical space. Both proteins have a similar solvent exposure even if their dihedral angle conformational space has mirror symmetry. Mirror symmetry explains to some extent the chemical inaccessibility of the spike protein SARS-CoV-2 as a receptor and the evasive but permanent chemical bonding in which it is involved."}
+{"text": "Foxc1, Foxc2, or both in mice worsen I/R\u2010induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R\u2010spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R\u2010induced intestinal damage in EC\u2010 and LEC\u2010Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia\u2010induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia\u2013reperfusion (I/R) injury. Vascular endothelial cell (EC)\u2010 and lymphatic EC (LEC)\u2010specific deletions of  The transcriptional activity of FOXC1 and FOXC2 in lymphatic and blood endothelial cells in the small intestine contributes to vascular repair and intestinal regeneration after ischemia\u2010reperfusion injury by regulating CXCL12 and R\u2010spondin3 signalling, respectively. Endothelial cells (ECs) present in the blood and lymphatic vessels are crucial participants in the vascular\u2010dependent processes that restore damaged tissue because they control the secretion of paracrine factors from both the vessels themselves and nearby cells  and other nonepithelial stromal cells express Wnt ligands in the ISC niche , is a homeostatic chemokine expressed in many cell types such as stromal cells, ECs, and fibroblasts in various tissues  defective repair of intestinal BECs and LECs, (ii) reduced expression of CXCL12 and RSPO3 in intestinal BECs and LECs, respectively, and (iii) decreased activation of the Wnt/\u03b2\u2010catenin pathway in ISCs. Importantly, treatment with either CXCL12 or RSPO3 partially rescues the defects in intestinal repair and regeneration associated with EC\u2010 and LEC\u2010Foxc1/c2 deficiency. Chromatin immunoprecipitation (ChIP) assays reveal that both FOXC1 and FOXC2 proteins bind to the regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Together, our data show a new role for FOXC1 and FOXC2 as key transcriptional regulators of paracrine signaling in the intestinal blood/lymphatic vessels during postischemic intestinal repair/regeneration, and our findings may have important implications for the treatment of ischemic bowel disease by modulating the vascular paracrine signaling pathways.In this study, we report that FOXC1 and FOXC2 in intestinal BECs and LECs contribute to vascular repair and intestinal regeneration after I/R injury by regulating the expression of paracrine signaling factors. Inducible, EC\u2010 and LEC\u2010specific, single and compound mutant mice for Foxc1 and Foxc2 mRNA expression in intestinal BECs and LECs was evaluated via quantitative PCR (qPCR) of BECs and LECs isolated from the small intestine in adult mice , which were used as a relative control , LYVE1 (LEC marker), and FOXC1 Fig\u00a0. In the Foxc2\u2010CreERT2 knock\u2010in mice  by oral gavage for 5 consecutive days and subjected to the I/R injury 12\u2009day post tamoxifen treatment. FOXC2\u2010GFP+ cells were detected mainly in the LECs in sham\u2010 and I/R\u2010intestines , which is characterized by gut microbiota induced intestinal inflammation and injury and intestinal ischemia arising from derangements in the intestinal microcirculation angiogenesis have been generally unsuccessful because global single and compound Foxc1/Foxc2\u2010mutant mice die perinatally with severe cardiovascular abnormalities  mice, which (after the mutation is induced) are referred to as EC\u2010Foxc\u2010DKO mice  and lymphatic (CD31+LYVE1+) vessels to confirm Cre\u2010mediated recombination in intestinal BECs and LECs  and their control littermates. Recovery from intestinal damage was impaired in both EC\u2010Foxc1\u2010KO and EC\u2010Foxc2\u2010KO mice 24\u2009h after I/R injury  were measured under homeostasis and at an early time point (3\u2009h) after I/R injury via qPCR. No significant difference was found in the levels of these regulators between sham\u2010operated mice  vs. the control mice but a significant increase vs. the sham\u2010operated EC\u2010Foxc\u2010DKO mice. Furthermore, EC\u2010specific loss of Foxc1/c2 significantly reduced the proliferative response of intestinal epithelial cells to I/R injury as assessed via immunostaining of EpCAM  and BrdU  by breeding conditional\u2010null Foxc1fl/fl and Foxc2fl/fl mice  intestinal epithelial cells  cells  regrow earlier than lacteals (LECs) in the villous stroma  analyses of distal jejuna from control and EC\u2010Foxc\u2010DKO mice 18.5\u2009h after I/R injury. Dimensionality reduction and clustering analysis identified 22 transcriptionally distinct cell clusters  after intestinal I/R injury  and human umbilical vein endothelial cells  by retro\u2010orbital injection 30\u2009min before intestinal ischemia. Quantification of Chiu scores was then performed 24\u2009h after I/R injury. In control mice, the rescue of intestinal mucosal damage was not significant after RSPO3 treatment at I/R\u201024\u2009h. However, in EC\u2010Foxc\u2010DKO mice, RSPO3 treatment could partially rescue the mucosal damage  and complement also contribute to intestinal I/R injury , as well as the lacteal length of EC\u2010Foxc\u2010DKO mice 24\u2009h after I/R injury  and stimulating Wnt signaling in the crypts of the intestinal epithelium.jury Fig\u00a0. Given emice Fig\u00a0. Moreovement Fig\u00a0. The numISCs Fig\u00a0 and celljury Fig\u00a0. However.5\u2009h Fig\u00a0. TogetheFoxc1/Foxc2 expression is crucial for the repair of the intestinal mucosa, BECs, and LECs after I/R injury and that the EC\u2010 and LEC\u2010Foxc\u2010DKO mutations in mice impair canonical Wnt/\u03b2\u2010catenin signaling in ISCs at the crypt base. Furthermore, our scRNA\u2010seq data indicate that RSPO3 expression is attenuated in LECs and stromal cells of the EC\u2010Foxc\u2010DKO mice after intestinal I/R injury, which is at least partially attributable to impairments in intestinal regeneration because ISC activity appears to be crucially dependent on Wnt/\u03b2\u2010catenin signaling in the subepithelial cellular microenvironment in the ISC niche , the extent of Foxc downregulation in the EC\u2010 and LEC\u2010specific mutant mouse lines may not be equal. However, the degree of impairments in intestinal repair is consistently greater in the EC\u2010Foxc\u2010DKO mutant line than in the LEC\u2010Foxc\u2010DKO mutant line, suggesting that Foxc1 and Foxc2 are required in both BECs and LECs for intestinal tissue repair. Equivalent experiments were also performed with adult mice carrying EC\u2010 (and LEC\u2010) specific KO mutations of each individual Foxc gene to determine the similarities and differences between the phenotypes associated with each deletion. While our qPCR and scRNA\u2010seq analyses show that expression levels of Foxc1 in intestinal ECs are higher than those of Foxc2, the phenotypic differences between EC\u2010 (or LEC\u2010) Foxc1\u2010KO and EC\u2010 (or LEC\u2010) Foxc2\u2010KO mice are not particularly distinct. Although the reason(s) for the phenotypic similarities remains unclear, recent evidence indicates that Foxc2 is essential for the maintenance of intestinal LECs and that treatment with antibiotics to deplete gut microbiota rescues the phenotype of LEC\u2010specific Foxc2 mutants, including lymphatic dilation in the small intestine \u2010specific Foxc genes in mice impair the regulation of RSPO3 in LECs  with Cdh5\u2010CreERT2;Foxc1fl/fl (EC\u2010Foxc1\u2010KO), Cdh5\u2010CreERT2;Foxc2fl/fl (EC\u2010Foxc2\u2010KO), Cdh5\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (EC\u2010Foxc\u2010DKO), Vegfr3\u2010CreERT2;Foxc1fl/fl (LEC\u2010Foxc1\u2010KO), Vegfr3\u2010CreERT2;Foxc2fl/fl (LEC\u2010Foxc2\u2010KO), Vegfr3\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (LEC\u2010Foxc\u2010DKO) males, respectively, as described previously , mTmG/+;Vegfr3\u2010CreERT2;Foxc1fl/fl;Foxc2fl/fl (mTmG/LEC\u2010Foxc\u2010DKO) and mTmG/+;Foxc2\u2010CreERT2 mice were generated by crossing mTmG females  with EC\u2010Foxc\u2010DKO, LEC\u2010Foxc\u2010DKO and Foxc2\u2010CreERT2 males, respectively. Genotyping of mice was performed by Transnetyx Inc.et\u00a0al,\u00a0For adult mice, Tamoxifen  was dissolved in corn oil (Sigma #C8267) at 40\u2009mg/ml by shaking at 37\u00b0C for 3\u20134\u2009h. 7\u20138\u2010week\u2010old male adult mice were treated with 150\u2009mg/kg Tm by oral gavage once daily for 5 consecutive days. For neonatal mice, each individual was treated with Tm  by oral gavage once daily from postnatal day 1 (P1) to day 5 (P5) , followed by dehydration in 30% sucrose, and OCT  embedding. Ten or 15\u2009\u03bcm cryosections were cut and immunostained with CD31 and/or LYVE1 antibody  surgery as previously described  by intraperitoneal (i.p.) injection 2 or 18.5\u2009h  before tissue dissection (at I/R\u201024\u2009h and I/R\u201018.5\u2009h respectively).et\u00a0al,\u00a0Distal jejunum was selected for this study because this segment has the most severe mucosal damage after intestinal I/R surgery compared with other segments according to pilot experiments. Different time points  were chosen for different analysis purposes. For histological analysis, transcardial perfusion was performed on the adult mice with cold PBS followed by 4% PFA after anesthesia. Distal jejunum was harvested and cut longitudinally to expose the lumen. After several washes with PBS, the intestine was postfixed in 4% PFA at 4\u00b0C for 4\u2009h (for frozen or whole\u2010mount samples) or for O/N (for paraffin\u2010embedded samples). For qPCR and western blot on whole tissue lysates of the small intestine, blood was removed by transcardial perfusion with cold PBS. Distal jejunum was harvested, opened longitudinally, and washed with cold PBS, then snap\u2010frozen in liquid nitrogen for RNA isolation and protein extraction. For the tissue collection for neonatal intestinal whole\u2010mount staining, the neonates were euthanized at P7 after Tm treatment from P1 to P5. The proximal jejuna were collected, washed with cold PBS, and fixed in 4% PFA at 4\u00b0C for 4\u2009h, then subjected to the whole\u2010mount staining protocol. Proximal jejunum was collected from neonatal mouse due to the ease of operation and similar lacteal length/blood capillary network length ratio between proximal and distal jejuna . Based on the H&E staining, Chiu Score (Chiu 3 in PBS) for 2\u2009h at 4\u00b0C, and then incubated with the indicated primary antibodies  according to the manufacturer's instructions.For IHC\u2010P, 4 or 15\u2009\u03bcm paraffin sections were deparaffinized, rehydrated, subjected to antigen retrieval, permeabilized with PBST, blocked with blocking buffer containing 5% donkey serum in PBST for 30\u2009min at room temperature (RT), and incubated with indicated antibodies Table\u00a0 in blockH&E staining images were acquired using an Olympus Vanox AHBT3 Research Microscope  and an Apple iPhone 12 Pro Max. Fluorescent images were acquired using a Zeiss AxioVision fluorescence microscope, a Nikon A1 Confocal Laser Microscope, or a Nikon AXR confocal microscope with the software of Zeiss AxioVision SE64 Rel. 4.9.1 or NIS\u2010Elements Viewer, respectively. Images were processed and analyzed with Adobe Photoshop, Imaris, and Fiji (ImageJ) software. Imaris imaging software was used to create videos Movies\u00a0 and EV2 et\u00a0al,\u00a0et\u00a0al,\u00a0The mouse lacteal permeability was assessed as previously described  in 100\u2009\u03bcl PBS  to get the CD45\u2212LYVE1+ LECs. The CD45\u2212LYVE1\u2212 cell suspension was incubated with Dynabeads labeled with CD31 antibody (BD #553369) to get the CD45\u2212LYVE1\u2212CD31+ BECs. Finally, the sorted Epis, LECs, and BECs were used for RNA isolation and qPCR analysis. For the detection of gene deletion in ECs in EC\u2010Foxc\u2010DKO mice, CD45\u2212CD31+ ECs (including BECs and LECs) were isolated by using Dynabeads labeled with CD45 antibody (depletion of CD45+ cells) followed by Dynabeads labeled with CD31 antibody (positive selection of CD45\u2212CD31+ cells).Epithelial cells (Epis), blood endothelial cells (BECs), and lymphatic endothelial cells (LECs) were isolated from the distal jejunum for further qPCR analysis as previously described (McCarthy A RNeasy Mini Kit (Qiagen #74104) was used for RNA extraction from cells. TRIzol\u2122 Reagent (Invitrogen #15596026) was used to isolate RNA from whole distal jejunum. The concentration of RNA was determined using NanoDrop\u2122 2000 Spectrophotometers (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio\u2010Rad #170\u20108891). qPCR was performed on triplicates of cDNA samples by using QuantStudio\u00ae 3 Real\u2010Time PCR System (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene\u2010specific primer sets. 18S (for mouse samples) and PPIA (for human cells) were used as internal standards for mRNA expression in mouse samples. Primer sequences are provided in Table\u00a0The frozen intestinal tissue was grinned using mortar and pestle chilled with liquid nitrogen, followed by lysis in RIPA buffer  containing protease inhibitors (Roche #4693116001). After centrifugation, the supernatant of the tissue lysates was collected and mixed with 5\u00d7 Protein Loading Buffer. Equal amount of total protein for each sample was loaded and run on an SDS\u2013PAGE gel. Samples were transferred to 0.45\u2009\u03bcm nitrocellulose (Invitrogen) and western blotted with the antibodies listed in Table\u00a0Foxc\u2010DKO I/R\u201018.5\u2009h. Briefly, mice were anesthetized by isoflurane. Blood was removed by cardiac perfusion with cold PBS. The distal jejunum was then dissected, washed with cold PBS, and cut into small pieces. The tissue was processed for scRNA\u2010seq upon dissociating into single\u2010cell suspension in a digestion buffer as mentioned above for 35\u2009min at 37\u00b0C, followed by filtration through a 70\u2009\u03bcm and a 40\u2009\u03bcm cell strainer. Cells were washed with washing buffer  for three times and resuspended in PBS with 0.04% BSA at a concentration of 1,200\u2009cells/\u03bcl (according to 10\u00d7 Genomics Document #CG00053 Rev B) before being passed through a 30\u2009\u03bcm MACS SmartStrainer. The cell viability was tested by using the Cellometer Auto 2000 Cell Viability Counter . The cell sample was processed for scRNA\u2010seq only when the cell viability was more than 70%. Average cell viability for samples was determined to be 80.96%.For single\u2010cell RNA sequencing, mouse distal jejunums were collected 18.5\u2009h after intestinal I/R surgery. Two mice were used for each group: control I/R\u201018.5\u2009h and EC\u2010Single\u2010cell 3\u2032 gene expression libraries were constructed by using the Chromium Next GEM Single\u2010Cell 3' Reagent Kits v3.1  according to the manufacturer's manual CG000204 Rev D. The single\u2010cell libraries were assessed for quality  and then run by using paired\u2010end 50\u2009bp sequencing on the Illumina HiSeq 4000 platform . Ten thousand cells were targeted for each sample with a sequencing depth of 20,000 read pairs per cell.Foxc\u2010DKO mice using CellRanger. The matrix files were then utilized for data processing and downstream analysis using the BIOMEX browser\u2010based software platform and its incorporated packages developed in R  using the CellRanger toolkit . Gene expression matrices were then generated from both control and EC\u2010FindClusters function in Seurat . Marker set analysis was then performed in BIOMEX on highly variable genes to identify the top 10 gene markers expressed in each initial cluster using a similar methodology described previously  and previously reported the data  package  were then auto\u2010scaled and summarized by principal component analysis (PCA), followed by visualization using Uniform Manifold Approximation and Projection ) to reduce the data into\u00a0a two\u2010dimensional space. Graph\u2010based clustering was then performed in BIOMEX to cluster cells according to their respective gene expression profile using a methodology similar to the Plotly software was used for UMAP and volcano plot visualization.BIOMEX implementation of the et\u00a0al,\u00a0https://www.ebi.ac.uk/gxa/sc/home) and was exported to check Lgr5 expression in mouse small intestinal ECs.The scRNA\u2010seq data for mouse small intestinal ECs from the publication entitled \u201cSingle\u2010Cell Transcriptome Atlas of Murine Endothelial Cells\u201d . Putative sites in the human genome were then searched against the Genome Reference Consortium Mouse Build 38 (mm10) genome using the Evolutionary Conserved Region (ECR) Browser (https://ecrbrowser.dcode.org) and rVista 2.0 tools to identify conserved and aligned putative binding sites between mouse and human sequences. Conserved and aligned putative FOX\u2010binding site sequences are underlined and bolded within the human RSPO3 and CXCL12 ECRs shown below:Putative FOX\u2010binding sites in the TATTTACAAAACGTCTTGGAATGAGAATGAGCTGCTTGTGGTTCCTGTGGCTGATTCAGGGATGGTTTCCTACAGGCAGAGGATGCTGGTCAACCGAATGACCTCTCTGTAACTAACCCGTGCACCCCTGTGGTAAGGCTGTTTGGTCTTATAGGTACCTCTTCTAACTAAGCTTGGAGGGATTTGTTTTTGTGGTAAAGAACTTAGTAATAACCAAACGTCACTGTAAAGACAGATTTAATAATGTTAAGGTCCATCAGAGCCTACTCCTTCTACTACCAACAAGAGAAGCCAGAAATACACTGGGATGCCTTTAGATTCCTGTGCATCAATCTTTCTTTCTCTAAGGATTATGGTCTTGAAATGGATAATGATAGATTCCAAACACATGGAAATCTCTTGCCCCTTTTACTTTTTAGGATCTTTGCAAGCTTACAATATGTACACGTTTTCTGTAAGTCACCAATGCTGAGTTACTGGCATGAAAAATGACCCTGTTACTTGGAAAGTAGTTTCACTTACAAGTCCCCCAGGCCCTGTAATGTCTAAACCTCCTGTGCCACTTTATGTGACTACCCCGCCCCCACAGAGGAGCATGCACAGGAAAAGCAGACTTCCCTTCCCCCACACATTTCCTTAGTTATAAATATAGATTTATGAGATACTTTAATGTTCTAAAACAAATGAAAACCACCCAAGAGGAGCCTCACCAAACCTGAGGTTGTCCAGATTGCATTGACTAAGATTAAGTAAAAGATCATTCATCTCCAGAGGTCATGCAATTAATCTCAGAGTGGGAGTTAAAGCAATGACTAAGCAGAAAAGGAAGCCAAATACAAGCTCGTAACAAAAGGTGCTGGGGCTCCAACATCAAGGAACTTGTTATTTCCTTTTTATTTATTTATTTTTTTTTAATAGACCTAAAACACTCATTCCTTACTACTGGTTTCTTTGGGTCCTAAAATTCCACTTGGTTAGGTCAGCTATTTTCCATGACTATTTTTGATACGGTCAAACAAATACAAAGAATAAGCTTTTAAAAAACAATACCAGTAAATGGGGACACATTATATTGAATAAGGGTATTGTTAGCCAAATTCTAAGATTCATCTTAAATTGTTTTCTTATAAGAATTGTGTATTTACCATTTTAAAAATCACTATTATTTTAAAACACTTAGAAAGTGAACATTTGAAAATGATGTGCCTTTGGATGCTCTGTAATGTTAAGCAGATCCAGACATAAAGACAAAAGTAAATTCCAGAGTATTTTTGTAGCCATGGAATCACCATAAAAAGGGGTTTTTGACCCCAATGTTACCGTAACATTGTCTTCAGCATTTCATATTTAATTACAGTAGATTACTCACCAATATATGTTTACTTCTCTGCATTTAATGAGCAGTTGTTAACATGACTGAAACCATCTGATGATTTTTACCAAATGGAAAAATCTGCCTACAGGGGCAATAAAATAAATATTCAGAATAGAGAGAGGCAGTCATAAAAGACATTACCCGGTTGTAAACGGAGGCGGGTGGTGGTGATCTATTACCCCTGCCTCGGCAGCTTTCAACAGAGTTCTGGAATTCCAGGAGGGGCCCTGACCCAAGGCAATATTTACTTCTGCGGCTTCTTCATCAGGTCAGCATGGGTATAATTCTGTCTACCAGTTGACTGGAGCTGAGGTTTCGAGCAGGAAGTGCAAACCCTGAGTGCTTATAACTCAGGAGGAGTGAGGCACCCCTTCCCAGAGTATGCCAAGAAAAGCACATTGTACTGTCCTGGCTGCAGGGGTGAGGCCCCTGCACACCCAGGCCATTATCAGCTTTGTGCCCTGGCCAACAGCGCCTCTGGTTGGTGCATTTGTCAGCTGTATTTTACCCTAGAGCTCTGGGAGGCTCATCCTTTTTTGGTATACCACCACGTGGAGAGAGCAGAGTTTTAATAGTGTGGCTGCATCAAAACCTTCACCTTTCTCTGCTGAAGGAATGGCCTTCTCTTATGGGCAGGGAGGGTTTCCTAGGGAAAGCCCACCCAGGCAGGAGATGAGGAGAGCAGCATCTGAGCACACTTCATCCCACAGTGCCCATCCCATGAGTATCCTCCATAAATTACAAAGAAAGAAAAAAAATAGGGAAAAAACAAACCTTTATTTCTCGTAAATAAATTCCCACATACAGTAGGACGTTTATACCATGAAACAATTAGCATTTTATTGCTAGTGCATATAATGTCACATTTGATACAATTTTAGTACAAGTGAAAAAATACACTGTGGCTAACATTGAAAAGCTGCAATCACATTTATATATCATATATATTTCTTTACAAATTGCCAGTAGTTTGAGATAATAGAGAAGTATAAACTACTGACATTCATATGGCTCCACTTCAAATATATGAATTGTTCGACTATAAATATATTTTGAAATACATTTGTTTTCTAAAGAAACGTAAAAAAAAATGTGCACAAAAATATATATAAAAAAATGCCTTGCAAAAAGTTACAAATACCACCAGGACCTTCTGTGGATCGCATTTATGCATGGAAATGTCACCTTGCCAACAGTTCTGATTGGAACCTGAAACCCTGCTGTGGCTTCAGGAGGGGGTAGTGGCAAGATGATGGTTTATTCACTGATTTTTTCGCTTCTGATTTCGGAAACCTCAGAGTTTGTTAGTGCCTC CATGGCATACATAGGCTGGGTTAAAAAAAAAAAAAAAAGATCCAAAAACTTGAGCTGCAGATCTAATCTGCTCGTGAGAAAAGCCCATACACTGTCACACATGGGCTGTGAGAAGGGGTCTCAGACACCTGACTGCAGGCAGGCTTAACTATATAAACCAGAAACGTCTATAAGCTCCATCACTAACAACTAATGAATTTTATTTCAGRspo3 and Cxcl12 ECRs corresponding to the human RSPO3 and CXCL12 ECRs are shown below:As comparison, in mouse, the conserved and aligned putative FOX\u2010binding sites (underlined and bolded) within the mouse sequences of ATAAATAAACTGAGGAAATGTGTGGGGGAAGGGAGGGCTGCTTTTCCTGTGCATGTTCTTCTGTAGGGTGGGGCAGTCCTATAAAGTGGGTGAGGAGGGTTTGCCATTATGGGACCTGAGGGATTTGTACCAGAAACTCCTTTCCAAGGAAGGTCATTTTTTATGACAGGGGACTAGGGGTGGGGGAGTGCTTTAGACATGGACACTATTAATCTGTATTTGCAATGATGTCTGATAATTAAGCTCTTTATTGCAGAAACAAAGCCCTGCAGTCTTACGATGCCACACCAAGAAGCAGAGAACAGGAGGAGAACCATGGTCTGGGAAATAAACTTCAAAAGTCCCATATTAATAATTTTTCTCCTCCAGCAAGGCCACACCTAAGGGCTCCATCTCTCCCCCAGATATTATCACCTACTAGCTAGGGATCAAGTATTCAAAACATGAGCCGGGAAGAATTATTTCATATTTCAAGTATAATATTAGTCTGTAATCACAGTGATAATTGGTTATTAAGCTCTTCATTGGAACAACAAAGCCCTCCAGGCACAGTCTGAAATGATACCCATAACAAAGATCCTTCCTGTTGGGGTATAGGAGTTAGCTCCATAGAGGTCATTCAGTTGACCCGCTTCCCTGGCCTGCAGGAAACCATCCCTGAATAAGCCACAGGAACCACAAGCAGCTCGCTCTCATTCCAAGATGTTTCTATTTATTATATTAGTGAATACTATACTACAATTAGATATGAAATGCTGAAGACAATGTTAAGGTGAGAGTGGGTTCACAAACCTCTATATTGGTAGCTTCAGAATTGCAAAAACATTTGTGGAATATTGGTTTTGTCTCTATGCCTGGATCTGTTTAACATAACATAGTATCCACAGCACACTCTTTCAGACAGGGACTTTGTGAATCCTCTAAAACAACCATCATTTCTAAATGCAACAATAAGGAGTATTTTAGGTTTATTCAGGAGGAGGAAAAAAAAAAGGAAATAGCTCCTTGATGTTGGAGGCCAGGCCCCTCTTGTTATGGGCTTGCATGCTGGCTTTCTTTTCTGCTTAATCATCGCCTGAACTCCCACTCTAAGATTGCTTACATAACCCTGGGAGATGAATGATCTTCTACTTAATTTCAGCCAGTGCAATCTGGACAACCTCAGGTTGGATTGGTGAGGCTCCTCTTGGGTGGTTTTCACTTGCTTTAAGAACATTAAAATATCTCATAAATCTAGTAAATATTGCCTTGGGTCAGATCCTCTCCTGGAATCCCAGAACTGTTGAAAGCTGCCAAGGCAGGGGTAATAGATCACCACGACCCGCCTCTGTTTACAACCGGGTAATGCCTTTTACGACCGCCTCTCTCTGTCCTGAATATTTATTTTATTGCCCCTGTAGGGAGATTTTTCCATTTGGTAAAAATCATCAGATGGTTTCAGTCATGTTAACAACTGTTCATTAAACGCAGAGGGTAAACAGAGATGGAAAGGTTTGTTTCTTTCCTTTTTCTCTATTTATTTTTAACTTATGGAGAAAACTCAGGGAGTGAACCCAGGGAGGGAGGCATGTTCAGATACCAGCCATCAGTACCGTCTGCCCAAGTACAAACAAGCTGTATATCCCACTGACAATGGCCTGGTTATGTGGGACCCTCACCTTGACAGCCAGGACGGTACCATGAGCTTTTCTTGGCACACCCTGGAAAGGGTGCCTTACTCTTCCTGAGTTATGAGTGCTTAGGGCTCTGGCTTCCTGCTCAAAACCTCAACTCCAGTCAACTGGTGGACAGAATTATACCCACAATGACCTGCCGAAAAAGCCACAGAATATTTGTAGTCAATTCATATATTTGAAGTGGAGCCATAGTAATGCCAGTAGATATCTCTATGATCTTGAGCTACTGGCAACTTGTAAAGAAATATATATGACATATAAATGTATTGTAGCTTTCCGGTGTCAGCCACGGTGTATTTTTCCACTTGGAATGAAATTGTATCAACTGTGACATTATATGCACTAGCAATAAAATGCTAATTGTTTCATGCTGTAAACCTCCTACCGTATGTGGGAATTTATTTACCTGAAATAAAATCTACTAGTTGTTAGATGGAGTGCACATACATTTCTGAAGATGGAGAAAAACAGGTGTGCCTGCTGATCAGGTGCTGTGGGCTGAAGCCACAGTGGGGATTCTGGGTTCCAATCAGAAATGGAGACAAGATAAAACTTGCATACATTCTTATGATCACAGACGGCCCTGGTGGTTTTTGGTAACTATTTACAAGGCATTTTTTTACATATATTTTTGTGCACTTTTTATGTTTCTTTGGAAGACAAATGTATTTCAGAATAet\u00a0al,\u00a0CXCL12 promoters  according to the prediction of JASPAR, as well as the binding sites at the ECRs of CXCL12 and RSPO3 mentioned above. The ChIP primers are listed below:The ChIP experiment was performed as previously described  and LPS (5\u2009mg/kg) to perturb the normal intestinal colonization process; (ii) gavage with formula every 3\u2009h ; and (iii) exposure to brief episodes of hypoxia (60\u2009s in 100% N2) followed immediately by cold stress (10\u2009min at 4\u00b0C) twice daily. This protocol induces intestinal injuries ranging from epithelial injury to transmural necrosis resembling human NEC, which typically develop after 36\u2009h and has been widely used to study NEC pathogenesis  per sample were analyzed for the fluorescent intensity (FI) of \u03b2\u2010catenin within the ISC by ImageJ. For quantification of FI of FOXC1 and FOXC2 in intestinal BECs and LECs, about 40\u2009~\u200950 cells were quantified by ImageJ. For analysis of BEC and LEC proliferation and apoptosis, confocal Z\u2010stacks were acquired using a 20\u00d7 objective from about 4\u2009~\u20098 different fields of 15\u2009\u03bcm paraffin sections for each sample. Area of blood vessels (CD31+LYVE1\u2212) and lymphatic vessels (CD31+LYVE1+) were measured using ImageJ software. Then, the number of BrdU+ or TUNEL+ cells per 0.1\u2009mm2 vessel areas was calculated and compared between groups. Measurements for the length of blood capillaries and lacteals were performed as previously described  package . All experimental protocols and procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University.Can Tan: Conceptualization; data curation; formal analysis; validation; investigation; visualization; methodology; writing \u2013 original draft; writing \u2013 review and editing. Pieter R Norden: Data curation; formal analysis; visualization; writing \u2013 original draft. Wei Yu: Data curation; formal analysis; investigation; visualization; methodology. Ting Liu: Data curation; investigation; methodology. Naoto Ujiie: Data curation; formal analysis; investigation. Sun Kyong Lee: Investigation. Xiaocai Yan: Investigation. Yaryna Dyakiv: Investigation. Kazushi Aoto: Resources. Sagrario Ortega: Resources. Isabelle G De Plaen: Resources; methodology. Venkatesh Sampath: Resources; funding acquisition. Tsutomu Kume: Conceptualization; supervision; funding acquisition; investigation; writing \u2013 original draft; project administration; writing \u2013 review and editing.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.PDF+Click here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.Source Data for Figure 9Click here for additional data file.Source Data for Figure 10Click here for additional data file."}
+{"text": "A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay.For complete details on the use and execution of this protocol, please refer to Williams et\u00a0al. (2023).  \u2022Linkage-specific enzymatic synthesis of ubiquitin chains\u2022Ubiquitin labeling with fluorescent dyes\u2022Positional attachment of fluorescent dyes to ubiquitin moieties in polyubiquitin chains\u2022FRET-based assay for ubiquitin unfolding by Ufd1/Npl4 and insertion into Cdc48 Publisher\u2019s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. We also describe how chain variants containing a substrate protein or a Met1-linked ubiquitin on the C-terminus of the most proximal ubiquitin are constructed. We used these specifically Cy3-labeled chains together with Cdc48-Ufd1/Npl4 (UN) complexes that had a Cy5 acceptor fluorophore conjugated at a position in the central processing channel of Cdc48 to monitor polyubiquitin-chain engagement in a FRET-based assay. Our studies revealed that Cdc48-UN unfolds and engages ubiquitins in a position- and linkage-specific manner.The protocol below describes the assembly of Lys-48-linked polyubiquitin chains containing a Cy3-donor fluorophore on the N-terminus of the most proximal ubiquitin  competent cells with the expression plasmids for tag-less wild-type S. cerevisiae ubiquitin or ubiquitin with a Met-Cys extension N-terminal to the native Met1 (MCM-Ub) by heat shock at 42\u00b0C in a water bath for 45 s. Recover cells for 1\u00a0h at 37\u00b0C in SOC medium.2.Prepare 50\u00a0mL starter cultures in dYT  with the appropriate antibiotic and grow 14\u00a0h at 37\u00b0C.3.Inoculate 1\u00a0L of Terrific Broth containing kanamycin  with 8\u00a0mL of starter culture and grow at 37\u00b0C while shaking at 180\u00a0rpm until OD600\u00a0= 1.0.4.Induce protein production for \u223c18\u00a0h at 18\u00b0C with 1\u00a0mM IPTG .5.Harvest cells by centrifugation at 3,500 rcf for 30\u00a0min at 18\u00b0C\u201320\u00b0C and resuspend the resulting pellets in NiA buffer  and clarify the lysate by centrifugation at 27,000 rcf for 30\u00a0min at 4\u00b0C.7.Add glacial acetic acid to the supernatant until a pH of 4.5 is reached, as monitored by pH paper.8.Stir the solution for 30\u00a0min at 18\u00b0C\u201320\u00b0C to precipitate proteins other than ubiquitin.9.Clarify the solution by centrifugation at 27,000 rcf for 30\u00a0min at 4\u00b0C.Note: Soluble ubiquitin will be found in the supernatant.10.Dialyze the supernatant against 4\u00a0L of cation buffer A  that has been equilibrated with cation buffer A. Elute ubiquitin from the column with a 0\u00a0mM\u2013350\u00a0mM NaCl gradient over 20 column volumes using cation buffer A and cation buffer B on a Cytiva \u00c4KTA Pure FPLC or equivalent  equilibrated in GF buffer  and cysteine-ubiquitin (MCM-Ub-His) were expressed and cells lysed using the same steps 1\u20136 as above.C-terminally His14.Following lysis, supernatants were incubated with Ni-NTA beads  and batch bound for 30\u00a0min.15.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350\u00a0mM imidazole.16.Eluates were concentrated with a 3\u00a0kDa Amicon spin filter  and subjected to size-exclusion chromatography using a Superdex 75 16/600 column  equilibrated in GF buffer.CRITICAL: TCEP at 1\u00a0mM was included in all purification steps above for MCM-Ub-His to keep the cysteine reduced for subsequent labeling with Cy3-maleimide.Timing: 3\u00a0days17.Ube1, the E2-E3 chimera gp78RING-Ube2g2, and PreScission protease were expressed and cells lysed using the same steps 1\u20136 as above for Ub and MCM-Ub.18.Following lysis, supernatants were incubated with Ni-NTA beads and batch bound for 30\u00a0min.19.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350\u00a0mM imidazole.20.6-tag. The high-imidazole buffer was exchanged for NiA buffer by repeated concentration and dilution, and a subtractive step with Ni-NTA beads was used to remove uncleaved gp78RING-Ube2g2 and His-tagged TEV protease.The eluate for gp78RING-Ube2g2 was treated with TEV protease (at a TEV-to-E2-E3 w/w ratio of 1:200) for 14\u201316\u00a0h at 4\u00b0C to cleave the N-terminal His21.The flow-through containing gp78RING-Ube2g2 as well as the Ube1 and PreScission elutions from Ni-NTA beads were concentrated with spin filters and subjected to size-exclusion chromatography using a Superdex 75 16/600 (gp78RING-Ube2g2 and PreScission) or a Superdex 200 16/600 (Ube1) column equilibrated in GF buffer.Timing: 2\u20133 h22.MCM-Ub and MCM-Ub-His at 50\u00a0\u03bcM in GF buffer were incubated with 1\u00a0mM TCEP for 5\u00a0min to ensure cysteines are reduced for labeling.23.Sulfo-Cy3-maleimide  was added at a 10-fold molar excess (500\u00a0\u03bcM) and labeling occurred for 30\u00a0min at 18\u00b0C\u201320\u00b0C. See 24.A 1\u00a0M stock of dithiothreitol (DTT) was added at 5\u00a0mM to quench unreacted maleimide dye, and the Cy3-labeled proteins were subjected to size-exclusion chromatography with a Superdex 75 increase 10/300 column equilibrated in GF buffer. 25.Cy3Ub and Cy3Ub-His preparations was calculated by comparing the total protein concentration determined by a Bradford assay and the concentration of Cy3 dye determined by absorbance at the excitation wavelength maximum (555\u00a0nm). Labeling efficiency for CRITICAL: Ensure that purified protein stocks are free of thiol-containing contaminants that can react with maleimide. Quenching the labeling reaction with DTT is important, as unreacted Cy3-maleimide can damage chromatography columns.Ub: MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKSTLHLVLRLRGG.MCMUb:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG.MCMUbHis:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGLEVLFQGPHHHHHH.D602AzF:Cdc48MGSSHHHHHHSQDPLEVLFQGPMGEEHKPLLDASGVDPREEDKTATAILRRKKKDNMLLVDDAINDDNSVIAINSNTMDKLELFRGDTVLVKGKKRKDTVLIVLIDDELEDGACRINRVVRNNLRIRLGDLVTIHPCPDIKYATRISVLPIADTIEGITGNLFDVFLKPYFVEAYRPVRKGDHFVVRGGMRQVEFKVVDVEPEEYAVVAQDTIIHWEGEPINREDEENNMNEVGYDDIGGCRKQMAQIREMVELPLRHPQLFKAIGIKPPRGVLMYGPPGTGKTLMARAVANETGAFFFLINGPEVMSKMAGESESNLRKAFEEAEKNAPAIIFIDEIDSIAPKRDKTNGEVERRVVSQLLTLMDGMKARSNVVVIAATNRPNSIDPALRRFGRFDREVDIGIPDATGRLEVLRIHTKNMKLADDVDLEALAAETHGYVGADIASLCSEAAMQQIREKMDLIDLDEDEIDAEVLDSLGVTMDNFRFALGNSNPSALRETVVESVNVTWDDVGGLDEIKEELKETVEYPVLHPDQYTKFGLSPSKGVLFYGPPGTGKTLLAKAVATEVSANFISVKGPELLSMWYGESESNIRDIFDKARAAAPTVVFLDELDSIAKARGGSLG\u2217AGGASDRVVNQLLTEMDGMNAKKNVFVIGATNRPDQIDPAILRPGRLDQLIYVPLPDENARLSILNAQLRKTPLEPGLELTAIAKATQGFSGADLLYIVQRAAKYAIKDSIEAHRQHEAEKEVKVEGEDVEMTDEGAKAEQEPEVDPVPYITKEHFAEAMKTAKRSVSDAELRRYEAYSQQMKASRGQFSNFNFNDAPLGTTATDNANSNNSAPSGAGAAFGSNAEEDDDLYSGSGSGSGSGSGLNDIFEAQKIEWHE.6-TEV-gp78RING-Ube2g2:HisMGSSHHHHHHDYDIPTTENLYFQGHMEARFAVATPEELAVNNDDCAICWDSMQAARKLPCGHLFHNSCLRSWLEQDTSCPTCRMSLNIADNNRVREEGTGSHMAGTALKRLMAEYKQLTLNPPEGIVAGPMNEENFFEWEALIMGPEDTCFEFGVFPAILSFPLDYPLSPPKMRFTCEMFHPNIYPDGRVCISILHAPGDDPMGYESSAERWSPVQSVEKILLSVVSMLAEPNDESGANVDASKMWRDDREQFYKIAKQIVQKSLGL.Timing: 2\u20133 hCy3Ub-Ubn). The proximal ubiquitin is the ubiquitin closest to the substrate and in case of unanchored chains has a free C-terminal glycine, while the distal ubiquitin extends away from the substrate and represents the last moiety added to a ubiquitin chain . For studies with Cdc48-Ufd1/Npl4, we aimed to generate heterogenous chains centered around Ub5 in length. The C-terminal His-tag on unlabeled MCM-Ub-His serves the same purpose as in the major step above. Only unlabeled Ub will be present at the most proximal position in a chain. Formation of Ub-Cy3Ub-Ubn, however,\u00a0requires two steps. In this first step, limiting Cy3Ub is added to MCM-Ub-His to form Ub-Cy3Ub\u00a0and small amounts of Ub-Cy3Ub-Cy3Ub. After a Ni-NTA purification step, Ub-Cy3Ub is mixed with unlabeled Ub to form Ub-Cy3Ub-Ubn.8.Cy3Ub, 1\u00a0\u03bcM E1 and 20\u00a0\u03bcM of the E2-E3 chimera, gp78RING-Ube2g2, in ubiquitination buffer.Create a mixture of 50\u00a0\u03bcM Ub-His, 5\u00a0\u03bcM 9.Cy3Ub and unreacted Ub-His. Initiate elongation by adding ATP to 10\u00a0mM. Ideally, the major products will be Ub-CRITICAL: The goal of this part of the protocol is to add limiting amounts of Cy3Ub to the ubiquitination reaction such that the major product is elongation of Ub-His by a single addition of Cy3-labeled ubiquitin (to form Ub-Cy3Ub). Conditions that lead to elongation of Ub-His by multiple additions of Cy3-labeled ubiquitin Cy3Ub (forming Ub-Cy3Ubn) should be avoided. Small-scale test reactions can be performed using different molar ratios of Ub-His to Cy3Ub to determine optimal ubiquitination reaction conditions  in assay buffer  with and without 4\u00a0\u03bcM UN in assay buffer supplemented with 1\u00a0mg/mL BSA and 1\u00a0mM ATP\u03b3S. These are the 2X Cy5Cdc48 and Cy5Cdc48-UN samples.Create samples of 4\u00a0\u03bcM 17.Cy5Cdc48 or Cy5Cdc48-UN sample. Incubate at 30\u00b0C in a thermocycler. The final concentrations are 0.2\u00a0\u03bcM Cy3Ub-Ubn or Ub-Cy3Ub-Ubn, 2\u00a0\u03bcM Cy5Cdc48, 0\u00a0\u03bcM or 2\u00a0\u03bcM UN, and 0.5\u00a0mg/mL BSA.Create mixtures consisting of 6\u00a0\u03bcL of the 2X polyubiquitin sample and 6\u00a0\u03bcL of the 2X Note: Pore-insertion kinetics are rapid (\u223c3 s)ref.18.Transfer 10\u00a0\u03bcL of each mixture into wells of a 384-well black plate  pre-heated at 30\u00b0C and load into a microplate reader (BMG Labtech CLARIOstar Plus) equipped to measure fluorescence emission spectra of Cy3 and Cy5 following excitation at the absorbance maximum for Cy3.19.Incubate at 30\u00b0C and scan wells for fluorescence emission between 525\u00a0nm and 725\u00a0nm using an excitation wavelength of 480\u00a0nm CRITICAL: Since both Cy3Ub-Ubn and Ub-Cy3Ub-Ubn contain contaminating unlabeled polyubiquitin chains, pore-insertion experiments are performed with a 10-fold excess of Cy5Cdc48-UN to avoid any competition between labeled and unlabeled chains. However, these conditions lead to technical challenges due to some extent of direct Cy5-acceptor excitation when exciting the Cy3 donor, which is further exacerbated by the fact that Cdc48 homohexamers can be modified with multiple Cy5 fluorophores depending on the labeling conditions and efficiency. If there is excessive direct excitation of Cy5, the Cy5 emission peak will dominate the spectrum and, due to spectral overlap, introduce some background fluorescence in the region of the Cy3 emission peak as well. To minimize Cy5 fluorescence emission resulting from direct excitation, experiments should be performed with an Cy3-donor excitation wavelength of 480\u00a0nm or lower.20.Cy5Cdc48 and UN  are synthesized in a single step in which unlabeled ubiquitin is added progressively to Cy3-labeled ubiquitin with a C-terminal His-tag in the presence of ubiquitination machinery. The C-terminal His-tag on the fluorophore-labeled species is critical, because it restricts the labeled ubiquitin to the first position in the chain by preventing conjugation of this moiety to itself or to other ubiquitins during the course of the reaction. A tight distribution of polyubiquitin chain lengths is expected  are synthesized in two steps. In the first step, a limiting amount of Cy3-labeled ubiquitin is added to unlabeled ubiquitin whose C-terminus is blocked with a His6-tag to yield a ubiquitin dimer with the labeled species in the second position (Ub-Cy3Ub). The molar ratio of these species should be optimized such that the single addition of Cy3-labeled ubiquitin to unlabeled ubiquitin is the major product  that can react with maleimide conjugated fluorophores. See Perform a dialysis or desalting step with a 7K MWCO spin desalting column  to exchange for buffer compatible with maleimide labeling.Following conjugation, my cysteine-ubiquitin sample did not elute from the size-exclusion chromatography column or eluted in the void peak. See Attachment of hydrophobic fluorophores to ubiquitin can greatly alter its properties and lead to aggregation or adsorption to filter membranes. We have achieved the best results with sulfonated fluorophores that are more hydrophilic and water soluble. The stability of the fluorophore-conjugated ubiquitin can also depend on the labeling position. Ubiquitin labeled at an N-terminal cysteine may be more well-behaved than ubiquitin labeled at an engineered internal cysteine.My fluorophore labeling efficiency is low. See Ensure that the cysteine-containing ubiquitin sample is in a buffer free of thiol-containing reagents. Oxidation of cysteine during the purification steps prior to conjugation will decrease the labeling efficiency. Include a reducing agent  throughout the purification to keep cysteines reduced.Cy3Ub-His, a diffuse distribution of chains was formed. See During the first round of chain extension on Decrease the amount of unlabeled ubiquitin in the reaction. If the concentration of unlabeled ubiquitin is too high, the processive E2-E3 ligase gp78RING-Ube2g2 will form a broad length distribution and distinct bands will not be visible on the gel.Cy3Ub is a ubiquitin trimer (Ub-Cy3Ub-Cy3Ub) or longer chain (Ub-Cy3Ub-Cy3Ubn). See The major product of the elongation of Ub-His with Cy3Ub to Ub-His.As shown in the example gel A, the maThe Cy3 fluorescence emission peak is masked by the Cy5 fluorescence emission peak originating from direct excitation of excess Cy5 fluorophores. See Cy5Cdc48 could be used in the assay to decrease the size of the Cy5 emission peak, but doing so may compromise the extent of donor quenching if the motor is no longer at concentrations that are saturating for polyubiquitin-chain binding. Another potential solution is to decrease the number of Cy5 fluorophores conjugated to Cdc48 hexamers by performing labeling reactions with lower concentrations of Cy5-maleimide.A lower concentration of a.martin@berkeley.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Andreas Martin (This study did not generate new unique reagents."}
+{"text": "AbstractPageBreakCatharylla Zeller, 1863  is redescribed. Catharylla contiguella Zeller, 1872, C. interrupta Zeller, 1866 and Myelois sericina Zeller, 1881, included by Munroe (1995) in Catharylla, are moved to Argyria H\u00fcbner. Catharylla paulella Schaus, 1922 and C. tenellus  are redescribed. Six new species are described by L\u00e9ger and Landry: C. bijuga, C. chelicerata, C. coronata, C. gigantea, C. mayrabonillae and C. serrabonita. The phylogenetic relationships were investigated using morphological as well as molecular data . The median and subterminal transverse lines of the forewing as well as the short anterior and posterior apophyses of the female genitalia are characteristic of the genus. The monophyly of Catharylla was recovered in all phylogenetic analyses of the molecular and the combined datasets, with three morphological apomorphies highlighted. Phylogenetic analyses of the morphology of the two sexes recovered three separate species groups within Catharylla: the chelicerata, the mayrabonillae, and the tenellus species groups. The possible position of Micrelephas Schaus, 1922 as sister to Catharylla, based on both morphological and molecular data, and the status of tribe Argyriini are discussed. The biogeographical data indicate that the chelicerata species group is restricted to the Guyanas and the Amazonian regions whereas the tenellus group is restricted to the Atlantic Forest in the South-Eastern part of Brazil. The mayrabonillae group is widespread from Costa Rica to South Bolivia with an allopatric distribution of the two species. COI barcode sequences indicate relatively strong divergence within C. bijuga, C. mayrabonillae, C. serrabonita and C. tenellus.The Neotropical genus  Pyraloidea is one of the largest superfamilies of the order Lepidoptera. The monophyly of the group and that of its two main lineages, the Pyraliformes and Crambiformes , are well supported by morphological characters , but these share no evident common characteristics other than the snow white color of the wings.Catharylla is revised using both morphological and molecular data. Phylogenetic relationships within Catharylla and with putatively related taxa as well as the distribution of each species along with biogeographical hypotheses are analysed.In this work, PageBreakCatharylla and outgroup taxa investigated were borrowed from museums and private collectors as listed in Several of the MHNG specimens were kindly given to this institution by collaborators mentioned in the acknowledgments section. The specimens were usually well preserved, the color being sometimes faded. Many specimens from the BMNH were dissected by S. Bleszynski. Unfortunately, his preparations were usually poorly made and badly mounted, sometimes hampering the investigation of genitalia characters.The dissection and slide mounting methods follow Catharylla species, four additional crambine species were included in the dataset for phylogenetic analyses. The material investigated to build the morphological matrix is reported below in Apart from the eight PageBreakPageBreakCatharylla contiguella Zeller, 1872, and Catharylla interrupta Zeller, 1866 could not be found. With the help of the descriptions and illustrations, they were excluded from Catharylla because of the forewing pattern, which is like that of Argyria H\u00fcbner, 1818, with only one median transverse line. For Catharylla sericina Zeller, 1881, based on the description and a photograph of the type in the BMNH, the species was rejected from Catharylla based on the elongated forewing shape and the silvery white pattern without transverse lines. For Catharylla paulella Schaus, 1922, a photograph of the habitus and the genitalia of the female type from the USNM allowed to find other specimens of the same species; the male and female were then associated based on wing pattern. For Catharylla tenellus Zeller, 1839, a photograph of the habitus and the genitalia of the female type were available and the male of the species was associated based on wing pattern. For the descriptions, we followed the nomenclature and terminology used by The types of the two species The following measurements were made with the use of an ocular micrometer: length of labial palpus (base of segment I to apex of segment III), diameter of eye , length of forewing (from base to apex), length of uncus (from tegumen-uncus junction to apex of uncus), length of tegumen connection , length of papillae anales , lengths of anterior and posterior apophyses (from base to apex).Regarding the holotype data, the information was copied exactly as found on the labels with vertical slashes to express changes of lines. Abbreviations are spelled out in square brackets. We assume that the labels are rectangular and white, and that the text is in black ink, otherwise differences are indicated in brackets. Paratype data are reported by country in alphabetical order and the information is recorded without indication of line change. Collecting localities are reported as written on labels, with a question-mark when the locality could not be recovered. Dates and collectors\u2019 information were standardized and the latter placed in parentheses. The specimen depositories are reported with the use of the corresponding acronyms.PageBreakhttp://earth-info.nga.mil/gns/html/). The localities were reported on a text file (*.txt) and loaded on a map using DIVA-GIS 7.4.0.1 . The localities that were not registered in Google Earth were localized more or less precisely with the help of internet search engines or with gazeeters from the GEOnet Names Server (GNS) of the National Geospatial Intelligence Agency  and the two nuclear genes wingless (353 bp) and EF-1\u03b1 (679 bp). These genes show different rates of substitution through time, with COI >> wingless > EF-1\u03b1 . COI peridoptera .Specimens used for molecular investigations are listed in Catharylla specimens collected no more than twenty years ago, a leg was sent to the Canadian Centre for DNA Barcoding (CCDB) at the Biodiversity Institute of Ontario (BIO) in Guelph. The barcode sequences of Catharylla are reported in each species description. The protocol for DNA extraction is found in the supplementary material of http://dev.ccdb.ca/docs/CCDB_Amplification.pdfFor DNA extractions were performed following the method of The primers used are recorded in PageBreakPageBreakPageBreakPCRs were performed using peqGOLD Taq DNA polymerase (PeqLab). In cases of weak or absent PCR result, a re-examination PCR was done using BIO-X-ACT Short Taq polymerase (Bioline). PCR protocols are given in Success of gene amplification was evaluated by an electrophoresis with 1% agarose gel, subsequent gel dying with GelRed and analysis under ultraviolet light. PCR products were purified using ExoSAP-IT (USB Corporation).Sequence PCR was done with the BigDye Terminator-Kit of Applied Biosystems. The amount of each product is reported in Sequence alignment was done with BIOEDIT 7.1.3  and PhyDhttp://www.ebi.ac.uk/ena/data/view/HG793012-HG793020; http://www.ebi.ac.uk/ena/data/view/HG793012-HG793020PageBreakGenetic distances between barcoding sequences of COI are given in Crambus pascuella and Crambus uliginosellus set as outgroups. Maximum parsimony analyses were performed using the Branch-and-bound method as searching algorithm with parameters left unchanged. The bootstrap resampling method with 1000 replicates was used. The resulting tree was a 50% majority-rule consensus tree with bootstrap (BS) values assigned to each node. BS supports are reported on the tree of The 21 characters are listed in PageBreakCatharylla bijuga and Catharylla coronata, and the whole COI gene for Catharylla paulella. For Catharylla bijuga, we used the sequence BC MTD 1840 to build the datasets as this sequence performed better than BC MTD 1839 in phylogenetic analyses. The COI sequence of LEP 972 was combined with that of wingless for LEP 973 given that the two samples come from the same population and are genetically very similar as attest the barcode sequences. We generated four different datasets from the sequence data: a complete dataset with all three genes sequences available for the 12 taxa (mol_1), a 12-taxa dataset with the 3rd codon position of COI deleted (mol_2), a dataset restricted to the 7 taxa for which the sequence of the COI gene and at least of one nuclear gene were available (nucl_1), and the same data as nucl_1 but with the 3rd codon position of the COI deleted (nucl_2). We used the programme RAxML  over 0.95 are considered well supported. PP values are reported on the tree  the morpho-matrix , (2) COIthe tree .Zeller, 1863http://species-id.net/wiki/CatharyllaCatharyllaCrambus tenellus Zeller, 1839, by subsequent designation by PageBreakCatharylla species have snow white to creamy white wings and short labial palpi. They can be separated from other Argyriini by the presence on the forewing of median and subterminal thin transverse lines, slightly curved, convex on costal 1/3. The labial palpi are also shorter in comparison to those of Vaxi. The highly variable male genitalia do not show any synapomorphy or generic diagnostic character. In females, a possible synapomorphy is the strongly reduced anterior and posterior apophyses of abdominal segments VIII and IX, but this is shared with some Crambini and a few other Crambinae (see inae see .Catharylla chelicerata)  : R1 presicerata)  backgrouTympanal organs : TransveCatharylla tenellus group, strongly developed. Juxta medially curved downward, narrowing toward rounded apex, slightly directed downward apically, basally triangular, sometimes with additional lobes at base or ventro-lateral projections. Vinculum of medium width; saccus short, rounded, directed anterad and slightly upward. Phallus straight or curved, usually more strongly sclerotized at apex; vesica without cornuti, with one cornutus, or with crest of cornuti.Male genitalia \u201333: UncuPageBreakrior apophyses 0.25\u20130.45 \u00d7 length of papillae anales, straight, regularly thin. Tergite VIII narrow, with postero-dorsal line of setae. Anterior apophyses reduced, 0.01\u20130.1 \u00d7 length of papillae anales. Sternite VIII about twice length of tergite, not connecting ventrally in tenellus species group. Sterigma present, strongly sclerotized except in tenellus species group, usually forming pockets of variable shape; reduced to sclerotized lamella antevaginalis in Catharylla bijuga. Ductus seminalis connecting posteriorly at base of ductus bursae. Ductus bursae long, at least 2 \u00d7 length of corpus, wide, basally curved. Corpus bursae usually rounded, egg-shaped, often enlarging progressively from ductus bursae, usually with one signum, sometimes without; corpus and ductus bursae covered with minute spicules.Female genitalia : PapillaThe genus is restricted to the Neotropical Region, from Costa Rica to Santa Catarina, Brazil, from sea level to 1300 m \u201346.Catharylla serrabonita in its environment, i.e. forested hills up to about 950 m in elevation, surrounded by cacao or coffee plantations in the lowlands. The moths were coming to light, usually very late (after 23:00).The biology of the species remains unknown. In the Serra Bonita Reserve in march 2011, we observed Catharylla has been placed in the Argyriini , Amazonas, P[ar]q.[ue] Nac.[ional] do Ja\u00fa, Rio Ja\u00fa, bg. Miratuc\u00fa, 1\u00b057'S, / 61\u00b049'W, 26\u201327.vii.1995, U[ltra] V[iolet] light sheet (R. W. Hutchings) (USNM). FRENCH GUIANA: 2 \u2642  with same data as holotype (BMNH); 5 \u2642, 1 \u2640  with same locality as holotype except i.1918 (1 \u2642), ii.1918  (S. M. Klages) ; 1 \u2642, 2 \u2640, Parcelles CIRAD de Combi, plantations exp\u00e9rimentales pk 1.5, 5\u00b018'N, 52\u00b055'30 W, 4.iii.2011, pi\u00e8ge lumineux [light trap] (B. Hermier)  (MHNG); 3 \u2642, 2 \u2640 , Saint-Jean-du-Maroni (Le Moult) (BMNH); PageBreak1 \u2642, Saint-Laurent-du-Maroni (USNM); 1 \u2642  Roura, 3.6km E[ast] Roura at r[oa]d to Crique Gabrielle, 50m, 20.iv.1994, at light (J. S. Miller & C. Snyder) (AMNH); 1 \u2640 , Cayenne, iii.1917 (CMNH). GUYANA: 1 \u2642, New River 1938 (C.A. Hudson) (BMNH); 1 \u2642, Mallali [sic] (USNM). SURINAME: 1 \u2640 , Geldersland, Surinam River (USNM); 1 \u2640 , Sipaliwini Distr[ict], Thibiti area, Kabo Creek, partly swampy, primary forest on hilly slopes, ca. 2km from river, 29.v.1989 (J. Beerlink) (Schouten Coll.).COI barcode sequence of paratype BC MTD 01839 (654 bp): ACATTATATTTTATCTTCGGAATTTGAGCAGGAATAGTTGGAACATCCCTAAGACTTTTAATTCGAGCAGAATTAGGTAATCCAGGTTCTCTTATTGGTGACGACCAAATTTATAATACTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATGCCAATTATAATTGGAGGATTCGGTAATTGATTAGTTCCATTAATATTGGGAGCACCAGATATAGCATTCCCACGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAATCCTATTAATTTCTAGAAGAGTTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCAAATATTGCTCATAGTGGTAGATCTGTAGATTTAGCAATTTTTTCTCTACACTTAGCAGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTCTTAATATACGAATTAATGGTTTATCTTTCGATCAAATACCTTTATTTGTTTGATCTGTAGGAATTACAGCTTTACTTCTTCTCTTATCCTTACCCGTATTAGCTGGTGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCTATCCTTTACCAACACTTAOn the forewing , the sevPageBreakunderside dull white to light ochreous along costal margin, with marginal dashes pronounced. Hindwing snow white, veins slightly ochreous, with shiny aspect; marginal PageBreakline thin, brown, pronounced up to CuA1, then shiny white; fringes white; underside white, with same margin as on recto.Male (n = 17) : AntennaTympanal organs (n = 8): Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic drum elongate, more or less oval, postero-laterally extended beyond transverse ridge.Male genitalia (n = 8) , 12: UncFemale (n = 10): Labial palpi: 1.1\u20131.4 mm long. Forewing length: 11\u201315 mm; frenulum triple.Female genitalia (n = 6) : PapillaThe species occurs in lowlands in the three Guianas and Brazil .bijugus, a, um which means \u201cyoked together, double\u201d, in reference to the bifid costal arm of the male genitalia.Bijuga comes from the Latin PageBreakCatharylla, he gave the manuscript name Catharylla ramona to this species, but never published it. The comparison of the tip of the tubular costal arm of the male genitalia and the female lateral projections of sternite VIII shows rather nicely that the male hooks the female genitalia during the mating process. The specimen collected in Parque Nacional do Ja\u00fa, Brazil, shows a divergence in COI barcode sequence of 5.05% with that of Roura, French Guiana. In morphology we find no significant difference corroborating this divergence. The relationships of this species to the others remain uncertain in our phylogenetic analyses.In some paratypes from French Guiana the collecting data mention a \u201cpk\u201d (=\u201dpoint kilom\u00e9trique\u201d). This kilometric marker refers to the distance of the collecting spot on the forest road to the nearest main road. CIRAD  refers to the name of the research institution leading agronomical research on the Combi site. the Combi site. When S. Bleszynski looked into Diagnosis. The synapomorphies of the group are the quadrangular valva with a truncated apex and the hook-shaped gnathos in the male genitalia. The chelicerata species group can be separated from the other Catharylla species based on additional diagnostic characters. Externally, the forewing has a clear, dark brown costal band, and its length is usually over 14 mm. In male genitalia, the apex of the uncus is regularly rounded with a short, narrow projection medially and the vesica shows one large, curved, pointed cornutus, preceded by a string of 13\u201314 smaller cornuti increasing in size toward apex. In female genitalia, the ductus bursae shows a pronounced, tongue-shaped projection postero-ventrally.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the group as sister-group of the mayrabonillae species group , Amazonas, Rio Negro, Mirapinima, 8.iv.1972  (CNC); 9 \u2642, 1 \u2640, Reserva Ducke, km 26 Manaus\u2013Itacoatiara Highway, 15.iv.1972 (1 \u2642), 18.iv.1972 , 21.iv.1972 , 16.v.1972 (2 \u2642), 17.v.1972 , 18.v.1972   (CNC). FRENCH GUIANA: 5 \u2642, 1 \u2640, 36km SE, Roura (Camp Patawa), 21.xi.2007 , 29\u201330.xi.2007 , 30.xi.2007 (1 \u2642) (MHNG); 1 \u2640  with same data as holotype; 1 \u2642, same data as holotype except 2.ix.2011 (Hermier n\u00b0 24755); 1 \u2640, same data as holotype except /604/ and 4.iii.2011 (Hermier n\u00b0 24344); 2 \u2642, Beaus\u00e9jour, N[ationale] 1 pk 28.5, 4\u00b042'30\"N, 52\u00b023'30\"W, 3.vi.2011, pi\u00e8ge lumineux (Hermier n\u00b0 24545 & 24546) (B. Hermier) (MHNG); 1 \u2642, Route d\u2019Apatou pk 25.5 spk 2+4.4, 1.x.2011, pi\u00e8ge lumineux (B. Hermier) (Hermier n\u00b0 24956) (MHNG); 1 \u2642 , R[ou]te foresti\u00e8re de Saut L\u00e9odate pk 4.5, 4\u00b055'N, 52\u00b033'W, 31.x.1995 pi\u00e8ge lumineux (B. Hermier) (Hermier n\u00b0 8457) (MHNG).PageBreakOther specimens. 1 \u2640 , Nova Olinda, Rio Purus, v.1922 (S. M. Klages) (CMNH); 1 \u2640 , Teff\u00e9 [sic], vi.1906 (W. Hoffmanns) (BMNH).COI barcode sequence of paratype BC MTD 01703 (654 bp): ACTTTATATTTTATCTTTGGAATTTGAGCAGGAATAATTGGAACATCCTTAAGACTACTAATTCGAGCAGAATTAGGTAATCCTGGATCTCTTATCGGGGATGACCAAATTTATAACACTATTGTTACTGCTCATGCATTTGTAATAATCTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGATTAGTACCTTTAATGCTAGGGGCACCAGATATAGCATTCCCTCGTATAAATAATATAAGATTTTGACTTCTTCCCCCCTCTTTAACCCTATTAATTTCAAGTAGAATTGTAGAAAATGGGGCAGGAACAGGATGAACCGTTTATCCACCTTTATCATCTAATATTGCCCATGGAGGCAGATCAGTAGATCTGGCAATTTTTTCACTACATTTAGCTGGAATTTCATCAATTTTAGGGGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATCTTTCATTTGATCAAATACCCCTATTTGTTTGATCAGTAGGTATTACAGCATTACTATTACTTCTATCTTTACCAGTATTGGCGGGAGCTATTACCATACTTCTAACTGACCGAAATCTCAATACTTCCTTTTTTGATCCAGCAGGGGGGGGAGACCCTATTTTATATCAACACCTACatharylla gigantea, Catharylla chelicerata differs in having the male costal arm hook shaped, longer, and thinner than in Catharylla gigantea, and the juxta is strongly downcurved, apically conical whereas it is long, almost straight, without apical conical projection downward in Catharylla gigantea. In female genitalia the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities, opened anterad in Catharylla chelicerata whereas it forms a pair of shallow pockets opened posterad in Catharylla gigantea.From Male (n = 21) , 9: HeadPageBreaksclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped, posteriorly reaching transverse ridge or slightly beyond.Tympanal organs (n = 9): Transverse ridge almost straight medially. Tympanic pockets conical, extending slightly beyond transverse ridge. Tympanic bridge lightly PageBreakshaped, forming angle of about 100\u00b0 with axis of basal arms, about 1/4 longer than uncus. Tegumen arms narrow at base, enlarging progressively toward dorsum to 2 \u00d7 basal width, projected dorsally with bump at connection, connecting at distal 1/6. Cucculus densely setose, slightly directed upward on distal 1/3, apically truncated; basal 2/3 of costa of valva dorso-ventrally and laterally widened; costal arm hook-shaped, PageBreakPageBreakstrongly sclerotized, directed upward at about 45\u00b0 from costal arm base. Juxta triangular, curved downward, tip rounded and sac-like, basal lateral lobes curved ventrally. Saccus short, curved upward medially. Phallus narrow, S-shaped; vesica covered with tiny spicules, with one large, curved, pointed cornutus apically, preceded by string of 13\u201314 smaller cornuti increasing in size toward apex.Male genitalia (n = 9) , 14: UncFemale (n = 4): Labial palpi: 1.8\u20132.2 mm long. Forewing: 15\u201319.5 mm. Frenulum quadruple.PageBreakPageBreakanterior margin; covered with minute punctuation. Ventro-basal section of ductus bursae tongue shaped, strongly sclerotized; ductus bursae long, ventrally sclerotized, widened and looped in basal half; enlarging progressively into corpus bursae. Corpus bursae egg-shaped with one signum.Female genitalia (n = 3) : PapillaThe species was found in French Guiana and Brazil (Amazonas) .Chelicerata\u201d refers to the shape of the costal arms of the male valva, which look like mygalomorph chelicerae.\u201cCatharylla robustella  and Catharylla tenellina  by S. Bleszynski, as indicated on labels, but these names were never published. These two specimens are probably Catharylla chelicerata, but the bad genitalia preparations do not allow to see details, and therefore they are not included as paratypes.Two females included here have been named T. L\u00e9ger & B. Landrysp. n.http://zoobank.org/06B7837B-A5DE-4347-824E-B74127F028E2http://species-id.net/wiki/Catharylla_giganteaHolotype. \u2642, with labels as follows: \u201cBrazil: Amazonas, Manaus, | Reserva Ducke, AM-010, k[ilo]m[eter]. 26 | 2\u00b055'S, 59\u00b059'W, Dec[ember].13, 1993 | J. Bolling Sullivan & | Roger W. Hutchings | U[ltra]V[iolet] Light (Plateau Hut)\u201d; \u201cHOLOTYPE | Catharylla gigantea | T. L\u00e9ger & B. Landry\u201d [red label]; \u201cBL 1747 \u2642\u201d [light green label]. Deposited in USNM.Paratypes. 5\u2642, 2 \u2640. BRAZIL: 1 \u2642, Amazonas, Reserva Ducke, km. 26, Manaus\u2013Itacoatiara Highway, 15.v.1972  (CNC). FRENCH GUIANA: 1 \u2642, 1 \u2640 , Saint-Jean-du-Maroni (E. Le Moult) (BMNH); 1 \u2642 , Oyapok [sic] River, Pied Saut, iii.1918 (S. M. Klages) (CMNH). GUYANA: 2 \u2642, 1 \u2640 , Potaro, ii.1908 (2 \u2642), v.1908 (1 \u2640) (S. M. Klages) (BMNH).Catharylla chelicerata, Catharylla gigantea differs in having the male costal arm shorter, basally wide and tooth shaped while it is long, narrow throughout and hook shaped in Catharylla chelicerata. The juxta is long, tongue shaped, almost straight, and apically rounded, whereas it is downcurved and apically conical in Catharylla chelicerata. In female genitalia, the sterigma forms a pair of shallow pockets opened posterad whereas in Catharylla chelicerata the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities opened anterad.From PageBreakbrown scales at half of length, white tipped. Thorax with some brown at collar. Foreleg coxa white, femur white, ashen brown dorsally; tibia and tarsomeres brown-ochreous, distally ringed with dark brown. Midleg white with tibia-femur joint and base of tibia ashen; tarsomeres ochreous to brown ochreous with upperside brown to dark brown, white tipped. Hindleg white with tarsomeres II\u2013V ochreous to brown ochreous, upperside brown, with white tips. Forewing length: 13.5\u201314.5 mm; snow white with wide brown to dark brown costal line from base to apex; median and subterminal transverse lines faded brown; dark brown spots on termen forming more or less continuous line; fringes brass colored; underside white, with costal margin brown ochreous, outer margin with subtriangular spots. Hindwing snow white; marginal spots dark brown between R5, M1, M2, M3, and CuA1; fringe white; underside snow white, with same spots as on upperside.Male (n = 6) : Head wiTympanal organs (n = 5): Transverse ridge medially convex. Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic bridge lightly sclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped.Male genitalia (n = 5) , 16: UncFemale (n = 2): Labial palpi: 2.5\u20133.1 mm long. Forewing length: 17.5\u201322 mm; frenulum triple.Female genitalia (n = 2) : PapillaCatharylla gigantea has been found in French Guiana, Guyana, and Brazil (Amazonas) (mazonas) .giganteus, a, um meaning very large.The name comes from the Latin The name was given to the species on manuscript labels by S. Blezynski, probably in reference to the large size of the female.PageBreakDiagnosis. The synapomorphies of the group are the dorsal furrow on the uncus, the uncus apex slightly bifid, the presence of a transtilla in male genitalia, and the absence of a ventral connection of sternite VIII in female genitalia. The tenellus species group can also be separated from the other Catharylla species based on the following additional diagnostic characters: the hindwings are creamy-white, and in female genitalia, the papillae anales are not produced.Notes. This group includes three species, including two new ones. Catharylla serrabonita and Catharylla tenellus form a monophyletic group within the tenellus species group http://species-id.net/wiki/Catharylla_tenellusCrambus tenellus Zeller, 1839: 174\u2013175Catharylla tenella : Argyria tenella : Platytes tenella : Holotype. \u2640, \u201cType\u201d [red ringed]; \u201cCatharylla | tenella Z[eller]. | Mon[ograph]. p[age]. 50 Am. anftr.\u201d [not clearly readable]; \u201cZell[er]. Coll[ection]. | 1884\u201d; \u201c\u2640 | Pyralidae | Brit[ish]. Mus[eum]. | Slide N\u00b0 | 1094\u201d. Deposited in BMNH.Other specimens examined. 20 \u2642, 7 \u2640. BRAZIL: 3 \u2642 , S\u00e3o Paulo, Ubatuba, Picinguaba, 23\u00b022'S, 44\u00b050'W, 2\u201320m, 22\u201324.ix.2001 (V. O. Becker n\u00b0132820) (Becker Coll.); 2 \u2642 with same data except 10\u201312.xi.2001 (V. O. Becker n\u00b0133712) (Becker Coll.); 2 \u2642, 1 \u2640 , S\u00e3o Paulo, Bertioga, 5 m, 5.xi.1995 (V. O. Becker n\u00b099090) (USNM); 1 \u2642  with same data (Becker Coll.); 1 \u2642  with same data except 15\u201317.v.1996 (V. O. Becker n\u00b0 99386) (Becker Coll.); 1 \u2642 with same data except 7\u20139.x.1996 (V. O. Becker Coll. n\u00b099757) (Becker Coll.); 1 \u2642 , S\u00e3o Paulo, S\u00e3o Luiz do Paraitinga, 23\u00b020'S, 45\u00b006'W, 900 m, 13\u201320.iii.2001 (V. O. Becker n\u00b0132356) (Becker Coll.); 1 \u2642 , S\u00e3o Paulo, 700 m (E. D. Jones) (BMNH); 1 \u2642 , Minas Gerais, Cara\u00e7a, 1300 m, 1\u20132.iv.1992 (V. O. Becker n\u00b085081) (USNM); 1 \u2640  with same data (Becker Coll.); 1 \u2642  with same data except 25.x.1994  (Becker Coll.); 1 \u2642, 1 \u2640 , Bahia, Porto Seguro, A. d\u2019Ajuda, 16\u00b027'S, 39\u00b003'W, 20 m, 12.vii.2009 (V. O. Becker n\u00b0144140) (Becker Coll.); 2 \u2642  with same data except 15.viii.2008 (V. O. Becker n\u00b0140808) (Becker Coll.); 1 \u2642  with same data except 1\u20133.v.2009 (V. O. Becker n\u00b0142784) (Becker Coll.); 1 \u2642, Paran\u00e0, Castro (USNM); 1 \u2640 , Rio de Janeiro, xi[day and year data missing] (H. H. Smith) (CMNH); 1 \u2640 , Rio de Janeiro, Corcovado, 457 m, 26.xii.1958 (E. P. Wiltshire) (BMNH). No locality data: 1 \u2642, 1 \u2640 ; 1 \u2640 , 1869 (NMW).COI barcode sequence of specimen BC MTD 1710 (654 bp): ACTCTATATTTTATCTTTGGAATTTGATCAGGAATAATTGGAACATCTTTAAGATTATTAATTCGAGCAGAATTAGGGAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAACACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATGGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTGGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAACATAAGATTTTGGTTATTACCCCCTTCCTTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACGGTCTACCCCCCCCTTTCATCTAATATTGCCCATAGTGGAAGATCTGTAGATTTAGCAATCTTTTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAGTAATTTATCTTTTGATCAAATACCTTTATTTGTTTGATCAGTCGGTATTACAGCTTTACTTCTTCTTCTATCTTTACCTGTATTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCTATCTTATATCAACATTTACatharylla serrabonita and Catharylla coronata, Catharylla tenellus can be separated by the median transverse line, which is faintly convex towards costa, whereas it is more strongly convex in Catharylla coronata and Catharylla serrabonita. The male genitalia provide the best diagnostic characters. The most obvious refers to the transtilla, which forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, with, in between, a pair of brushes directed medio-ventrally, whereas it forms a pair of arms pointing posterad with a string of spines ventrally in Catharylla serrabonita and Catharylla coronata. In female genitalia, the anterior angle of sternite VIII is directed downward into a more or less rounded projection covered with short spinules of same length, whereas it is projected anterad in Catharylla serrabonita, and it is not projected in Catharylla coronata.From PageBreaktips. Thorax slightly ochreous at collar. Foreleg coxa white; femur ochreous, dorsally dark brown; tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg light ochreous with tibia-femur joint brown; tarsomeres II\u2013V dark brown on upperside, with white ringed tips. Hindleg white; tarsomeres as midleg. Forewing length: 10.5\u201312 mm; snow white; costal line ochreous, lightly pronounced from base to apex; median and subterminal transverse lines ochreous, median transverse line faintly convex towards costa; outer margin ochreous with 7 dark brown spots often triangular, strongly pronounced; fringes brass colored; underside ochreous with costal margin pronounced in basal half and marginal spots pronounced. Hindwing cream-coloured; outer margin with small ochreous brown spots forming more or less continuous line between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2; underside light ochreous, with marginal spots pronounced; fringes white.Male (n = 20) : Head whTympanal organs (n = 13): Transverse ridge more or less regularly rounded, medially more straight. Tympanic pocket extending slightly beyond transverse ridge. Tympanic drum ovoid, posteriorly not extended beyond transverse ridge. Tympanic bridge faintly sclerotized.Male genitalia (n = 13) , 27\u201329: Female (n = 7): Labial palpi: 1.6\u20132 mm; forewing length 12\u201316 mm; frenulum triple.Female genitalia (n = 7) : PapillaPageBreakThe species is known from Brazil in the Atlantic Forest  .Notes. The species was described from \u201cone female collected in Brazil, near Rio de Janeiro\u201d. Hence, the lectotype designated by a label by S. Bleszynski is not warranted. This designation is presumably based on the fact that Catharylla tenellus, or that there is indeed a deep divergence in the COI barcode between populations of this species.Specimens from Porto Seguro, Brazil show a divergence of 3.34% in COI barcode sequences with the specimen from Ubatuba, Brazil. In morphology, differences in male genitalia are also observed: in the specimens from Bertioga, Cara\u00e7a, S\u00e3o Paulo and Ubatuba the costal arm of the valva is wide and 1/3 of the length of the cucullus, almost reaching its tip, and the dorsal edge at base is slightly produced . In the T. L\u00e9ger & B. Landrysp. n.http://zoobank.org/7E0EB0BE-44C4-42EC-9F4D-2C923E9299E6http://species-id.net/wiki/Catharylla_coronataHolotype. \u2642, with labels as follows: \u201cCol. BECKER | 81552\u201d; \u201cBRASIL:ES | Linhares, 40m | 20-29.ii.1992 | V.O.Becker Col\u201d; \u201cHOLOTYPE | Catharylla | coronata | L\u00e9ger & Landry\u201d [red label]. Deposited in Becker Collection.Paratypes. 21 \u2642, 4 \u2640. BRAZIL: 5 \u2642 with same data as holotype ; 2 \u2642 with same data as holotype (1 used for DNA barcoding BC MTD 01891) except 05\u201309.iv.1992 (V. O. Becker n\u00b082486); 6 \u2642, 1 \u2640 , Paran\u00e0, Rio Negro, 900 m, 8.ii.1973 (2 \u2642), 10.ii.1973 (1 \u2642), 11.ii.1973 (3 \u2642), 13.ii.1973 (1 \u2640) (A. & J. Razowski) (ISZP); 2 \u2642, 2 \u2640, Paran\u00e0, Curitiba, 920 m, 17.ii.1975 (1 \u2642) (V. O. Becker n\u00b010167), 20.ii.1975  (V. O. Becker n\u00b010168), 12.iii.1975  (V. O. Becker n\u00b010166), 10.x.1975 (1 \u2642) (V. O. Becker n\u00b04010) (Becker Coll.); 1 \u2642 , Paran\u00e0, Castro, 950 m (E. D. Jones) (BMNH); 1 \u2642, Paran\u00e0, Quatro Barras, 850 m, 27.ii.1970 (Laroca & Becker) (V. O. Becker n\u00b015442) (Becker Coll.); 1 \u2642  Rio de Janeiro, Novo Friburgo (BMNH); 1 \u2642  Sao Paulo, 700 m (E. D. Jones) (BMNH); 1 \u2642, 1 \u2640 , Santa Catarina, Rio Vermelho, 968 m, 18.ii.1973 (\u2642), 28. ii. 1973 (\u2640) (A. & J. Razowski) (ISZP); 1 \u2642, no locality data (V. O. Becker) (Becker Coll.).PageBreakCOI barcode sequence of paratype BC MTD 01890 (654 bp): ACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACATCATTAAGATTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTCTTATTGGAGATGATCAAATCTATAATACTATTGTAACCGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTAGTTCCCTTAATATTAGGAGCACCAGATATAGCTTTTCCTCGAATAAATAACATAAGATTTTGATTATTACCCCCCTCTTTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCCCATAGTGGAAGATCCGTAGATTTAGCAATCTTTTCCCTTCATTTAGCTGGAATTTCTTCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATCAATAATCTTTCATTTGATCAAATACCTCTTTTTGTTTGATCAGTAGGAATTACAGCTTTACTTCTTCTTTTATCATTACCAGTATTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCCGCAGGAGGAGGAGATCCTATTTTATATCAACATTTACatharylla serrabonita and Catharylla tenellus, Catharylla coronata can be separated with characters of the male genitalia: the uncus is apically bifid and grooved on distal 1/5 in Catharylla coronata whereas it is only indented medially at apex in Catharylla serrabonita and Catharylla tenellus; the costal arm of the valva is short and the apex is curved inward in Catharylla coronata whereas the costal arm is longer and points postero-dorsally in the other two species; the transtilla forms a pair of sclerotized arms slightly bent inward distally, ventrally with a row of short spines increasing in size from base to apex whereas it forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus and a pair of sclerotized arms strongly bent inward on distal 1/4 and with a string of long spines of same length medially along it in Catharylla serrabonita; the juxta is shorter than in Catharylla tenellus, and regularly narrowing toward apex whereas it is strongly narrowing on distal 1/4 in Catharylla serrabonita; the ventral projections of the juxta form a pair of shallow pockets whereas they are bell-shaped in Catharylla serrabonita and thumb-like in Catharylla tenellus; the vesica has a row of 6\u20137 cornuti in Catharylla coronata whereas it does not show any cornuti in Catharylla serrabonita and Catharylla tenellus. In the female genitalia of Catharylla coronata, the anterior angle of sternite VIII is not projected whereas it is rounded, projected anterad and covered with short spinules in Catharylla serrabonita, and projected downward in Catharylla tenellus. The anterior apophyses are quadrangular, anvil shaped whereas they are spine like in the other two species.From PageBreakline thin, light ochreous, apically faded; median transverse line light ochreous, concave on costal half, more or less disrupted; subterminal transverse line ochreous, curving toward base on costal half; R5 vein faintly marked apically with ochreous; outer margin ochreous with 7 pronounced dark brown spots more or less triangular between veins, sometimes connecting; fringes brass colored; underside white ochreous to ochreous, costal margin basally brown; outer margin with pronounced spots. Hindwing white to creamy white, usually with marginal brown spots between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2, forming more or less continuous line; fringes white; underside light ochreous, with dark brown marginal spots pronounced.Male (n = 21) : Head whTympanal organs (n = 7): Transverse ridge more or less regularly rounded. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 7) , 20: UncFemale (n = 4): Labial palpi: 1.6\u20132.2 mm long. Forewing length 14\u201316 mm. Frenulum triple.Female genitalia (n = 4) : PapillaThe species occurs in Brazil in the following states: Bahia, Espirito Santo, Paran\u00e0, Rio de Janeiro, Santa Catarina, Sa\u00f5 Paulo .PageBreakcoronatus, a, um: crowned, referring to the longitudinal string of short spines of the transtilla in the male genitalia.The name comes from the latin Catharylla coronata is the sister species of the Catharylla tenellus + Catharylla serrabonita pair , Esp\u00edrito Santo, Linhares, 40m, 25\u201330.i.1998 (V. O. Becker n\u00b0113929) ; 2 \u2642, 1 \u2640  with same except 20\u201329.ii.1992 (V. O. Becker n\u00b081552) ; 2 \u2642 with same data as holotype except 05\u201309.iv.1992 (V. O. Becker n\u00b082486) (USNM); 10 \u2642  Bahia, Camacan, Serra Bonita Reserve, 15\u00b023'S, 39\u00b033'W, 800 m, B. Landry, V. O. Becker, 1.iv.2011 (1 \u2642), 2.iv.2011 (2 \u2642), 3.iv.2011 , 5.iv.2011 (3 \u2642), 6.iv.2011 (1 \u2642), 7.iv.2011 (1 \u2642) (MHNG); 1 \u2642 with same data except vii.2010 (V. O. Becker) (Becker Coll.); 1 \u2642  Bahia, Porto Seguro, A. d\u2019Ajuda, 16\u00b027'S, 39\u00b003'W, 20 m, 12.vii.2009 (V. O. Becker n\u00b0144140) (Becker Coll.).COI barcode sequence of holotype LEP 979 (516 bp): TAGTTGGAACATCATTAAGACTATTAATTCGAGSAGAGTTAGGGAATCCTGGATCTCTTATTGGAGATGATCAAATTTATAATACTATTGKAACAGCTCATGSATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGACTAGTTCCATTAATATTAGGAGCCCCAGACATAGCTTTCCCCCGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAACCCTTTTAATTTCCAGAAGAATTGTAGAGAATGGAGCTGGAACAGGATGAACGGTTTACCCCCCCCTTTCATCTAATATTGCTCATAGKGGAAGATCTGTAGATTTAGCAATTTTTTCTCTTCATTTAGSAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCTTTTGATCAAATACCGTTATTTGTCTGATCAGTTGGTATTACAGCTTTACTCCTTCTTTTATCTTTACPageBreakCatharylla coronata and Catharylla tenellus, Catharylla serrabonita can be separated by the zigzagging median transverse line with the short triangular dent at CuA2 and the pronounced creamy color of the hindwing. The male genitalia provide the best discriminant characters: in Catharylla serrabonita, the transtilla forms a pair of sclerotized arms bent inward in distal 1/4 and with a string of long spines of same length medially along it, whereas it forms a pair of short, narrow sclerotized arms with pointed tips projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus, and two sclerotized arms slightly bent inward distally, with a row of short spines increasing in size from base to apex in Catharylla coronata, and the juxta is apically narrow and pointed whereas it is triangular and regularly narrowed in Catharylla coronata and Catharylla tenellus. In female genitalia, the anterior angle of sternite VIII is projected anterad into a rounded protrusion covered with short spinules in Catharylla serrabonita, whereas it is projected downward in Catharylla tenellus and it is not projected in Catharylla coronata.From Male (n = 21) : Head whTympanal organs (n = 4): Transverse ridge more or less rounded, medially slightly flattened. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 4) \u201326: UncuFemale (n = 1): Labial palpi: 1.9 mm long. Forewing length: 14 mm. Frenulum triple.PageBreakbetween segment VIII and IX covered with microspines. Sternite VIII laterally about 5/3 length of tergite VIII; posterior margin of tergite VIII with line of setae; sternite VIII forming 2 triangular lobes regularly narrowing downward, not connected, densely covered with short spinules of same length; anterior angle of sternite VIII slightly projected anterad, rounded, covered with short spinules of same length. Anterior apophyses 0.03 \u00d7 length of papillae anales. Sterigma membranous, covered with spinules. Ductus bursae about 3 \u00d7 length of corpus bursae, narrow, basally directed downward and then bent upward. Corpus bursae elongate, ovoid, with one tiny signum.Female genitalia (n = 1) : PapillaThe species occurs in Brazil   & 46.The name comes from that of the Serra Bonita Reserve founded by Vitor O. Becker and Clemira de Souza. It is managed by Instituto Uira\u00e7u in the State of Bahia, Brazil.Serra Bonita Reserve is located in the Atlantic Forest, in a hilly region of cacao plantations and scattered forest. Adults came late to light, usually after 23:00. Our molecular analysis of the COI barcode sequences highlighted that specimens from Serra Bonita respectively show 3.24 and 2.21 % base differences with those of Porto Seguro and Linhares. This divergence is possibly associated with slight morphological differences in male genitalia as shown in Diagnosis. We have not recovered any obvious synapomorphy for this group. It can be separated from the other Catharylla species based on the shorter forewing length, usually between 7.5 and 9.0 mm (maximum 10.5 mm). In male genitalia, the tegumen connection is more than two times longer than the uncus, the uncus is beak-shaped, with the apex narrowing to a point, the gnathos is bent at an angle of about 90\u00b0. In female genitalia, the basal line along the anal papillae is ventrally expanded onto a triangle, and the sterigma forms a pair of sclerotized pockets on each side of the middle, covered with short spines or spicules. The sterigma does not bear tiny setae on the ventral membrane of segment VIII.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the mayrabonillae group as sister to the chelicerata group , [Acre] Rio Branco, 1924 (Dengler) (SMNS); 2 \u2640, Amazonas, Manaus, Reserva Ducke, AM-010, km 26, 2\u00b055'S, 59\u00b059'W, 15.xii.1993, U[ltra]V[iolet] Light (J. B. Sullivan & R. W. Hutchings) (USNM); 1 \u2642 , Amazonas, Fonte Boa, ix.1906 (S. M. Klages) (BMNH); 1 \u2640 , Federal District, Esta\u00e7ao Florestal, Cabeca do Vedao, 1100m, 18.x.1971  (CNC); 2 \u2642, Maranh\u00e3o, Feira Nova, Faz[enda]. Retiro, 480m, 07\u00b000'S, 46\u00b026'W, 1\u20133.xii.2011 (V. O. Becker n\u00b0148263) (Becker Coll.); 2 \u2640, Par\u00e1, Bel\u00e9m, 20m, i.1984 (V. O. Becker n\u00b046981) (Becker Coll.); 1 \u2640, Par\u00e1, Capitao Poco, 25\u201331.i.1984 (V. O. Becker n\u00b097880) (Becker Coll.); 1 \u2642, Rondonia, Cacaul\u00e3ndia, 140m, xi.1991 (V. O. Becker n\u00b079592) (Becker Coll.); 1 \u2640, Rondonia, 62km S[outh] Ariquemes, Fazenda Rancho Grande, 165m, 10\u00b032'S, 62\u00b048'W, 18\u201326.iv.1991 (R. Leuschner) (USNM). COLOMBIA: 1 \u2642, Valle, J[un]ct[ion]. Old B\u2019[uena]v[en]tura R[oa]d. and Rio Dagua, 50m, 8.ii.1989 (J. B. Sullivan) (USNM). COSTA RICA: 1 \u2640 , Alajuela, Area de Conservacion Guanacaste, Estacion Caribe, 12.xi.2007 (S. Rios & H. Cambronero) (INBio); 1 \u2640, Alajuela, Area de Conservacion Guanacaste, Rio Negro, 25.i.2009 (H. Cambronero & F. Quesada) (INBio); 2 \u2642 , Alajuela, San Carlos, Arenal National Park, Send[ero] Pil\u00f3n, Rio Celeste, 700m, light trap, 17\u201319.x.2001 (G. Rodriguez) (INBio); 1 \u2642, Prov[incia] Guanacaste, F[in]ca Pasmompa, Est[acion] Pitilla, 5km SO S[an]ta Cecilia, 400m, xii.1990 (P. Rios & C. Moraga) (INBio). ECUADOR: 4 \u2640 with same data and deposition as holotype; 4 \u2642, 8 \u2640 (1 \u2642 used for DNA barcoding BC MTD 01844) with same data (USNM); 1 \u2640 , Napo, 6km NW Tena, Lumu Caspi, 0\u00b054'37\"S, 77\u00b049'32\"W, 590m, 29.ix.2002 (Schouten Coll.); 1 \u2640 , Pastaza, 1km N Santa Clara, 1\u00b016'02\"S, 77\u00b052'57\"W, 630m, 28.ix.2002 (Schouten Coll.). FRENCH GUIANA: 1 \u2642, 4 \u2640 , Saint Jean de Maroni (E. Le Moult) (BMNH); 2 \u2640, Piste Nancibo, km 6, 4\u00b041'N,; 52\u00b025'W, in logged rain forest, at 125W[atts] mer[cury]-vapor light and 15W[atts] U[ltra]V[iolet], 11.i.1985 (J.[-]F. Landry) (USNM); 1 \u2642, Roura, Montagne des Chevaux, xii.2008 (S. Delmas) (MHNG); 1 \u2642 , Cayen[ne] (BMNH). GUYANA: 1 \u2640 , Omai, vi.1908 (S. M. Klages) (BMNH); 1 \u2640 , Potaro i.1908 (S. M. Klages) (BMNH). PANAMA: 1 \u2640, Rio Trinidad, 12.iii [no year data] (A. Busck) (USNM). PERU: 1 \u2642 , Agnaytia, Huallaga, 400m, ix.1961  (CNC); 1 \u2640 , Yurimaguas, PageBreakHuallaga 14.iv.[19]20 (CMNH). SURINAME: 2 \u2640 , Kabo, 5\u00b016'N, 55\u00b044'W, Saramaca, black light, respectively 15\u201316.iii.1983 and 13\u201314.i.1983 (K.E.Neerling) (Schouten Coll.); 1 \u2640 , Sipaliwini Distr[ict]., Tibiti area, Kabo Creek, partly swampy primary forest on hilly slopes, ca 2km from river, vi.1989 (J. Beerlink) (Schouten Coll.).Other specimen examined. 1\u2640 (used for DNA sequencing Lep 1126), Peru, Hu\u00e1nuco, Rio Llullapichis, Panguana, 74,945\u00b0W / 9,614\u00b0S, 23.9.\u201310.10.2011 (SMTD).COI barcode sequence of paratype 07-SRNP-113921 (654 bp): ACATTATATTTTATTTTCGGGATTTGAGCAGGTATAGTAGGAACTTCACTTAGATTATTAATTCGTGCTGAATTAGGTAACCCTGGCTCTCTTATTGGAGATGATCAAATTTATAATACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATCGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCACCAGATATAGCTTTCCCTCGAATAAATAACATAAGATTTTGATTATTACCACCATCATTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTATCCACCTTTATCATCTAATATTGCCCATGGGGGTAGATCTGTAGATTTAACAATTTTTTCATTACATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCATTTGATCAATTATCATTATTTATTTGATCAGTAGGAATTACTGCTTTACTTTTATTATTATCATTACCAGTTTTAGCTGGGGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCAGCAGGAGGAGGAGATCCAATTTTATATCAACATTTAmayrabonillae group are the shape of the forewing outer margin, which is slightly produced apically in Catharylla mayrabonillae and not produced in Catharylla paulella, and the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3 in Catharylla paulella, whereas these spots are lacking in Catharylla mayrabonillae. The hindwing of Catharylla mayrabonillae has a faded subterminal transverse line on costal half whereas the hindwing of Catharylla paulella lacks this marking. In male genitalia, the heavily sclerotized sacculus bears a dorso-lateral sclerotized string of short spines on distal 1/4 whereas the two processes of the costa are S-shaped in Catharylla paulella, and the apex of the phallus is trifid, rounded medially, shortly triangular laterally, whereas it is simply rounded in Catharylla paulella. In female genitalia, the sterigma forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae, whereas the sterigma forms a pair of shallow rounded pockets on each side of middle and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella.The best discriminant characters externally between the two species of the PageBreakdorsally ashen brown, tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg femur white, tibia light ochreous, basally brown, tarsomeres II\u2013V ochreous with tips ringed white. Hindleg white, except tarsomeres, as in midleg. Abdomen dull white. Forewing length: 7.5\u20138.5 mm; with apex slightly produced; costal line thin, ochreous or white in basal half, white in apical half; median transverse line ochreous, slightly undulated; subterminal transverse line ochreous; transverse lines enlarging into brown spot on costal margin with ochreous bar on costa following subterminal transverse line; terminal sector with light ochreous between veins, margin with thin, dark brown line from apex to CuA1, with two dark brown spots in cubital sector, with spot between CuA1 and CuA2 slightly displaced toward base; fringes brass colored; underside light ochreous with some brownish scales, with thin brown margin. Hindwing white with thin transverse subterminal line faded ochreous, in continuity with forewing median transverse line; outer margin line pronounced, dark brown; underside dull white with thin faded brown margin; fringes white.Male (n = 17) : Head win=7): Transverse ridge regularly rounded, medially slightly flattened. Tympanic pockets broadly rounded, extended widely beyond transverse ridge, connected medially at base of praecinctorium. Tympanic drum bean-shaped, elongated, extended beyond tympanic pockets.Tympanal organs  , 31: UncFemale (n = 37): Labial palpi length: 1.1\u20131.3 mm. Forewing length: 9.5\u201310.5 mm; frenulum triple.PageBreakside of enlargement and posterior section. Corpus bursae circular to elongate, about as long as tergite VII; single signum faintly pronounced.Female genitalia (n = 16) : PapillaCatharylla and the only one so far found in Central America and in Venezuela, Columbia, Ecuador and Peru.The species has been found so far in Panama, Costa Rica, Colombia, Venezuela, Guyana, Suriname, French Guiana, Ecuador, Peru and Brazil  . It is tCatharylla mayrabonillae is named in honor of Ms. Mayra Bonilla of San Jose, Costa Rica, in recognition of her artistic portrayal of the biodiversity and ecosystems of Costa Rica and her many years of support for the existence of the rain forest in Area de Conservacion Guanacaste.The relatively strong COI barcode divergence of 4.34% between samples LEP 1126 from Peru and 07-SRNP-113921 from Costa Rica  is notabSchaus, 1922http://species-id.net/wiki/Catharylla_paulellaCatharylla paulella Schaus, 1922: 131; Holotype. \u2640, with labels as follows: \u201cSao Paulo | S.E. Brazil.\u201d; \u201cCollection | W[illia]mSchaus\u201d; \u201cType No. | 25533 | U.S.N.M.\u201d [orange label]; \u201cSLIDE | SB \u2640 | No.4641\u201d [light blue label]; \u201cCatharylla | paulella | type Sch[au]s\u201d [hand written]; \u201cGenitalia Slide | By SB | USNM 111,535\u201d [green rectangular label with thin black line submarginally]. Deposited in USNM.Other specimens examined. 2 \u2642, 7 \u2640. BOLIVIA: 2 \u2642 , Prov.[incia] del Sara, 450 m, iv.1910 (J. Steinbach) . BRAZIL: 1 \u2640 , Federal District, Planaltina, 15\u00b035'S, 47\u00b042'W, 1000 m, 3.xi.1977 (V. O. Becker n\u00b022055) (Becker Coll.), 1 \u2640  with same locality, 16.x.1990 (V. O. Becker n\u00b096854) (Becker Coll.); 1 \u2640, Maranh\u00e3o, Feira Nova, Faz[enda]. Retiro, 480m, 07\u00b000'S, 46\u00b026'W, 1\u20133.xii.2011 (V. O. Becker n\u00b0148263) (Becker Coll.); 1 \u2640 , Mato Grosso, Urucum, 15 miles S[outh]. of Columb\u00e1, 650 f[ee]t, 19. iv. [19]27, at light (C. L. Collenette) (BMNH); 1 \u2640, Par\u00e0, Bel\u00e9m, 20m, i.1984 (V. O. Becker n\u00b046993) (Becker Coll.); 1 \u2640 , S\u00e3o Paulo (BMNH); 1 \u2640 , S\u00e3o Paulo, S\u00e3o Luiz do Paraitinga, 23\u00b020'S, 45\u00b006'W, 900 m, 13\u201320.iii.2001 (V. O. Becker n\u00b0132357) (Becker Coll.).PageBreakTATTAATTCGTGCTGAATTAGGTAATCCTGGATCTCTTATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGTGCACCAGATATAGCTTTCCCTCGAATAAATAATATGAGATTTTGATTATTACCCCCATCATTAACTCTTTTATTTT?TAGAAGAATTGTCGAAAATGGAACTGGAACAGGATGAACAGTTTACCCACCCTTATCATCCAATATTGCTCATAGAGGTAGATCAGTAGATCTAGCAATTTTTTCTTTACATTTGGCTGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGAATTAATAATTTATCTTTTGATCAATTATCATTATTTATTTGATCTGTAGGTATTACAGCTTTACTTTTATTATTATCATTACCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCTGCAGGAGGAGGTGATCCTATCTTGTATCAACATTTACOI barcode sequence of specimen LEP 965 (654 bp): ACATTATATTTTATTTTTGGAATTTGAGCAGGTATACTAGGAACTTCACTTAGATCatharylla species by the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3. The forewing is also sparkled with dark brown scales, which is unique in the genus. In male genitalia, the two S-like projections of the costal arm of the valva discriminate this species from the other species of Catharylla. In female genitalia, the sterigma forms a pair of shallow rounded pockets on each side of middle, and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella, whereas it forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae.This species can be easily separated from the other Male (n = 2): Head with ochreous chaetosemata. Antenna ochreous with white scales, with patch of dark brown scales at base. Maxillary palpus light ochreous, white tipped. Labial palpus: 1.4\u20131.7 mm long, light ochreous, white tipped. Thorax light ochreous at collar. Foreleg coxa whitish ochreous, femur light ochreous, dorsally ochreous; tibia and tarsomeres greyish brown, distally ringed with dark brown. Midleg and hindleg whitish ochreous; midleg tibia basally brown, hindleg tibia white; midleg and hindleg tarsomeres with white tips. Forewing length: 7\u20138 mm; costal line thin, brown or dirty white; median transverse line ochreous, with two dark brown strongly pronounced spots at 1/3 and 2/3; subterminal transverse line thin, ochreous, with small triangular spot on costal margin; with ochreous bar on costal margin following subterminal transverse line; outer margin ochreous with short dark brown lunules or dashes; fringes brass colored; underside light ochreous with brownish suffusion; with pronounced marginal spots. Hindwing white; outer margin with thin ochreous line in apical half; fringes white; underside dull white with dark brown marginal spots more or less connected on apical half.PageBreaklus with apex more thickly sclerotized, with blunt apical margin, with short triangular ventral projection; vesica covered on basal 1/4 with tiny spicules, with barely visible microspicules all along, with one wide and curved cornutus at about 1/4 length of phallus.Male genitalia (n = 2) , 33: UncFemale (n = 7) , 10: LabTympanal organs (n = 5) : TransveFemale genitalia (n = 5) : PapillaThe species has been found in Brazil  and in Bolivia .Catharylla hibisca to specimens that appear to be Catharylla paulella. The BMNH S\u00e3o Paulo specimen is associated with slide n\u00b0 17692, but the genitalia on this slide seem to be wrongly associated, given the inscription \u201cwrong abdomen?\u201d on the label, as well as the size of the abdomen, which is much bigger than those of Catharylla paulella. Therefore, this specimen cannot be identified with certainty. An error is possible in the association of the sexes of this species as there are no series of both sexes from the same locality or other means of associating them with 100% confidence.The original description doesn\u2019t mention the original number or sex of the specimens but it is assumed that there was only one. S. Bleszynski gave the new name of The number of bases obtained for each barcode sequence is given in Catharylla species. We observe a relatively high divergence between different barcode haplotypes in Catharylla bijuga (5.05%), Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%), sometimes possibly associated with differences in morphological characters  and with geographical distances. The divergence between Catharylla chelicerata samples LEP 1703 and LEP 1704 is of 0.15% (1 base) because they are issued from the same population. Sample LEP 1290 differs from samples LEP 1703 and LEP 1704 respectively by 0.62 and 0.46%. We observe no variation between samples LEP 1708, LEP 1709, LEP 1710 and LEP 1888 because they are all issued from the same population. The inter-specific variation in COI barcode sequences (6.29\u201316.84%) is always higher than the intraspecific variation (0.15\u20135.05%).As most of the data were restricted to few sequence samples per species, and some of the sequences came from the same populations, we don\u2019t have the definitive picture of the intraspecific variation in the COI barcode sequences of The 21 analysed characters are listed in PageBreakPageBreakPageBreakCatharylla is supported by the analysis of mol_1 and the combined Bayesian analysis  and by the analyses of nucl_1 and nucl_2 . Three synapomorphies  and one non-unique apomorphy  support the group. The mayrabonillae group is well supported in all analyses  except in the morphology-based analysis (BS support = 60), because no clear synapomorphy was found for the group. However, two non-unique synapomorphies and one reversal (observed only once in Catharylla) are observed . The tenellus group is well supported by the morphology-based analysis (BS support = 97) with four clear synapomorphies  and two reversals , but show no support in other analyses, probably because only the barcode sequence was available for Catharylla coronata. The closer relationship between Catharylla serrabonita and Catharylla tenellus within the tenellus group is supported by the combined Bayesian analysis and by one non-unique apomorphy (but unique in Catharylla). The tenellus group as represented in the analyses of nucl_1 and nucl_2 (Catharylla serrabonita + Catharylla tenellus) is well supported . The chelicerata group is well supported by the morphology-based analysis (BS support = 96) and the combined Bayesian analysis (PP = 1.00). The group shows two synapomorphies , one non-unique apomorphy (7:1) and one reversal (11:0). Unfortunately, no sequence was available for Catharylla gigantea, thus we cannot compare the morphology with the molecular-based analyses. The closer relationships between the chelicerata and the mayrabonillae groups is strongly supported by the nucl_1 analysis (BS support = 100) and the combined Bayesian analysis (PP = 1.00). Two reversals  support the group. The position of Catharylla bijuga as sister group of the other Catharylla species is weakly supported by the combined Bayesian analysis (PP = 0.90), and show no support in other analyses. The position of Micrelephas pictellus as sister group of Catharylla is supported by the analysis of mol_2 and nucl_2 (respective BS supports of 91 and 100). This node is supported by one synapomorphy . Node 10 (Argyria lacteella+ Micrelephas pictellus+ Catharylla) is supported by the analysis of mol_1 (BS support = 100) and the combined Bayesian analysis (PP = 1.00). One synapomorphy (2:1) supports the group. The monophyly of the two Crambus species chosen as outgroups is well supported in the mol_1 analysis (BS support of 100). The settings of MrBayes do not allow to choose two outgroups, therefore the monophyly of the two Crambus species is not supported by the combined Bayesian analysis. Three non-unique synapomorphies support the genus based on these two species . The synapomorphies of the different groups highlighted by the phylogeny are reported here below:The monophyly of Chelicerata group (node 3)8:1. Apex of valva quadriangular, truncated9:2. Gnathos regularly curvedTenellus group (node 5)5:1. Presence of a dorsal furrow on the uncus7:3. Apex of uncus slightly bifid10:1. Transtilla present20:1. Postvaginal sterigma absentCatharylla (node 8)11:1. Lateroventral projections on juxta17:1. Posterior apophyses/papillae anales < 0.519:1. Ventral membrane of segment VIII with tiny setaeCatharylla + Micrelephas (node 9)6:1. Uncus dorsally bare, or with few setaeCatharylla + Micrelephas + Argyria (node 10)2:1. Length of labial palpi/eye diameter < 2/1PageBreakPageBreakPageBreakCatharylla occurs northward from Middle America with locality records in Costa Rica (Area de Conservacion Guanacaste) at a latitude of 10\u00b054 N, southward to Rio Vermelho  at a latitude of 27\u00b030 S, from sea level up to 1300 m . Within Catharylla, species and species groups show distinct distribution patterns. The chelicerata group is widespread in the Northern Amazonian rainforest of Brazil, and in the three Guyanas , whereas Catharylla paulella is widespread from Feira Nova (07\u00b000 S) down to S\u00e3o Paulo (23\u00b035 S)  , in the Chaco formation   while haplotype BC MTD 1840 is from the Humid Guyana province . Catharylla mayrabonillae barcode haplotype 07-SRNP-113921 is found in the Carribean subregion, whereas haplotype LEP 1126 is found in the Amazonian subregion. The barcode haplotypes of the Catharylla serrabonita populations from the two coastal localities of Linhares and Porto Seguro are more closely related, whereas the haplotype from the population of the forested hills of the Serra Bonita Reserve clearly diverges from the other two  . They arFederal) , 44, wheFederal) . CatharyBolivia) , 44, whiBolivia) . Catharydiagonal  formed bdiagonal , constitther two .PageBreakCatharylla (nodes 7 & 8). The reduced number of taxa , as well as the quality of the datasets (complete sets of genes for nucl_1 and nucl_2 vs partly complete sets of genes in mol_1 and mol_2) explain the better results obtained in nucl_1 and nucl_2, because taxa with incomplete data tend to lower the resolution and the bootstrap supports of the tree  because of the great divergence of these sequences from those of other Catharylla species. However, the clear support brought by the morphological analysis places Catharylla coronata together with Catharylla serrabonita and Catharylla tenellus. The neighbor-joining (NJ) analysis of barcode sequences of Catharylla species (not represented here) places Catharylla coronata together with Catharylla serrabonita (divergence of 7.59% with the sequence BC MTD 1843) and Catharylla tenellus (divergence of 7.3% with the sequence BC MTD 1842), and thus corroborates the findings of the morphology-based analysis. Moreover, the species is morphologically very similar to Catharylla serrabonita especially regarding the transtilla in the male genitalia. The basal position of Catharylla bijuga in Catharylla, as sister taxon of other Catharylla species, is doubtful (PP = 0.90) and may be an artefact due to the great divergence of the barcode sequence (the lowest divergence with other Catharylla species is of 9.7% with sample BC MTD 1843 of Catharylla serrabonita). The supports brought by the combined Bayesian analysis have to be carefully considered since the results are sensitive to small variations of the taxon and character sampling, and the posterior probabilities tend to overestimate the strongness of the nodes  along with Argyria, Urola and Vaxi, and Micrelephas in Crambini , Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%) could therefore suggest that these different barcode haplotypes represent different species. Some morphological variation in male genitalia was found in Catharylla serrabonita and Catharylla tenellus , as well as Catharylla serrabonita and Catharylla tenellus (both collected in Porto Seguro), while Catharylla coronata and Catharylla serrabonita are vicariant, Catharylla serrabonita being distributed north of Linhares, whereas Catharylla coronata is found south of this locality. The patterns of distribution suggest that the diversification of the tenellus group might have occurred in the Atlantic Forest, or have occurred in a wider area that reduced afterwards. In Catharylla tenellus, two forms of male valva linked to different COI barcode sequences seem to be geographically separated, with the form from Ubatuba (associated to barcode sequence BC MTD 1842), Bertioga, S\u00e3o Paulo (S\u00e3o Paulo State), and Cara\u00e7a (Minas Gerais) occurring more to the south than the form from Porto Seguro . The third form, observed in Cara\u00e7a (Minas Gerais) suggests that this locality could represent a point of contact between the two other forms. Regarding Catharylla serrabonita, the locality of Serra Bonita is a moist forest of middle elevation (800 m)  , while t"}
+{"text": "Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of  Drosophila models of SCA3. Hsp104 has no homologue in animals, but has an unusual ability to dissolve PolyQ aggregates in vitro, an activity that could be harnessed therapeutically. Indeed, Hsp104 suppressed degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, which accumulates in disease. However, Hsp104 enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 rescued forms of MJD unable to engage polyubiquitin or with a deletion in the deubiquitylase domain indicating that MJD-ubiquitin interactions hinder protective Hsp104 activities. Importantly, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics for any of the neurodegenerative disorders caused by expanded polyglutamine (PolyQ) tracts including Spinocerebellar Ataxia Type-3 (SCA3). These disorders are connected with the misfolding and aggregation of proteins bearing expanded PolyQ tracts in the neurons of affected individuals. In SCA3, ataxin-3 (MJD) is the protein that bears the PolyQ expansion and forms insoluble aggregates. Here, as a therapeutic strategy we introduce Hsp104, a powerful protein disaggregase from yeast, into  Many neurodegenerative diseases, such as Alzheimer's Disease, Parkinson's Disease (PD), prion disease, and the collection of polyglutamine (PolyQ) disorders, including Huntington's Disease (HD) and the Spinal Cerebellar Ataxias (SCAs), are characterized by the formation of protein inclusions in the nervous system Despite the extraordinary structural stability of amyloid, a protein disaggregase from yeast, Hsp104, can rapidly solubilize amyloid. Hsp104 is a hexameric AAA+ (ATPases Associated with diverse cellular Activities) protein that couples ATP hydrolysis to translocation of substrate through a central pore, thus prying individual monomers from the amyloid fiber Curiously, Hsp104 has no homologue in metazoa. Indeed, until recently it was unclear whether the metazoan proteostasis network possessed any coupled protein disaggregase and reactivation machinery. It is now clear that Hsp110, Hsp70, and Hsp40 collaborate to promote the dissolution and reactivation of disordered aggregates in vitro, with the Josephin domain forming SDS-soluble linear polymers that then convert into SDS-insoluble PolyQ-driven amyloid fibers Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease (MJD/SCA3) is the most prevalent dominantly inherited ataxia C. elegans, Hsp104 prevented aggregation and toxicity of GFP-tagged PolyQ in vivo after aggregates have already formed and degeneration has begun; a situation likely to mimic an actual therapy. Therefore, we created novel Hsp104 Drosophila lines to exploit well-characterized models of disease in combination with powerful genetic tools to temporally control the expression of Hsp104 after disease-associated aggregation and degeneration has begun.Hsp104 has been introduced to combat protein-aggregation disease in metazoan systems with various levels of success in vivo.Our studies reveal surprisingly distinct interactions of Hsp104 with the full-length versus a truncated version of the MJD protein. Importantly, we establish that Hsp104 possesses the ability to suppress the progression of degeneration when activated subsequent to onset of expression of the disease protein. These data indicate that protein context is central in Hsp104 interactions, and that Hsp104 displays the ability to halt the progression of pre-established disease Drosophila to evaluate its ability to prevent and potentially reverse aggregation of disease-associated human proteins, readily available in various fly models of disease. To achieve strong expression of the Hsp104 protein in the fruit fly, we codon-optimized the transgene for Drosophila (see ACAAA) before the start codon Drosophila using the GAL4/UAS system glass gene element for driving GAL4 expression, we instead used a driver line with reduced expression  and the truncated C-terminal region of the protein containing the expanded glutamine tract (MJDtrQ78) To probe the mechanism underlying the dichotomous results found for the Hsp104 interaction with MJDtrQ78 and MJDnQ78, an in-depth investigation of the protein aggregates was performed. To slow protein aggregation such that we could analyze underlying protein accumulations in detail, we expressed the transgenes in the eye with an adult-onset driver rhodopsin1(rh1)-GAL4. Analysis of the PolyQ protein accumulations showed that Hsp104 altered the kinetics of inclusion formation for both MJD protein isoforms. By cryosectioning and subsequent immunohistochemistry (IHC), MJDtrQ78 formed compact inclusions that increased in size over time , top rowTo examine protein accumulation by biochemical methods, we used SDD-AGE (Semi-Denaturing Detergent\u2013Agarose Gel Electrophoresis), a protein agarose gel technique that can resolve amyloid aggregates in vitroNext, we assessed MJDnQ78 misfolding. In contrast to the truncated MJDtrQ78 isoform, the pathogenic full-length MJDnQ78 initially formed amorphous inclusions that did not become more numerous after day 1 . Thus, tWhile it is known that in select conditions, Hsp104 promotes amyloid formation of specific yeast prions We also examined variants lacking DUB activity through mutation of the active site in the Josephin domain, MJD-Q88-C14A , which cTo uncover additional mechanistic insight into the interactions with Hsp104, we examined inclusion formation and kinetics with adult-onset rh1-GAL4 expression. By IHC, the MJD variants formed accumulations in a manner roughly consistent with severity of eye degeneration , precludA summary of the effect of Hsp104 on MJD variants is presented in DPLDWB), which is structurally identical to wild-type but functionally inactive DPLDWB were innocuous  into Hsp104 to ensure that Hsp104 could not engage substrate or hydrolyze ATP, creating the mutant known as Double Pore Loop Double Walker B  the toxic MJDtrQ78 protein driven directly by a gmr element such that the disease-associated protein was constitutively expressed in the eye; (2) a drug-inducible gmr-GAL4 driver known as \u201cGeneSwitch\u201d (gmr-GS) to activate GAL4 expression only in the presence of the drug RU486 (mifepristone) Hsp104 is unique in its capacity to reverse pre-existing amyloids in yeast and DPLDWB had no effect  and progressed in severity to d7 , Fig. 10o effect . Hsp104 Next, we examined the underlying protein aggregates by SDD-AGE and Western immunoblot. We observed that gmr-MJDtrQ78 had high levels of amyloid, and this was lessened with time  . When tuin vivo, which is its ability to mitigate the course of protein-aggregation disease even after it has already initiated. Indeed, our studies show that Hsp104 is able to mitigate disease progression once it has begun, unlike the classical metazoan chaperone Hsp70. These studies provide new insight into the in vivo effects of Hsp104 in the context of a therapeutic agent.Here, we reveal key novel insights into the efficacy and interactions of Hsp104 with the pathogenic PolyQ protein MJD. Our studies reveal the surprising finding that Hsp104 interacts differentially with different forms of the MJD protein. Hsp104 is a potent suppressor of toxicity of the truncated protein, but an enhancer of toxicity of the full-length protein. These differences are determined by specific domains of MJD that are not directly implicated in aggregation. Our findings have also uncovered a heretofore unrecognized and important application of Hsp104 Our detailed investigations of the effects of Hsp104 on the MJD protein led to the unexpected result that Hsp104 has opposite effects on the toxicity of different versions of the MJD protein (MJDtrQ78 and MJDnQ78), despite the fact that these proteins contain the same pathogenic PolyQ stretch. These disparate actions indicate that Hsp104 might be a useful probe to understand the nature of aggregates and the toxicity imposed by them. Hsp70 suppressed both MJDtrQ78 aggregation and toxicity , 3C. By What might these biochemical changes be? Hsp104 can disrupt toxic soluble oligomers of various proteins, including Sup35, which may help explain why Sup35 prion formation is not intrinsically toxic to yeast In other settings, it has been suggested that chaperone-initiated formation of large, insoluble amyloid aggregates can actually be protective by sequestering potentially toxic pre-fibrillar conformers Moreover, our findings demonstrate that an agent with a mitigating effect on the truncated version of the MJD protein may act in a different manner against the full-length MJD protein. Thus, what is good for one may not be beneficial for the other. In MJD/SCA3, as well as other neurodegenerative diseases, fragmentation of the disease protein may initiate aggregation and this process is critical for disease progression Although Hsp104 enhances MJDnQ78 amyloidogenesis and toxicity, we found that elimination of functional domains not implicated in PolyQ aggregation facilitated the ability of Hsp104 to suppress MJD-associated degeneration. Elimination of UIM functionality or removal of a component of the Josephin domain (exon 2) restored the remodeling capacity of Hsp104. This suggests that MJDnQ78 pathogenicity is not intrinsically intractable, but is capable of being suppressed by Hsp104 if other domains of the protein are inactivated . Alternatively, potentiated or MJDnQ78-optimized Hsp104 variants might be developed that are able to overcome these hindrances via increased unfolding power Our studies underscore the importance of protein context in studying protein-misfolding diseases. Within the protein itself, neighboring domains not thought to be involved in aggregation may be impacting accumulation kinetics and the biochemical properties of inclusions in vitro have characterized aggregation of the full-length, pathogenic MJD protein as a two-step process in which the protein assembles first into SDS-soluble fibrillar polymers associating via the Josephin domain, and then converts to SDS-insoluble amyloid fibers driven by the PolyQ domain in vivo as well. Indeed, it is consistent with our observation that full-length MJDnQ78 forms amorphous accumulations that appear visually by IHC before they can be observed as SDS-insoluble amyloid aggregates by SDD-AGE  may increase efficiency of such interactions and enable Hsp104 to rescue disease phenotypes. Moreover, if UIM binding to poly-ub chains is impairing access of Hsp104 to MJD, this suggests that co-administering an agent to modulate function of a neighboring domain may affect the access of a treatment to the aggregation-prone domain. Indeed, increasing global DUB activity coupled with Hsp104 induction could overcome antagonism due to poly-ub chains.DrosophilaChaperone treatment, and examination of Hsp70 in particular, has been an exciting avenue of research in the battle to combat and contain neurodegenerative disease An inducible system is particularly well suited for Hsp104 because of its unique ability to rapidly dismantle pre-existing amyloid aggregates. Since metazoan chaperones can only very slowly depolymerize amyloid Our experimental paradigm offers the exciting possibility to address the efficacy of Hsp104 (or other molecules) in a more genuine therapeutic setting. Indeed, we found that turning on Hsp104 was able to significantly suppress disease-associated degeneration. Interestingly, however, Hsp104 did not disaggregate MJDtrQ78 amyloid in these experiments . Thus, Hin vitro and in the most appropriate animal models. Moreover, the fact that Hsp104 is well tolerated by mammalian systems is encouraging Naturally, several barriers must be surmounted to translate Hsp104 into a therapeutic agent for human neurodegenerative disease Clostridium botulinum, as a therapeutic agent. Despite being a deadly toxin, botulinum toxin variants have found key clinical applications due to their highly potent and selective ability to cleave SNARE proteins and prevent secretion Finally, the concept of using a yeast protein as the basis for a therapeutic agent might at first glance seem implausible. However, it must also have seemed equally implausible to use a lethal protein toxin from the bacterium, DPLDWB were generated by standard techniques using the pUAST vector Drosophila and a Kozak sequence (ACAAA) was added prior to the start codon Transgenic flies expressing UAS-Hsp104 and UAS-Hsp104The full sequence of codon-optimized Hsp104 is:ATGAACGATCAGACCCAGTTCACCGAGCGCGCCCTGACCATCCTGACCCTGGCCCAGAAGCTGGCCAGCGATCACCAGCACCCCCAGCTGCAGCCCATCCACATCCTGGCCGCCTTCATCGAGACCCCCGAGGATGGCAGCGTGCCCTACCTGCAGAACCTGATCGAGAAGGGCCGCTACGATTACGATCTGTTCAAGAAGGTGGTGAACCGCAACCTGGTGCGCATCCCCCAGCAGCAGCCAGCCCCAGCCGAGATCACCCCAAGCTACGCCCTGGGCAAGGTGCTGCAGGATGCCGCCAAGATCCAGAAGCAGCAGAAGGATAGCTTCATCGCCCAGGATCACATCCTGTTCGCCCTGTTCAACGATAGCAGCATCCAGCAAATCTTCAAGGAGGCCCAGGTGGATATCGAGGCCATCAAGCAGCAGGCCCTGGAGCTGCGCGGAAACACCCGCATCGATAGCCGCGGAGCCGATACCAACACCCCCCTGGAGTACCTGAGCAAGTACGCCATCGATATGACCGAGCAGGCCCGCCAGGGAAAGCTGGACCCAGTGATCGGACGCGAGGAGGAGATCCGCAGCACCATCCGCGTGCTGGCCCGCCGCATCAAGAGCAACCCATGCCTGATCGGAGAGCCAGGAATCGGCAAGACCGCCATCATCGAGGGAGTGGCCCAGCGCATCATCGATGATGATGTGCCAACCATCCTGCAGGGAGCCAAGCTGTTCAGCCTGGATCTGGCCGCCCTGACCGCCGGCGCCAAGTACAAGGGCGATTTCGAGGAGCGCTTCAAGGGCGTGCTGAAGGAGATCGAGGAGAGCAAGACCCTGATCGTGCTGTTCATCGATGAGATCCACATGCTGATGGGCAACGGCAAGGATGATGCCGCCAACATCCTGAAGCCAGCCCTGAGCCGCGGACAGCTGAAGGTCATCGGAGCCACCACCAACAACGAGTACCGCAGCATCGTGGAGAAGGATGGAGCCTTCGAGCGCCGCTTCCAGAAGATCGAGGTGGCCGAGCCAAGCGTGCGCCAGACCGTGGCCATCCTGCGCGGACTGCAGCCCAAGTACGAGATCCACCACGGCGTGCGCATCCTGGATAGCGCCCTGGTGACCGCCGCCCAGCTGGCCAAGCGCTACCTGCCATACCGCCGCCTGCCAGATAGCGCCCTGGATCTGGTGGATATCAGCTGCGCCGGAGTGGCCGTGGCCCGCGATAGCAAGCCAGAGGAGCTGGATAGCAAGGAGCGCCAGCTGCAGCTGATCCAGGTGGAGATCAAGGCCCTGGAGCGCGATGAGGATGCCGATAGCACCACCAAGGATCGCCTGAAGCTGGCCCGCCAGAAGGAGGCCAGCCTGCAGGAGGAGCTGGAGCCACTGCGCCAGCGCTACAACGAGGAGAAGCACGGCCACGAGGAGCTGACCCAGGCTAAGAAAAAGCTGGATGAGCTGGAGAACAAGGCCCTGGATGCCGAGCGCCGCTACGATACCGCCACCGCCGCCGATCTGCGCTACTTCGCCATCCCCGATATCAAGAAGCAGATCGAGAAGCTGGAGGATCAGGTGGCCGAGGAGGAGCGCCGCGCCGGCGCCAACAGCATGATCCAGAACGTGGTGGATAGCGATACCATCAGCGAGACCGCCGCCCGCCTGACCGGCATCCCCGTGAAGAAGCTGAGCGAGAGCGAGAACGAGAAGCTGATCCACATGGAGCGCGATCTGAGCAGCGAGGTGGTGGGCCAGATGGATGCCATCAAGGCCGTGAGCAACGCCGTGCGCCTGAGCCGCAGCGGACTGGCCAACCCACGCCAGCCAGCCAGCTTCCTGTTCCTGGGCCTGAGCGGCAGCGGCAAGACCGAGCTGGCCAAGAAGGTGGCCGGCTTCCTGTTCAACGATGAGGATATGATGATCCGCGTGGATTGCAGCGAGCTGAGCGAGAAGTACGCCGTGAGCAAGCTGCTGGGCACCACCGCCGGCTACGTGGGCTACGATGAGGGCGGCTTCCTGACCAACCAGCTGCAGTACAAGCCCTACAGCGTGCTGCTGTTCGATGAGGTGGAGAAGGCCCACCCCGATGTGCTGACCGTGATGCTGCAGATGCTGGATGATGGCCGCATCACCAGCGGCCAGGGCAAGACCATCGATTGCAGCAACTGCATCGTGATCATGACCAGCAACCTGGGCGCCGAGTTCATCAACAGCCAGCAGGGCAGCAAGATCCAGGAGAGCACCAAGAACCTGGTCATGGGCGCCGTGCGCCAGCACTTCCGCCCCGAGTTCCTGAACCGCATCAGCAGCATCGTGATCTTCAACAAGCTGAGCCGCAAGGCCATCCACAAGATCGTGGATATCCGCCTGAAGGAGATTGAGGAGCGCTTCGAGCAGAACGATAAGCACTACAAGCTGAACCTGACCCAGGAGGCCAAGGATTTCCTGGCCAAGTACGGCTACAGCGATGATATGGGCGCCCGCCCCCTGAACCGCCTGATCCAGAACGAGATCCTGAACAAGCTGGCCCTGCGCATCCTGAAGAACGAGATCAAGGATAAGGAGACCGTGAACGTGGTGCTGAAGAAGGGCAAGAGCCGCGATGAGAACGTGCCAGAGGAGGCCGAGGAGTGCCTGGAGGTGCTGCCAAACCACGAGGCCACCATCGGAGCCGATACCCTGGGCGATGATGATAACGAGGATAGCATGGAGATCGATGATGATCTGGATTAA5\u2032-TCGAACCCAGTGGAAACCCTTGAAATGCCTTTAACTCGAGACGG-3\u2032 and 5\u2032-GTACCCGTCTCGAGTTAAAGGCATTTCAAGGGTTTCCACTGGGT-3\u2032), with a single copy of the 31 bp glass-binding site from the Rh1 proximal enhancer, were duplexed and ligated into the vector, producing p1\u00d7GR (1 copy of glass reporter). This plasmid was then modified to introduce the GAL4 coding sequence, excised from pGaTN Multiple insertion lines were characterized for each transgene. To create the 1\u00d7gr-GAL4 driver line, the pGMR (glass multimer reporter) vector 2)  area was selected; particle analysis was performed with ImageJ and statistics performed with one-way ANOVA and unpaired t-test.Heads were frozen in Tissue Freezing Medium (Electron Microscopy Sciences) and sectioned at 12 \u00b5m by cryotome, and the tissue sections were then fixed with 4% paraformaldehyde. Immunohistochemistry was performed according to standard procedures using primary antibodies anti-HA 5B1D10  or anti-myc 9E10  (both mouse) alongside either anti-Hsp104  or anti-Hsp70  (both rabbit). Hsp70 staining was confirmed with human-specific anti-Hsp70  (mouse) alongside anti-HA Y11  or anti-myc A14  (both rabbit). Rabbit primary antibodies were preadsorbed at 1\u223625 with fixed, dissected wild-type larvae. Secondary antibodies were Alexa Fluor 594 Goat-anti-Mouse IgG , Alexa Fluor 488 Goat-anti-Rabbit IgG , Alexa Fluor 594 Goat-anti-Rabbit IgG , and Alexa Fluor 488 Goat-anti-Mouse IgG . Sections were co-stained with Hoechst nuclear dye  and viewed with a Leica fluorescence microscope. A 75 \u00b5m\u00d775 \u00b5m square  and anti-actin  with secondary antibody Goat-anti-Rabbit-HRP . For MJD aggregation analysis through SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis) and accompanying Western immunoblots, heads were ground in lysis buffer  A 4.0 mg/ml stock solution of RU486 (Sigma M8046) was prepared in 100% ethanol, and then 50 \u00b5l (200 \u00b5g) was added to pre-prepared food vials containing \u223c12 ml of food and gently shaken overnight"}
+{"text": "Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of +iph1 promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in  Human Insulin Degrading Enzyme (hIDE) or insulinase belongs to the M16A family of peptidases, which comprises large zinc-dependent metalloproteases found in all prokaryotic and eukaryotic organisms examined The budding yeast orthologue of hIDE, Ste23p, displays similar substrate specificity as mammalian IDE and, together with Axl1, the second yeast M16A metalloprotease, cleaves the precursor of the mating pheromone a-factor Cellular proteins are subjected to continuous damage and maintenance of protein homeostasis is central to all biological processes. A cellular compartment particularly susceptible to protein damage is the endoplasmatic reticulum (ER). Accumulation of misfolded proteins in the ER induces the Unfolded Protein Response (UPR) that increases the level of chaperones, stimulates retro-translocation of misfolded proteins to the cytosolic proteolytic system and attenuates general translation and transcription. If this response cannot resolve the ER stress, apoptotic pathways are engaged Schizosaccharomyces pombe has two: the non-essential +tor1 gene encodes the kinase forming the TORC2 complex, while the essential +tor2 gene encodes the kinase present in the TORC1 complex tor2-S1837E mutant, which is predicted to prevent interaction with the FKBP12-rapamycin complex in vitro, similarly to other organisms The generation and resolution of cellular stress is intimately linked to the evolutionary conserved target of rapamycin (TOR) kinase, which regulates cell growth according to nutrient and energy availability Unbridled activation of mammalian TOR exacerbates cellular stress and is linked to diabetes, cancer and a shorter life span S. pombe homologue of mammalian IDE as model.Several facts concerning IDE \u2013 its preference for amyloidogenic substrates able to cause cellular stress, its high expression in beta cells subjected to permanent ER stress, its dead-end chaperone function, indirect evidence for unidentified functions that might underlie its ubiquitous expression \u2013 prompted us to speculate that IDE might be implicated in the response to proteotoxic stress. To address this hypothesis, we took advantage of its evolutionary conservation and used the Strains are listed in iph1 with KanR marker: (5\u2032TGGCCTCTAAACAGTAATGCCTACGTACTGTGTGTATGTAAACACATAATTCAACCTATTGCCATATTTCTTACATATTACGGATCCCCGGGTTAATTAA-3\u2032) and (5\u2032CTAGCAGAAGAGTAGGTCTCGTCACACTTGTTTGGATAGCGAGAAAAACCGCAGTGCCAGAATGCAAAACTGAAATTAAGGAATTCGAGCTCGTTTAAAC3\u2032).Gene disruption and replacement were performed by PCR-based gene targeting iph1 with ura4+ marker:Primers used to to disrupt (5\u2032TATTACCCTTTTTTTGGGTGTAATAGCAGTAGTCAGAATTCTGGGTTGTTTTATCTTTTCCTTTCATAAATAAAAACGCCAGGGTTTTCCCAGTCACGAC-3\u2032) and (5\u2032GTGCCAGAATGCAAAACTGAAATTAAGATGAGAATATAAAATCAGTAAATTTGAGAATCGGATTAGGGAAAAAAAAAGCGGATAACAATTTCACACAGGA).tor1 with ura4+ marker in strain iph1-d ura4-D18: (5\u2032TGGAAGAATTGAACACCGCGACTATTAGAAAGTCTATCGTTTCACTCGCTCTCTTTGATTCATGGAGTATTTTAGTCGCCAGGGTTTTCCCAGTCACGAC 3\u2032) and (5\u2032TAAATTAATAACAACACGAAAAAAATTATCATAATCTCAAAAAACAGAAAACATCATTACCAAAAACTACACCATCAGCGGATAACAATTTCACACAGGA3\u2032).Primers used to disrupt iph1 coding sequence between position 188 and position 280 by the ura4+ gene to obtain strain iph1-M:Primers used to replace the (5\u2032GAGAGATCCGGAAACAGATAATGCAAGTGCAGCTATTGACGTTCACATCGGCAGTCAAAGCAATCCACGAGAGTTGCGCCAGGGTTTTCCCAGTCACGAC 3\u2032) and (5\u2032ATAGAGCATCATGAGACACTTCGAAGTAATAATTTGTATTATTAGAGGCTGTATAGGCGTTTGAAATTCCATTATGAGCGGATAACAATTTCACACAGGA 3\u2032).iph1-E71D was obtained by replacing the ura4+ gene in strain iph1-M with a DNA fragment of iph1 gene mutated at codon 71 (GAA to GAT). This fragment was synthesized, cloned and sequenced by GeneArt .Mutant The cDNA coding the hIDE protein was cloned into pCRBlunt plasmid. Site direct mutagenesis was used to change nt A390 to C to obtain the hIDE-E111D mutant as described in (5\u2032ATTCAACCTATTGCCATATTTCTTACATATTACCCTTTTTTTGGGTGTAATAGCAGTAGTCAGAATTCTGGGTTGTTTTATCTTTTCCTTTCATAAATAAAAAATGCGGTACCGGCTAGCG 3\u2032) and (5\u2032TGTTTGGATAGCGAGAAAAACCGCAGTGCCAGAATGCAAAACTGAAATTAAGATGAGAATATAAAATCAGTAAATTTGAGAATCGGATTAGGGAAAAAAAACTAGAGTTTTGCAGCCATGAAGTTAATATG-3\u2032).iph1-d ura+ strain and transformants were selected on standard 5 fluoroorotic acid (5-FOA) medium. Stable transformants expressing hIDE and hIDE-E111D from the iph1 promoter were selected.PCR- fragments were used to transform the Alignment was performed with Clustalw software. The tridimensional structure of Iph1 was performed using the Modeller software A full length cDNA of 2911 bp encoding Iph1 was amplified using a high fidelity enzyme  and primers encoding a C-terminal extension by six histidine residues, inserted into pCRBlunt , sequenced completely to confirm the absence of errors, and transferred as XbaI/PstI fragment into the baculovirus transfer vector pVL1393 (Invitrogen). To produce the E71D mutant, site-directed mutagenesis was performed directly on the pVL1393-Iph1 plasmid. The primers used for the mutagenesis PCR were (5\u2032GGATTGGCGCACTTTTGTGATCATCTGTTGTTTATGGGGAC3\u2032) and (5\u2032GTCCCCATAAACAACAGATGATCACAAAAGTGCGCCAATCC3\u2032). After the PCR, the reaction mixture was digested with Dpn I and this material was used for transformation. Successful mutagenesis was confirmed by sequencing. Recombinant baculoviruses encoding wt Iph1 and Iph1-E71D were produced by co-transfection of Sf9 insect cells with the resulting plasmids and BaculoGold\u2122 virus DNA, followed by a plaque assay and plaque selection by PCR. Recombinant Iph1 and Iph1-E71D were produced in Hi5 insect cells after infection with the recombinant baculoviruses. Cells were harvested 72 h post-infection and lysed on ice for 30 min in 25 mM Tris, 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, pH 8.0 in the presence of protease inhibitors. The lysis supernatant was harvested by centrifugation for 10 min at 14000 rpm. Then it was transferred on Ni-NTA beads (Invitrogen) equilibrated in 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0 and left to bind on a turning wheel at 4\u00b0C overnight. The resin was washed with the same buffer containing 10 mM imidazole, then with 25 mM imidazole and the protein was eluted with buffer containing 300 mM imidazole. The recombinant protein was used directly for enzymatic assays. Insect cell-expressed recombinant hIDE carrying an N-terminal extension by 7 His residues was purchased from R&D Systems .Enzymatic activity towards the fluorogenic substrate Mca-RPPGFSAFK(Dnp)  was measured by following the time-dependent increase in the fluorescence signal at 405 nm after excitation at 340 nm on a Mithras LB 940 plate reader . 10 \u00b5M of substrate were incubated with 10 ng of hIDE or 20 ng of Iph1 in 50 mM Tris, 150 mM NaCl pH 7.4 at 25\u00b0C and fluorescence was recorded for 10 min. The resulting time slope was used to calculate the digestion rate of the fluorogenic substrate.10 \u00b5g of insulin were incubated with 2\u2013500 ng hIDE or 8\u2013800 ng Iph1 in 50 mM Tris, 150 mM NaCl pH 7.4 for 3 h 20 min or 16 h in 300 \u00b5l final volume. Reactions were stopped by the addition of 30 \u00b5l 10% formic acid. Analysis of insulin digestions was performed by reversed phase HPLC on a \u03bcRPC C2/C18 ST 4.6/100 column . Digestion products were eluted using a 20\u201350% acetonitrile gradient, while monitoring the absorbance at 215 nm. For analysis by SDS-PAGE on 15% Tris-Tricine gels, 185 ng insulin were digested for 16 h with Iph1 WT, hIDE or Iph1 E71D . Bands were visualized using SYPRO Orange Protein Gel Stain .2O and spotted onto the indicated medium. TU, dithiotreitol  and rapamycin  treatments were performed on mid-log cultures grown in YNB. For each time point or drug concentration two to three dilutions were plated on YNB in triplicates and plates incubated at the appropriated temperature for 3\u20135 days. Colonies formed were counted and percent of survival calculated against time 0. All experiments shown were performed at least three times. TU was suspended in dimethyl sulfoxide (DMSO) at 10 mg/mL and used at 10 \u00b5g/mL otherwise stated. RA was suspended in DMSO at 0.5 mg/mL, and used at the final concentration of 300 ng/mL. DTT was suspended in H2O at 1 M and used at the final concentration of 50 mM.In drop tests, cells from exponentially growing cultures in either EMM or YNB were treated or not with tunicamycin  for 45 min, serially diluted in HProtein extracts were prepared as described in Cells were treated with 10 \u00b5g/ml of TU for 45 min, collected, suspended in fresh medium without TU and incubated at 30\u00b0C with agitation. At 0, 4 and 6 hours from release, cells were processed as described in S. pombe encodes five putative metallopeptidases belonging to the M16 peptidase family that are potential orthologues of budding yeast Ste23p. Among these, the SPACUNK4.12c ORF on chromosome 1 encodes a protein sharing the highest degree of identity (37%) with hIDE .In order to compare the protease activity of Iph1 with hIDE we expressed recombinant wt Iph1 and a mutant Iph1 protein with a substitution of residue Glu71 in the catalytic site by Asp, both tagged by six C-terminal His residues, using the baculovirus system. The equivalent mutation in hIDE (E111D) decreases enzyme activity to<1% without affecting substrate binding Next, we sought to determine whether Iph1 is able to cleave insulin, the hallmark natural substrate of hIDE cleaved with high efficiency by it. We performed insulin digestions with various amounts of enzyme and analyzed the results by column and gel chromatography. After 16 h of digestion with either 500 ng hIDE or 800 ng Iph1, the insulin peak was no longer detectable in reversed phase chromatography and various new product peaks appeared on the chromatogram . Analysiiph1 ORF was replaced by the selectable marker KanR. Cells disrupted for iph1 (iph1-d) did not show any obvious phenotype and, in contrast to budding yeast ste23 null mutant, cells were not sterile.To study Iph1 function we constructed a haploid strain where the iph1-d) to wild-type (wt) cells. TU is an ER stressor that blocks protein N-glycosylation in the ER, leading to accumulation of misfolded proteins.IDE is highly expressed in murine pancreatic beta cells (our unpublished observation), in which the UPR is constitutively activated to handle glucose-triggered bursts of insulin synthesis that challenge the ER protein folding capacity iph1-d cells were more resistant to induced ER stress than wt  was constructed by replacing in the haploid strain the +iph1 sequence with iph1-E71D. As shown in iph1-d cells is due to lack of the protein but not to lack of its protease activity. Thus, Iph1 has a function in the ER stress response that is unrelated to substrate degradation.Given that proteins belonging to the insulinase family have protease activity, we asked if increased survival to ER stress of tor1 (tor1-d) were slightly more sensitive than wt cells to TU in all experiments, however differences in survival between wt and tor1-d were at the limit of statistical significance (p\u200a=\u200a0.05) , indicating that the observed phenotype might be specific to ER stress.Fission yeast Tor1 kinase is required for the response to a wide range of stresses including heat stress and DNA damaging conditions \u200a=\u200a0.05) . In contng agent . The hig-d cells , indicat-d cells . Thus, rwt and tor1-d cells by iph1 deletion might depend on the Tor2 kinase that participates in the formation of the RA sensitive TORC1 complex as shown in wt cells and of tor1-d cells .Next to activating the UPR, ER stress is an inducer of autophagy that plays a cytoprotective role by preventing accumulation of misfolded proteins in apoptosis proficient cells We have shown that hIDE shares the protease independent function of Iph1 at least in yeast cells. Our results suggest that knock-out of mammalian IDE might also lead to ER stress resistance at least under certain metabolic conditions. This might be particularly relevant for pancreatic beta cells, in which excessive ER stress is linked to functional failure and diabetes development. The known capacity of IDE to degrade insulin is commonly viewed as mechanistic underpinning of its genetic link to diabetes. Our results suggest considering an additional or alternative possibility, namely that an implication of IDE in the response to proteotoxic stress might affect the survival of pancreatic beta cells in this pathology."}
+{"text": "This paper aims to present a new genetic approach that uses rank distance for solving two known NP-hard problems, and to compare rank distance with other distance measures for strings. The two NP-hard problems we are trying to solve are closest string and closest substring. For each problem we build a genetic algorithm and we describe the genetic operations involved. Both genetic algorithms use a fitness function based on rank distance. We compare our algorithms with other genetic algorithms that use different distance measures, such as Hamming distance or Levenshtein distance, on real DNA sequences. Our experiments show that the genetic algorithms based on rank distance have the best results. In many important problems in computational biology a common task is to compare a new DNA sequence with sequences that are already well studied and annotated. Sequences that are similar would probably have the same function, or, if two sequences from different organisms are similar, there may be a common ancestor sequence In computational biology the problem that deals with this task is known as the closest string problem (CSP): given a set The standard method used in computational biology for sequence comparison is by sequence alignment. Sequence alignment is the procedure of comparing two sequences  or more sequences  by searching for a series of individual characters or characters patterns that are in the same order in the sequences. Algorithmically, the standard pairwise alignment method is based on dynamic programming; the method compares every pair of characters of the two sequences and generates an alignment and a score, which is dependent on the scoring scheme used, i.e. a scoring matrix for the different base-pair combinations, match and mismatch scores, or a scheme for insertion or deletion (gap) penalties.Although dynamic programming for sequence alignment is mathematically optimal, it is far too slow for comparing a large number of bases, and too slow to be performed in a reasonable time.Also, since some of the search solutions are inaccurate from a biological point of view, alternative approaches periodically are explored in computational biology. This important problem, known also as DNA sequence comparison, is ranked in the top of two lists with major open problems in bioinformatics The standard distances with respect to the alignment principle are edit (Levenshtein) distance rank distance (RD)To measure the similarity between strings Dinu proposes a new distance measure, termed To measure the distance between two strings with RD we scan (from left to right) both strings and for each letter from the first string we count the number of elements between its position in the first string and the position of its first occurrence in the second string. Finally, we sum up all these scores and obtain the rank distance. In other words, the rank distance measures the \u201cgap\u201d between the positions of a letter in the two given strings, and then sums up these values. Intuitively, the rank distance gives us the total non-alignment score between two sequences.ad hoc extension to arbitrary strings, without affecting the low computational complexity. In contrast, the extensions of Hamming distance are mathematically optimal but computationally too heavy, and lead to the edit-distance, which is the base of the standard alignment principle. Thus, the rank distance sides with Hamming distance rather than Levenshtein distance as far as computational complexity is concerned: a significant indicator is the fact that in the Hamming and rank distance case the median string problem is tractable Clearly, the rank distance gives a score zero only to letters which are in the same position in both strings, as Hamming distance does . On the other hand, an important aspect is the reduced sensitivity of the rank distance with respect to deletions and insertions. Reduced sensitivity is of paramount importance, since it allows the RD is easy to implement, does not use the standard alignment principle, and has an extremely good computational behavior. Another advantage of RD is that it imposes minimal hardware demands: it runs in optimal conditions on modest computers, reducing the costs and increasing the number of possible users. For example, the time needed to compare a DNA string of Traditionally, the Closest String Problem (CSP) is related to Hamming distance and it tries to find a minimal integer In When CSP emerged in bioinformatics, the problem was investigated from many points of view. These investigations implied the use of different distances. The most intensive studied approach was the one based on edit distance. In In many practical situations the alphabet is of fixed constant size . For some applications, one needs to encode the DNA or protein sequences on a binary alphabet that expresses only a binary property of the molecule, e.g. hydrophoby  via rank distance are NP-hard. In this paper we use an approach based on genetic algorithms to propose an approximation of CSP and CSSP via rank distance.In In this section we introduce notation and mathematical preliminaries. We first introduce the rank distance and then we define closest string and closest substring problems.A ranking is an ordered list and is the result of applying an ordering criterion to a set of objects. Formally,Definition 1. Let A ranking defines a partial function on full rankings, while the others are partial rankings. We define the order of an object The rankings that contain all the objects of an universe Definition 2. Given two partial rankings In Definition 3. If The rank distance is naturally extended to strings. The following observation is immediate: if a string does not contain identical symbols, it can be transformed directly into a ranking (the rank of each symbol is its position in the string). Conversely, each ranking can be viewed as a string, over an alphabet equal to the universe of the objects in the ranking. The next definition formalizes the transformation of strings that have identical symbols into rankings.Definition 4. Let Example 1. If Observe that given Definition 5. Given Example 2. Consider the following two strings The computation of the RD between two rankings can be done in linear time in the cardinality of the universe. Our universe has precisely Let Problem 1 (Closest string via rank distance). Let The CSSP is a generalization of CSP where the objective is to find a string similar to substrings of the input.Problem 2 (Closest substring via rank distance). Let Homo sapiens, V00662), common chimpanzee , gorilla , donkey , rat , mouse , fat dormouse , and cow . Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria. The DNA sequence of mtDNA has been determined from a large number of organisms and individuals, and the comparison of those DNA sequences represents a mainstay of phylogenetics, in that it allows biologists to elucidate the evolutionary relationships among species. In mammals, each double-stranded circular mtDNA molecule consists of We test the genetic algorithm using mitochondrial DNA sequences extracted from several mammals available in the EMBL database: human  we design two similar experiments. We have another artificial experiment for CSSP, and another experiment for CSP with great interest for biologist.For the first experiment we use the human, chimpanzee and donkey genomes. We want to find the closest string (or substring) of nucleotides between the human and chimpanzee DNAs on one hand, and between the human and donkey DNAs on the other hand. The goal of this experiment is to compare the distances obtained for the two strings (or substrings). Note that the donkey belongs to the Perissodactylae branch, while the human and the chimpanzee belong to the Primates branch. Since the human and the chimpanzee are both primates, the human-chimpanzee distance should be smaller than the human-donkey distance. In other words, we expect the biological classification of mammals to be reflected in the DNA.For the second experiment we use the rat, house mouse, fat dormouse and cow genomes. As in the former case, we want to find the closest string (or substring) of nucleotides between the rat and house mouse DNAs, between the rat and fat dormouse DNAs, and between the rat and cow DNAs. The goal of this experiment is to compare the distances obtained for the three strings (or substrings). Note that the cow belongs to the Cetartiodactylae branch, while the rat, the house mouse, and the fat dormouse belong to the Rodentia branch. We expect the rat-house mouse distance and the rat-fat dormouse distance to be smaller than the rat-cow distance. We have chosen this experiment because in We also use an artificial test case for the closest substring problem to point out our optimization of the genetic algorithm presented in Note that another study that shows experiments using Hamming distance for CSP and CSSP is Each of our experiments are performed using three different metrics: rank distance, Hamming distance and Levenshtein distance. We want to compare the results for each distance measure. We show graphs of the best candidate evolution for each metric used.After we determine the metric that has the best results, we will perform another experiment (using only this metric) with great interest for biologists. At present, no definitive agreement on either the correct branching order or differential rates of evolution among the higher primates exists, despite the research in this area. Joining human with chimpanzee and the gorilla with the orangutan is currently favoured, but the alternatives that group humans with either gorillas or the orangutan rather than with chimpanzees also have support With two experiments and three distance measures for the closest string problem, we have six test cases with associated graphs. For the closest substring there is an extra artificial experiment, generating nine test cases and six graphs associated to the real DNA experiments. In our latest experiment we use the distance measure that has the best performance on the former test cases. We investigate only the closest strings for DNA sequences of variable lengths and we present three more graphs.For each experiment we give the input strings, then we present the results obtained by using rank distance, Hamming distance and Levenshtein distance, respectively. An input string is a DNA sequence. The algorithm designed for CSRD needs at least two DNAs (of same length) to produce an output DNA sequence. The output DNA is the closest string to the input strings computed with rank distance. Using Hamming or Levenshtein distance in the selection process of the genetic algorithm is analogous. The algorithm designed for CSSRD need two DNAs (not necessary of same length) to produce the output DNA that represents the closest substring.Let us describe the genetic algorithm parameters and the format of the input and output data. The population size represents the number of chromosomes in a single generation. The crossover probability represents the percent of chromosomes (from a single generation) that get involved in the crossover operation. The mutation probability is similar to the crossover probability only that the chromosomes are mutated. The number of strings (DNA sequences) gives the number of input strings. The size of each DNA sequence is the number of nucleotides in every DNA sequence. We use different input parameters for each problem that we are trying to solve. The input parameters used for one experiment are the same for every investigated metric. We want to compare only the metrics used, without changing the genetic algorithm parameters or the genetic operations involved.The average time represents the mean time for There are two different settings for this experiment corresponding to CSP and CSSP, respectively. We present the test cases and results separately for each setting.In this setting we use the first 1. RANK DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 22 seconds; Distance achieved: 3698; Closest string: G T A C T A C G C G T T T A C T C T A C C A A A C G C A T A C T G A C A A A T G T C T G T T A G A T G G A T C C A T C T C C G C G T G T A C T G T C T A A A A G C G T A G C G T C A C G T A C G T C A A G C A G T G T T T C A G T C C C A C A A T C C A T T G C A C A T T A C T G A G C T C T C C A T T C G T C T C A C T C T T T T T A C G A A C A A T A T T A T C A A T G C A A A C G T G G G C C C T C T T C.HUMAN-DONKEY RESULT: Average time: 22 seconds; Distance achieved: 5001; Closest string: T G A A G A G C A T T C C A T A T C T A A C T C C T G A A G T A C A C G A A C G G A T A T G C A C T T T G C T T C G T T A C A C T A G C G T G G A C G T A C A T T C T C G G C T G A C C T T G G G C A T A T A A T A T T A A A G T A A C G G A G T C T A C A T C T A A T A T C A T C G T A A C C C A T A G A A T G T T A T A C C C T C A T C G T C C T T C G C C C A A G T G C C C T G C T T A A C T T C T C A T.2. HAMMING DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 1 min 14 seconds; Distance achieved: 73; Closest string: G A T C A T G T G G C T A T C A C C C T C A A A G C C A C T C A C G G G A A C T G T T C A G A C A T T T T T A C A T T A C C C C A T G A A G A T A T G C G C G T G G T A C T A T T C T G T C A A G C A G C A G T C A G A A A A C T C A C T C T T G C A A T A A C T G T C T T T G C T T G C T T C A T C A T C T T A A T C T C T A T C A T C A C T A G G T A C A A T A A T A C A G G C C G A C G C A C T G C A G C.HUMAN-DONKEY RESULT: Average time: 1 min 13 seconds; Distance achieved: 77; Closest string: G A T C A C A G A G C T A A A A G A C A A C A A A C C A C G C A C C T G A A A A T G C C A A G A T T T T G G T T C C T A C G C C T T G G G C A T A T A C A C T C G A T C C C G T T C T G A T A C T C T G T A A C C G G T G C A A C C A C T C A T G C A A G A T T C G T C A T T C C T G C C T G A A T G A T C T C T T T A T T G A A C T C T C C G A T C T T A A G G A G C A G G T G C G A A C A A A A T T A C T A.3. LEVENSHTEIN DISTANCE TEST CASE 1: Population size: 2500; number of generations: 300; crossover probability: 0.36; mutation probability: 0.002; size of each DNA sequence: 200.HUMAN-CHIMPANZEE RESULT: Average time: 24 min 12 seconds; Distance achieved: 63; Closest string: G T A T A C A C A G C T C T A C C C C C T A A A G C A A T A C C A C G G A A G A T C T T C C A T G G A T T T A T A T C A T C C T C T A A G C A A C A T G C A T G G T A G C C T T G C G A T T C G A T T G A G C T C G T G A G A C C C T A T A T C G C A T A C T G A T C C C C G A T C C T G G T C A T C C T A T T A A T C A T C C A T G T A A A G T T A C A A G T A T T A C A G C G C G C A G C A A T T A C A A C.HUMAN-DONKEY RESULT: Average time: 24 min 11 seconds; Distance achieved: 59; Closest string: G T T C A A T G T A C T A T C A C G A T A T A A A T C A A G G A G C T G T C A A T G C A C T T G G T A G T T T C C T C T G C G C T A T G C A C A C A T A G G G C A T T G C G A C C T G G A G C C T T A T T A T T A C T A T G A A G C A G A T T A A C A T G C A T T G A T T C C T G C C T C C C C A T A T A A T C C T C T A A A T C G C A C T C T A G A T C A A A T T A C A G G C G A A C A A G A C T C T A C T A.In this setting we use the first 300 nucleotides extracted from the human, chimpanzee and donkey genomes. We want to determine the human-chimpanzee and human-donkey closest substrings of 24 nucleotides.Here the substring size input parameter represents the desired length of the best substring .1. RANK DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 39 seconds; Distance achieved: 26; Closest substring: C T T A G T A A C T A T A T C G A G A C A A G C.HUMAN-DONKEY RESULT: Average time: 40 seconds; Distance achieved: 34; Closest substring: A C A T G C C T A T C T A C C C G T A A T A C C.2. HAMMING DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 1 min 36 seconds; Distance achieved: 7; Closest substring: C T A C A C A C G C A A G C C T T C C C T G C A.HUMAN-DONKEY RESULT: Average time: 1 min 38 second; Distance achieved: 7; Closest substring: A C G T A C G A A C C A T A C T A C A A G C T A.3. LEVENSHTEIN DISTANCE TEST CASE 2: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of each DNA sequence: 300; substring size: 24.HUMAN-CHIMPANZEE RESULT: Average time: 7 min 4 seconds; Distance achieved: 4; Closest substring: T T G A T T C C T G C C T A T C T A T T A G C T.HUMAN-DONKEY RESULT: Average time: 7 min 3 seconds; Distance achieved: 4; Closest substring: A T G C T A C T C T T A A T C G C A C C T A C G.First, we must point out that rank distance, Hamming distance and Levenshtein distance use different scales, i.e. a rank distance of 100 is not equivalent to a Hamming distance of 100, nor a Hamming distance of 100 to a Levenshtein distance of 100. We would also like to point out that rank distance has a finer scale, possibly being able to detect subtle differences between DNA strings.As one might expect, the results indicate that the human genome is closer to the chimpanzee genome, than it is to the donkey genome.In the CSP setting, rank distance shows a great difference between the human-chimpanzee closest string and the human-donkey closest string. Levenshtein distance indicates that humans are closer related to donkeys than to chimpanzees, while Hamming gives the expected result as rank distance does. Both Hamming and Levenshtein distances show small differences between the two analysed strings. The evolution of the best closest string candidate for each distance measure is given in In the CSSP setting, RD is the only distance that can catch the subtle difference between the human-chimpanzee closest substring and the human-donkey closest substring, even if we use only In both CSP and CSSP settings, rank distance clearly outperforms Hamming and Levenshtein distances see  and 2.As for the Human-Chimpanzee-Donkey experiment, there are two different settings corresponding to CSP and CSSP. We present the test cases and results separately for each setting.In this setting we use the first 150 nucleotides extracted from each of the rat, house mouse, fat dormouse and cow DNA sequences. We want to determine the rat-house mouse, rat-fat dormouse and rat-cow closest strings which also have 150 nucleotides.1. RANK DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 12 seconds; Distance achieved: 454; Closest string: G T T G A A T C G T T A A T A T A C A A A G C A A G T A C A T G A A T C A G A A G T G A T A T T C T A A A A G C T T A G C A A C C A T C A A A T A T G T G G C C G T G T T C T A C A T T T A A G T G A A G A T G T A A A T C A A A C C T A A G C A T C A T G A C A T G C G A A T C A A G C A T A C C T A T T.RAT-FAT DORMOUSE RESULT: Average time: 12 seconds; Distance achieved: 1209; Closest string: G T A T A C T G T A G T A T A A A A A A T C T G A G A C C A T G A T A A T G T A C A G T A G G A T A C A T A C C T A A C C G C A A C A A T T G A T G C C G T G T A C G C T T A A T T T C A A T G T C T C T A G C A G G A A G A A A A T T T G C A A A C T T C C A A C G A A A G T C G C T A A A T G T C C A T.RAT-COW RESULT: Average time: 12 seconds; Distance achieved: 3321; Closest string: G T A T A A C A T G T C A C T G A A C C G A A T A C T A G T A A T G A A A A T T C G G C T C T T A T G C A A G A C T T A T A C T T T C A G G A G G A T C G A T T T T A G A A C A T G A A A A T G C T A G G C T G T A G T G G C G T A G A T C A C T A G G C A G C T G C T T G T T C T T T T G T C A A C T G G.2. HAMMING DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 42 seconds; Distance achieved: 45; Closest string: G T T A A T G T A G C T T A T T A A C A A G G A A A G G A A T T G A A A A T G T T T A G T G G G T T C A A T A T T C C C A A T A A C C C A A A G G G T T G G T C C C G G G C C T G T A A A T A A A T T A A G G G T A G A A T A A A C A T T C A A A A C C C C C A A A A A C C G G G T T A A A A C C C T T T A.RAT-FAT DORMOUSE RESULT: Average time: 41 seconds; Distance achieved: 43; Closest string: G T T A A T G T A G C T T A T A A T A A G C A A A A C C A T T A A A A A G C T T T G G A T G G A A T C T A A A A C C C C T A A A A C A A A A A G T T T G G G C C C A G G C T T T T T A A T T G T T T G T A G G A A A A A T A A A C A T T G C A A C A A T C A C G A C A C C G G T A T A A A A C C C T T T A C.RAT-COW RESULT: Average time: 41 seconds; Distance achieved: 56; Closest string: A T T A A T G G A T A A T C T G C T A A T G C A A A G A C A T G A C A A T G C T G T G A T A G A T T T A G A A A T T C T A T A A T C A G G A A G G T T T T G G C A T T C A G C T A T G G T T G A C T G A G G G T A T G A T T C G A C A C A T A A A C T T C A A T A G G C C T T A G C A G A A T C T T T A G A.3. LEVENSHTEIN DISTANCE TEST CASE 3: Population size: 1800; number of generations: 300; crossover probability: 0.36; mutation probability: 0.005; size of each DNA sequence: 150.RAT-HOUSE MOUSE RESULT: Average time: 9 min 28 seconds; Distance achieved: 14; Closest string: G T T A A T G T A G C T T A T A A T A A A G C A A A G C A C T G A A A A G C T T A G A T G G A T C A A A T G A T C C C A T A A A C A C A A A G G T T T G G T C C T G G C C T A A A T A A T T A G A G G T A A A G A T C T A C A C A T G C A A A C C T C C A T A G A C C G G T G T A A A C A T C C C G T T A A.RAT-FAT DORMOUSE RESULT: Average time: 9 min 29 seconds; Distance achieved: 28; Closest string: T T A A T G A G C T T A A A A G C A A A G C A A C T G A A A T G C T T A G A T G G T A G C A A A T A T C C C A T A A A C A C A A A G G T T C T G G T C C C A G C C T T C T A T T A A T T A G A T T G T A T A G C A A G A T T A C A C A T G C A A C A T C A T G A A C C T G G T G T A A G A A T C C C T T A A.RAT-COW RESULT: Average time: 9 min 29 seconds; Distance achieved: 46; Closest string: G A C T A A T G G C T A T C A G A A T G C A A A G C A C A T G A A C A T G C T G C T G A G A T A G A T T T G A A A A T C T T T A A T A C T G G A A G G G T T G C T C C T G G A C T C A T A G C T A T G G A C G T A A G G C T T G A C A C A G C A T A C A T T G T A C C G G A G T A A A A T G C A C T T A A G.In this setting we use the first 300 nucleotides extracted from the rat, house mouse, fat dormouse and cow genomes. We want to determine the rat-house mouse, rat-fat dormouse and rat-cow closest substrings of 24 nucleotides.The substring size parameter is the desired length of the best substring.1. RANK DISTANCE TEST CASE 4: Population size: 700; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 1 min 25 seconds; Distance achieved: 0; Closest substring: A A A G C A A A G C A C T G A A A A T G C T T A.RAT-FAT DORMOUSE RESULT: Average time: 1 min 24 seconds; Distance achieved: 4; Closest substring: A T A A G A C A A G C A C T G A A A A T G C T T.RAT-COW RESULT: Average time: 1 min 25 seconds; Distance achieved: 22; Closest substring: A G A T A C G T T C A G T A C A T G A G T A C C.2. HAMMING DISTANCE TEST CASE 4: Population size: 600; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 2 min 5 seconds; Distance achieved: 0; Closest substring: T C A G C A G T G A T A A A T A T T A A G C A A.RAT-FAT DORMOUSE RESULT: Average time: 2 min 4 seconds; Distance achieved: 1; Closest substring: C C C C A T A A A C A C A A A G G T T T G G T C.RAT-COW RESULT: Average time: 2 min 4 seconds; Distance achieved: 7; Closest substring: G T A A T T G G A C A T A A A T T T T C A C A T.3. LEVENSHTEIN DISTANCE TEST CASE 4: Population size: 700; number of generations: 110; crossover probability: 0.36; mutation probability: 0.03; size of each DNA sequence: 300; substring size: 24.RAT-HOUSE MOUSE RESULT: Average time: 13 min 18 seconds; Distance achieved: 1; Closest substring: T A A A A A A G C A A A G C A C T G A A A A T G.RAT-FAT DORMOUSE RESULT: Average time: 13 min 19 seconds; Distance achieved: 1; Closest substring: T A A A C G A A A G T T T G A C T A A G C T A G.RAT-COW RESULT: Average time: 13 min 19 seconds; Distance achieved: 6; Closest substring: C A A A C A T C T A C C A C C C G G T T A A A A.The expected result for this experiment should indicate that the rat is closer to the house mouse and fat dormouse, than the cow. We would also like to catch even a finer difference between the rat-house mouse distance and the rat-fat dormouse distance.In the CSP setting, rank distance shows again a great difference between the rat-house mouse closest string, the rat-fat dormouse and the rat-cow closest string. Hamming is able to distinguish the rat from the cow genome, but it doesn\u2019t catch the difference between the rat-house mouse closest string and the rat-fat dormouse closest string. The rat-fat dormouse Hamming distance appears to be smaller than the rat-house mouse Hamming distance, which is wrong. Levenshtein distance works as good as rank distance in this case, giving the expected result. Our observations are supported by the graphs shown in In the CSSP setting, all distances perform very good and are able to put the rat genome near the house mouse and fat dormouse genomes rather than the cow genome. However, the rat-house mouse Hamming distance is very close to the rat-fat dormouse Hamming distance (the closest substrings differ only by one letter). The Levenshtein distance is the same for rat-house mouse and rat-fat dormouse closest substrings. The associated graphs are given in In both CSP and CSSP settings, all distances are able to put the rat near the house mouse and fat dormouse rather than the cow, which is the expected result see  and 4. OFor this experiment we use only the CSSP setting. The goal of this experiment is to show the time improvement obtained by optimizing the genetic algorithm introduced in 1. TEST CASE 5: Population size: 500; number of generations: 100; crossover probability: 0.36; mutation probability: 0.02; size of DNA sequence 1\u223690; size of DNA sequence 2\u223690; substring size: 30.DNA Sequence 1: A A A A A A A A A A A A T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G A A A A A A A A A A A A A A A A A A A A A A A A A A A G G G G G G G G G G T T T T T A A A A A A A A A A A A A A A A.DNA Sequence 2: C C C C C C C C C C G G G G G G G G G G T T T T T C C C C C C C C C C C C C C C C C C T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G C C C C C C C C C C C C C C C C C.RANK DISTANCE RESULT: Average time: 10 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.HAMMING DISTANCE RESULT: Average time: 35 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.LEVENSHTEIN DISTANCE RESULT: Average time: 3 min 22 seconds; Distance achieved: 0; Closest substring: T T T T T T T T T T T T T T T T T T T T T T T T T G G G G G.Using an algorithm to compute rank distance in linear time and a hash table to store precomputed distances between DNA sequences, we are able to report a great improvement in terms of speed. The algorithm that computes rank distance in linear time was introduced in At the selection step, the genetic algorithm needs to sort the chromosomes in each generation by distance. In order to sort the chromosomes we must compare distances that are computed (or recomputed) between chromosomes and input sequences. Instead of computing the distances each time, we store the precomputed distances in a hash table. It is much faster to access a distance value stored in a hash table instead of computing it in linear time. Note that we also used the hash table optimization for Hamming and Levenshtein distances. This optimization helps us reduce the number of distances to be computed from For this test case, in We obtained the same closest substring for each of the three metrics. This result shows that if an exact common substrings exists, the genetic algorithm can find it disregading the metric used. This shows that the genetic algorithm is robust and it can find the optimal solution if the input parameters are properly set.We designed simple and clear experiments that can show the differences of the compared distances. In order to keep things simple, we used the genetic algorithms to determine the closest string or substring for only two DNA sequences. Of course, the algorithms work as well with multiple sequences at once, since the CSP and CSSP problems are generally defined for sets of strings.We mention that the results obtained are not influenced by the fact that the DNA strings are part of coding or non-coding sequences or within genes or part of intergenic regions. The DNA strings used in our experiments were selected without taking into consideration these aspects so the strings may be part of any kind of region. However, it is important for DNA strings used in the same experiment to be extracted from the same position because the alignment matters. In other words, it doesn\u2019t have sense to compare DNA from different regions that have different significance.All our experiments show that RD can be computed 2 times faster than Hamming distance and 10 to 15 times faster than Levenshtein distance. As the closest string (or substring) size increases the Levenshtein distance takes more time to compute when compared to rank distance and Hamming distance.Although the Hamming distance computes almost as fast as rank distance, the downside is that is gives inaccurate results compared to RD. The Levenshtein distance can easily be dismissed because is takes longer to compute and it is also unable to detect the subtle differences that rank distance detects by having a finer scale.Neither Hamming distance or Levenshtein distance were able to give the right answer in all our experiments (Levenshtein distance is wrong in TEST CASE 1 and Hamming distance is wrong in TEST CASE 3). Only rank distance has the expected outcome in all the experiments. Overall, we believe that rank distance is best suited for finding closest strings or substrings on DNA sequences. Due to this observation we conducted the following experiment using only RD.The goal of this experiment is to see if the DNA information can lead to one of the three distinct unrooted phylogenetic trees of higher primates. For this experiment we use only the CSP setting: we want to find the human-chimp closest string and the human-gorilla closest string and compare the associated rank distances. We perform four tests using DNA sequences of variable length and different input parameters for the genetic algorithm. We show graphs for the last three test cases which are more relevant.In the first test case (TEST CASE 6) we use the first The difference between the human, chimpanzee and gorilla mtDNA is very small and We present only the distance achieved for each closest string, because the strings are too long to be presented here.1. RANK DISTANCE TEST CASE 6: Population size: 7000; number of generations: 500; crossover probability: 0.36; mutation probability: 0.001; size of each DNA sequence: 800.HUMAN-CHIMPANZEE RESULT: Average time: 7 min 3 seconds; Distance achieved: 43207.HUMAN-GORILLA RESULT: Average time: 7 min 6 seconds; Distance achieved: 45544.2. RANK DISTANCE TEST CASE 7: Population size: 33000; number of generations: 2000; crossover probability: 0.36; mutation probability: 0.0002; size of each DNA sequence: 5000.HUMAN-CHIMPANZEE RESULT: Average time: 13\u201314 hours; Distance achieved: 426232;HUMAN-GORILLA RESULT: Average time: 13\u201314 hours; Distance achieved: 358525.3. RANK DISTANCE TEST CASE 8: Population size: 40000; number of generations: 2400; crossover probability: 0.36; mutation probability: 0.0001; size of each DNA sequence: 7000.HUMAN-CHIMPANZEE RESULT: Average time: 27\u201328 hours; Distance achieved: 682664.HUMAN-GORILLA RESULT: Average time: 27\u201328 hours; Distance achieved: 656806;4. RANK DISTANCE TEST CASE 9: Population size: 55000; number of generations: 2800; crossover probability: 0.36; mutation probability: 0.00005; size of each DNA sequence: 16000.HUMAN-CHIMPANZEE RESULT: Average time: 5 days and 6\u20137 hours; Distance achieved: 2412780.HUMAN-GORILLA RESULT: Average time: 5 days and 6\u20137 hours; Distance achieved: 2089976.We adjusted the genetic algorithm parameters to obtain the best results disregarding the higher computational time needed to get these results for the first three test cases. The graphs show that the size of the population used in the genetic algorithm is much higher than necessary because the best chromosome evolves very fast during the first In TEST CASE 9 the parameters are rather ajusted for speed than accuracy. We can obtain better approximations of the closest strings by using a population larger than The results for TEST CASE 6 shows that according to rank distance the human is near the chimpanzee rather than the gorilla. The graphs corresponding to TEST CASE 7, 8 and 9 from It seems that Note that in our last test case we used almost all of the entire mtDNA which is approximately Overall, the DNA information that RD was able to extract during this experiment seems to support the theory favours the phylogenetic tree that joins the human with the gorilla In this paper we presented two genetic algorithms designed for solving the closest string problem and closest substring problem, respectively. The genetic operations for the closest string problem have a strong mathematical background and are only inspired from nature. The genetic algorithm designed for the closest substring problem uses standard genetic operations.We tested these two algorithms using several experiments that involve DNA sequences extracted from mammals genomes. Each of these experiments were performed using three different metrics: rank distance, Hamming distance and Levenshtein distance. In all our experiments rank distance clearly outperforms Hamming and Levenshtein distances. On top of this, rank distance is the only distance able to catch subtle differences between DNA strings.By comparing the results for each distance measure, we are able to conclude that RD is best suited for finding closest strings or substrings on DNA sequences.We used our genetic algorithm with rank distance to bring some light in a case disputed by biology scientists: which is the closest human relative, the chimpanzee or the gorilla? The DNA information extracted by rank distance supports the theory that says the human closest relative is the gorilla. We also showed the importance of using DNA sequences that are long enough to obtain conclusive results. Too short DNA sequences can lead to confusing results.In the near future we would like to compare our genetic algorithms based on RD with other approaches, such as dynamic programming techniques. We strongly believe that our approach is comparable, in terms of precision and speed, with other approaches.We also want to investigate a possible approach to obtain better results. This approach combines the results coming from several parallel executions of the genetic algorithm. The best candidates from these parallel executions may be taken to form the first generation of another genetic algorithm. The best candidates will evolve together until the final result is achieved. The final result is expected to be an optimal solution. This approach could work very good with very high-dimensional input data.Genetic algorithms are adaptive searching techniques based on the principles of genetics see . The firWe used the classic general form of the genetic algorithm. For each problem, we used a different set of operations. The set of operations used for the closest substring problem are classical. The crossover and the mutation operations are the same operations found in nature. For the closest string problem the operations are only inspired from biology, but they rely on a mathematical background. We will later describe the structure of the chromosomes and the operations applied on each generation.Algorithm 1General Form.Initialization: Generate a random population that represents the first generation.1: Loop: For a number of generations apply the next operations:2: 2.a 1em Apply the crossover according to the probability of having a crossover.2.b 1em Apply mutations according to the probability of having a mutation.2.c 1em Select the best candidates for the next generation using a density of probability.Termination: Choose the best individual from the last generation to be the optimal ranking.3: An individual chromosome is a permutation of ranks. Each chromosome is a possible candidate for the optimal ranking. We need to convert each input DNA to a permutation. Note that any string can be converted to a permutation. Each letter of the string can be annotated with an index that starts at There are three forms of crossover that are used by the algorithm. Each time the crossover must occur we apply all three forms of crossover.The first crossover operation keeps the first part (prefix) of the individuals and completes the rest of the permutation according to the order given by the complementary chromosome. The second operation uses the same principle, but applies it at the other end of the chromosomes. This crossover operation keeps the last part (suffix) of the individuals and completes the rest of the permutation according to the order given by the complementary chromosome.The third crossover is a natural combination of the previous two. This crossover keeps both the prefix and the suffix of the chromosomes but completes the middle part according to the order found in the complementary chromosome.In order to successfully apply the crossover operations a certain cutting point should be randomly generated. There are six new individuals after the recombination because each crossover operation generates two new individuals. The best two individuals are chosen to replace the parent chromosomes. The optimality condition is used as a criterion to choose the best individuals.We have chosen this model (with 3 types of crossover) because the use of a single crossover usually destroys certain parts of the two individuals involved in the operation. For example, the crossover that keeps the prefixes will have to reorder the components of the suffix. If this single type of crossover is used, we would be unable to evolve the suffix part of the chromosome. This will generate populations with similar individuals that tend to have a bad pattern. In this pattern a good part and a bad part always appear. With our model we ensure that individuals do not follow this pattern and get close to the optimal ranking, but in different ways.The mutation operation may be applied to any chromosome. The mutation only needs one chromosome. To apply a mutation on an individual two positions are randomly chosen. The values at the two positions are swaped.To select the individuals for the new generation from the current generation we use a density of probability function. The new generation is involved in the next iteration of the algorithm. The first step is to sort the individuals on the maximal distances from the input rankings criterion in descending order. Then we generate indexes from the top to the bottom of the list of candidates. The indexes close to the top of the list are more probable. Note that one index can be generated several times; this is the case with the best candidates. There are also indexes that may never be generated; this is the case of the candidates close to the bottom of the list. The density probability function used to select the candidates for the next generation is the normal distribution of mean The graph of this function is represented in Note that in the implementation of the algorithm the fitness function was statistically approximated.The motivation for using this fitness function is based on test results. This fitness functions reduces the number of generations that are required to obtain a close-to-optimal solution. Helped by the crossover and mutation operations, the fitness function has a good generalization capacity: it doesn\u2019t favour certain chromosomes which could narrow the solution space and lead to local minima solutions.Each chromosome is a sequence of DNA of fixed length that represents a possible candidate for the closest substring. Note that a sequence of DNA is simply a strand of nucleotides  that appear randomly in a sequence.The crossover operation between two chromosomes for the closest substring problem is straightforward. First, we need to generate a random cutting point. The prefixes of the two chromosomes remain in place, while the suffixes of the two chromosomes interchange. This is the standard crossover operation inspired directly from nature.To apply a mutation to a certain chromosome, one position is randomly chosen. The nucleotide found at that position will be changed with a new one. Multiple mutations may appear at the same chromosome, although this is very unlikely. This is the classic mutation operation that can also be found in nature.The selection operation used here is similar to the selection used for closest string problem and is based on the normal distribution of mean"}
+{"text": "We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a \u201cproof-of-concept\u201d for custom-designing promoters that are suitable for biotechnological and medical applications.The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur  Artificially engineered promoter sequences have the potential for use in industrial and biotechnological applications, such as recombinant protein production of biopharmaceuticals. Some human proteins require mammalian cell lines for proper production, with e.g. the Chinese hamster ovary (CHO) cell line being a widely used system for EPO, Interferon-\u03b2, Factor VIII, IX, etc The promoter is the genomic region around the transcription start site (TSS) of a gene, and acts as an essential component in gene regulation and transcription, its role being to interface with transcription factors (TFs) through protein-DNA binding. The TFs anchor the pre-initiation complex (PIC), specifying the exact point of initiation, and recruit RNA polymerase (Pol) II to start transcription SSRCGCC\u2019 General transcription factors (GTFs), organized in complexes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and TFIIJ, form a special class of TFs, in that they are ubiquitously present and both necessary and sufficient to enable Pol II transcription at significant levels, making these proteins desirable candidates as drivers of expression of artificial promoters. Only TFIIB and TFIID have been shown to exhibit sequence preference: the TATA-Binding Protein  is most well characterized and binds to the TATA-Box, thereby establishing the TSS 25\u201330 base pairs downstream of its location. However, only about 10% of human promoters rely on a TATA-Box nucleotide composition, rather than sequence motifs, where we define the term \u201cnucleotide composition\u201d as the frequency patterns of mono-nucleotides, di-nucleotides, tri-nucleotides etc. This composition varies over the human genome on a large scale, recognizable as isochores in vitro in mammalian cells, as well as how expression levels depend on highly localized features in these sequences.Designing artificial sequences that attract TFIIB and TFIID requires determining the features that capture the interactions between these proteins and the DNA. Generally, a complicating factor is the lack of a one-to-one relationship between TFs and exact instances of binding motifs, so that neither motif consensus of short sequences nor Position Weight Matrices (PWM) are ideal representations of binding sites in vitro expression. Here, we first recapitulate how highly active promoters (mostly associated with both housekeeping and strong tissue-specific genes) differ from the genome-wide distribution in GC and CpG, as well as in other di-nucleotides. We then exploit these \u201cun-genomic\u201d features by devising a measure that tracks the genome-wide frequency of each short motif: the less common genome-wide, the more likely it is to reflect properties of a promoter. We subsequently overlap a set of uncommon motifs to build \u201cpromoter-like\u201d contiguous sequence, which allows for editing existing promoters, as well as to construct entirely artificial ones that work in a number of mammalian cells. We last show that TFIIB and TFIID bind to these promoters, which is reflected in their stability of expression level across multiple cell lines.Highly active promoters exhibit nucleotide patterns that are different from the majority of the genome Trinity . The resulting assembly consists of 38 Mb of sequence, residing in 27,000 disjoint transcripts. We eliminated non-full-length transcript assemblies of less prominently expressed genes by requiring sequences to contain open reading frames of 500 bp or more, aligned the remaining sequences to the human genome, and selected only transcripts with the 5\u2032 end falling within 50 bp of an annotated TSS To capture a set of highly active promoters, we sequenced the mRNA of the most highly expressed genes in human cerebellum tissue. We constructed two cDNA libraries, one normalized and one un-normalized library (both filtered for poly-A tails), and sequenced both libraries on one lane of Illumina each, yielding a total of 2 billion base pairs in 71 bp long reads. We then assembled the reads from both libraries into contiguous transcripts using the transcriptome assembly program While only 127 (7.3%) of the 1,746 promoter sequences contain one or more instances of a TATA-Box in the correct orientation, the sequences are clearly distinct from the genome-wide average by their high G/C content (66% vs. 40% genome-wide) as well as the average frequency of CpGs (9% vs. 1% genome-wide). \u03b1 score\u201d, see \u03b1 yields a potential spatial resolution of tens of nucleotides. annotated TSSWe defined a measure that tracks with G/C and CpG content see , incorpo\u03b1 score for each possible 12-mer, including those not present in the human genome. We selected sequences from two quintiles: (a) the top 5% represent the most \u201cun-genomic\u201d (or promoter-like) 12-mers; and (b) the percentile between 45\u201350%, which contains 12-mers with di-, tri-nucleotide etc. frequencies close to the genomic median, representing more \u201cnormal\u201d (or non-promoter-like) sequences. The 12-mers from each set were then independently assembled into \u201cconcatomers\u201d  and non-promoter-like (for down-regulation) artificial constructs. As test case, we chose the promoter upstream of the TSS of the X-linked gene cancer/testis antigen 1A (CTAG1A), which exhibited strong in-vitro activity in human cell line HEK293. The CTAG1A promoter region contains three distinct regions of elevated \u03b1 scores  with size-matched, but of higher \u03b1 score, snippets from the promoter-like concatomer.We tested whether we could modulate in-vitro promoter activity in HEK293 cells relative to the original sequence, as does replacement with \u201cnon-promoter\u201d sequences with \u03b1 scores of approximately half the original (\u201chCTAG1A-replace\u201d). Replacement with \u201cpromoter-like\u201d sequences with an \u03b1 score roughly twice that of the original (\u201chCTAG1A-UP\u201d) increases activity beyond that of the original sequence. This indicates that highly localized changes in sequence composition can drive up- and down-regulation of in-vitro gene expression.in-vitro expression, we pulled sequences from the promoter-like concatomer using different criteria and of different lengths  in four mammalian cell lines: CHO (hamster ovary); P19 (mouse embryo); Vero (monkey kidney); and HEK293 (human kidney). Promoter strength of most constructs was comparable to, or exceeded activity of the SV40 core promoter, which is a routinely used viral promoter for recombinant protein expression in mammalian cell lines (Spodoptera frugiperda) for any of the constructs, suggesting fundamental differences in promoter mechanisms between insects and mammals .To create entirely artificial promoter constructs for ll lines . NotablyAll constructs contain at least one instance of a TATA-Box. In addition to two TATA-Boxes, ArS232 contains one perfect, and 12 imperfect  instances of BRE, and one Inr. To examine to what extent the TATA-box is needed to drive expression, we constructed three variations : (i) remD values, and that these readings are thus somewhat more difficult to interpret than the TFIIB binding results.The general transcription factors TFIIB and TBP bind to the artificial promoter constructs: we monitored real-time binding of TFIIB to constructs ArS110, ArS300, ArS201 and ArS232 through measurement of quantitative protein kinetics . Figure in vitro transcription depending of the abundance of the binding proteins that drive the promoter.Artificially engineered promoter sequences have potential for use in industrial, biotechnological and medical applications involving recombinant protein production and gene therapy, since they can be designed to have different activities and be adapted to the specific requirements (strong or weak expression). The method proposed here yields constructs that appear less variable and species specifically regulated than the viral promoter SV40, a feature that would increase stability across cell types and conditions. By extension, it should be possible to build artificial test beds to determine the behavior of known binding sites, and subsequently design promoters that are targeted and regulated by specific transcription factors. Preliminary results already show promise that this is, in fact, the case, and future experiments will help expand our \u201cvocabulary\u201d of promoter elements, and to predict their effect on in vivo expression, in presence of additional factors, such as methylation, degradation by miRNAs etc. While it might not be possible to accurately predict the behavior of artificially designed promoters in living organisms in the immediate short term, modifying short sub-sequences to adjust relative expression levels should be. In addition, emerging fields such as Synthetic Biology We do not address here how these results translate to This work might also be relevant for the study of expression regulation on a more general level: our findings suggest that different sequences can respond to transcription factors in very similar ways, even though they share no nucleotide sequence similarity; this would provide an explanation as to why promoters are generally not conserved across species over their entire length, but exhibit a pattern of conservation peaks and troughs \u03b1 score measures the \u201cun-genomicness\u201d of short (12 nucleotides long) sequences, taking into account the genome-wide frequencies of di-nucleotides, tri-nucleotides etc. within the sequence. Let N denote the number of k-mers  in the genome, and ki\u03d5 the genome wide occurrence count of the k-mer starting at position i (using zero-based counting) in the 12 base pair (bp) long sequence, then the score k\u03b1 iskThe For existing promoter templates as well as the CTAG1A promoter, we computed the scores for all overlapping 12 bp long sequences within, and assigned the score to the base at position 6.\u03b1 score, and extended the boundaries of each until its composite \u03b1 score became negative, which yields a 2% false positive rate on the promoter template set  were designed as described in the Constructs ArS 50, 110, 201, 232 and 300  were selected from the 160,000 nucleotide (nt) long promoter-like concatomer. For identification and spacing of TATA Boxes, we used a promoter prediction tool trained on fruit fly E. coli and purification using NucleoSpin Extrakt II  all plasmids were sequenced to confirm the original sequence. In case of CHO dhfr-, HEK293 and P19, 4\u00d710\u22276 cells were transfected with 10 \u00b5g of the firefly luciferase plasmids and co-transfected with 1 \u00b5g of the Renilla luciferase reporter vector pRL-SV40  as an internal standard using Amaxa's Nucleofector Kit V  according to the manufacturer's instructions. 3\u00d710\u22275 VERO and MDCK cells were transfected with 1 \u00b5g DNA of the firefly luciferase constructs and co-transfected with 0,25 \u00b5g pRL-SV40 using Dreamfect Gold and CombiMag . 3\u00d710\u22276 Sf9 cells were transfected with 1 \u00b5g of the firefly luciferase constructs using Cellfectin II  according to the manufacturer's instructions. Instead of the SV40 promoter the baculovirus derived immediate early promoter OplE2, which is active in insect cells was used for the positive control. Luciferase expression was measured 48 h post transfection on a Synergy 2 microplate reader  with the Gen5 software using the Dual-Glo luciferase assay system . To normalize transfection efficiency, promoter activities are expressed as the ratio of firefly and Renilla luciferase activity. The pGL3-Promoter plasmid , containing the SV40 promoter served as positive control. The promoter activity of this viral promoter was set to 100%. All other measurements refer to this value within the same cell-line. The promoterless pGL3-Basic Vector  was used as a negative control.All inserts have been assembled either by oligo synthesis  followed by annealing and PCR or by gene synthesis  and cloning into the reporter vector pGL3 Basic  upstream of a firefly luciferase gene. After propagation in on and koff) and affinities (KD) were calculated using the Octet Data Analysis Software Version 6.3.For the identification of sequence-specific DNA binding of the transcription factors TFIIB and TBP the binding kinetics were measured by biolayer interferometry on an Octet QK instrument (ForteBio Inc.), which provides continuous real-time display of biomolecular interactions. Streptavidin biosensors were loaded with biotinylated DNA fragments (25 \u00b5g/ml) of the promoter constructs ArS110, ArS201, ArS232 and ArS300, or with the promoter constructs ArS232, ArS232 dT1, ArS232 dT2 and ArS232 dT12, generated by PCR amplification using 5\u2032 biotinylated primer (Sigma-Aldrich). Binding was conducted in 1\u00d7 Kinetics Buffer (ForteBio Inc.) with a protein concentration of 285 nM for TBP  and 270 nM for TFIIB . Kinetic parameters (k>ArS 50ACGCACGCGGTATAAACGCGCGACCTATTCGCGACCGTATAGCGACCGGA>ArS 110CTACGCCGCGTAAATATCGCGCGCTAACGGTGCGCGTTAAAACGCCGACGCGTCATAAAGCGCCGGCGTATAAGCGCGCCGTACGTCGTCGAACCACGTTAGTCCGGACC>ArS 201AACGGTGCGCGTTAAAACGGCCGACGCGTCATAACCGCGACTCGTCGACGCAGCGCCGGCGTATAAGCGCGCCGTACGTCAACCGTCGACGTTAGTCCGACGATCGCGGCGTCTATACGCCGCGTCAATCGCGCGCGGTTCAACGTCGCGCTACGGGCGCGTATAAGTCGCGCGTATGGACCGCGTACGTCCTACGAGCGT>ArS 232TCGACGCGCGTATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCCCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCGCCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGCGTAACGGTGCGCGTTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGC>ArS 300AACGGTGCGCGTTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCAGCGCCGGCGTATAAGCGCGCCGTACGTCAACCGTCGACGTTAGTCCGACGATCGCGGCGTCTATACGCCGCGTCAATCGCGCGCGGTTCAACGTCGCGCTACGGGCGCGTATAAGTCGCGCGGTTAATACGCGCGGTGTACGCGGATGCCGGGGTCGCGTATAATCGGCGCGTATACCTCGCGCGTATACGCGGCGTATTACGGCCGCGTATAATTCGCGCGTATGGACCGCGTACGTCCTACGAGCGT>CTAG1A_originalCGGAGCACGTGACCGGTTCTCACCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCAGCCGGCTGTCCGCCGGACCCCTGTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGGACTGTGCGGCCATCGTCCCGGACCCTGCCTGCCCTGTCCGTCCTTGGGGGAGCGCCCAGGACAGACfCCCGGGGGGCAGGCCTCTAfACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCC>CTAG1A_replaceTTACCTAAAACAGCCCAAAAGAGCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCCCACCACCACCTCCACCACCACTGTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGCCAAAGAAGCCCAAAAAGGCCCAGGAAACCCAAACTTTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCC>CTAG1A_deltaTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCCCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGG>CTAG1A_upCGTTTGACGGACGCCGTTCGCAGTCAACCCCGCCCCTCCCCAAGAGAGCCCGGGCCGGAAGGTGGCCGCAATGCCAGCTTGGACCCCTCACCCCTGAGCCGGAGCACGTGACCGGTTCTCACTCCCGGGAGCCCTGCAGGGAGTCAGGCACTGCGGGGCCCAGCCTGTCCCATCCCCCGGGTCTCCCTCACATCGAGGAGCAAGACGGGCCTGGGAACACGGGGCCGGATCGCGCAGCGATCGACGCCGGATCAACGCGATACGGTCCGTCCTTGGGGGAGCGCCCAGGACAGACCCCGGGGGGCAGGCCTCTAACTGGGCTCAGCAGCCTCCGTCCCTGTCCTGGTCGCCCAGCTGGTGGGGTAGCTGGAACTGCATGTCTGGTCTCAGAGAGAAGGTCAGGGCCCACGAGGATGCGGAGGCAGAGAGGCTGCAGGAAGTTCCGCCCCCTGGCGTGAGATGGGCAGCCCGGGATCCTCAGGGCGCCTGCGCACAGGGGCCCTACTTCCGGCCCTGGGAGACCCCGAGTGAGCCCTATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCTCGACGCGCG232\u2003 60TCGACGCGCG-----CACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGC 55dT1\u2003TATAACACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGCTCGACGCGCGdT2\u2003 60TCGACGCGCG-----CACGCGAGCGGTTCGAACGTTGGCGCGCTAACGCGAGTCGTACGC 55dT12\u2003**********\u2003*********************************************CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 120232\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 115dT1\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 120dT2\u2003CCGTCAACGCGGATCAATCGCGCGACTTGTGCGCGACGTTAGACCGCCGATCGTCAAGCG 115dT12\u2003************************************************************CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 180232\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 175dT1\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 180dT2\u2003CCGATCGGTAATCGGACGATTCGGATACGCGAGTTCGGACGTACGAGCGTGATACGGCGC 175dT12\u2003************************************************************TTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCGTAACGGTGCGCG232\u2003 232TTAAAACGCCGACGCGTCATAACCGCGACTCGTCGACGCGTAACGGTGCGCGdT1\u2003 227GTAACGGTGCGCG------CGCCGACGCGTCATAACCGCGACTCGTCGACGC 226dT2\u2003GTAACGGTGCGCG------CGCCGACGCGTCATAACCGCGACTCGTCGACGC 221dT12\u2003*************\u2003*********************************"}
+{"text": "Fibroblast growth factor 19 (FGF19) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor \u03b2Klotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation. FGF19 (fibroblast growth factor 19) and its murine ortholog Fgf15 are the founding members of the endocrine FGF subfamily that also includes FGF21 and FGF23 In addition to the effects on lipid and glucose metabolism, FGF19/Fgf15 has also been implicated in the regulation of hepatic BA metabolism and hepatocyte proliferation. FGF19/Fgf15 expression in the intestine is transcriptionally regulated by the nuclear BA receptor Farnesoid X Receptor (FXR) FGF19/Fgf15 is believed to act by activating FGF receptor (FGFR) homodimers complexed with a membrane bound protein \u03b2Klotho (KLB) In order to determine which of the metabolic effects elicited by FGF19 are mediated by FGFR4, we treated HFD-fed WT or Fgfr4 KO mice with recombinant FGF19 or vehicle control and studied metabolic phenotypes and gene expression. To achieve sustained exposure to FGF19, mice were implanted with osmotic pumps to continuously infuse FGF19 at 1 ng/hr. This achieved an average FGF19 serum concentration of 26 ng/ml, as determined by ELISA, about 50- to 250-fold higher than circulating FGF19 concentrations in humans To evaluate changes in systemic BA regulation, serum BA composition was determined by liquid chromatography-mass spectrometry . AlthougBased upon the results described above, we hypothesized that if we could generate FGF19 variants with specifically reduced FGFR4 activity, such molecules would retain beneficial metabolic effects while losing FGFR4-dependent actions such as the induction of hepatocyte proliferation and altered BA homeostasis. In order to quantitatively evaluate specific activation of FGFRs by FGF19, an FGF-responsive GAL-Elk1 luciferase reporter assay was introduced into rat L6 cells One chimeric construct classified as a class II molecule, consisting of amino acids 1-20 of FGF21 and 25-194 of FGF19 , was selected for large scale synthesis in CHO cells and this variant is referred to as \u201cFGF19v\u201d. When compared with FGF19 using the luciferase reporter assay, FGF19v protein exhibited a similar dose-dependent activity to FGF19 in L6 cells cotransfected with KLB and FGFR1c . HoweverActivity of FGF19v was further tested in vivo in comparison with FGF19 and FGF21 by intravenously injection into overnight fasted FVB mice. Livers were harvested at 4 hours post injection and hepatic mRNA expression was determined by qPCR. Genes that were acutely induced by FGF19 but not by FGF21, such as Egr-1 and c-Fos, were not efficiently induced by FGF19v, consistent with the reduced FGFR4 activity of FGF19v . FGF19v It has been previously proposed that FGFR4 mediates the induction of hepatocyte proliferation by FGF19 The in vitro and in vivo results described above raised the question as to whether FGF19v, a variant of FGF19 with reduced FGFR4 activity and proliferative potential, could improve hyperglycemia in diabetic animals similar to FGF21. FGF21, FGF19v (1ng/hr) or vehicle control was continuously infused subcutaneously into ob/ob mice using osmotic mini-pumps. While infusion did not significantly affect body weight , both FGThe mechanism by which FGF21 and FGF19 ameliorate hyperglycemia in diabetic animals is not well understood. Since FGF21 and FGF19v show very similar anti-diabetic effects in ob/ob mice, we hypothesize that commonly regulated pathways may contribute to their anti-diabetic effects. We identified a number of genes exhibiting commonly altered expression in ob/ob mice treated with FGF21 and FGF19v. In the liver, both proteins induced IGFBP2 (a recently demonstrated anti-diabetic protein) Although FGF19 has been shown to activate multiple FGFRs in the presence of the coreceptor KLB in vitro, contribution of each FGFR to the in vivo activity of FGF19 has been poorly defined. Our findings in Fgfr4 KO mice as well as using a FGF19 variant protein with reduced FGFR4 activity have delineated pathways downstream of FGF19. We have shown that FGFR4 is required for regulation of BA biosynthesis and hepatocyte proliferation as previously proposed Intriguingly, we found that both FGF19 and FGF21 acutely reduce hepatic expression of Cyp7a1 even in Fgfr4 KO mice . This FgFgfr4 has also been implicated in the regulation of lipid metabolism and glucose tolerance and may indeed mediate regulation of fat metabolism by endogenously produced Fgf15 protein Previously, therapeutic potential for FGF19 in the treatment of obesity and diabetes has been proposed Each FGF family protein consists of the structurally conserved central globular domain, and the flanking N-terminal and C-terminal segments that are structurally flexible and are divergent in primary sequence. In X-ray crystal structures of multiple FGF/FGFR complexes, the N-terminal segment of the FGF molecule makes specific contact with the FGFR and is believed to play an important role determining the specificity of the FGF-FGFR interaction In conclusion, our study demonstrates that FGFR4 is not required for beneficial pharmacological activity of FGF19, and that an engineered FGF19 variant mimicking the specificity of FGF21 could successfully be generated. Given the pleiotropic activities of FGF19 and FGF21 on multiple receptors , further exploration into altering receptor specificity of FGF19 or FGF21 to achieve specific activation of a particular FGFR may provide a safer and more predictable approach to exploit endocrine FGF pathways and provide new therapeutic options for the epidemic of obesity associated-disorders such as type 2 diabetes, nonalcoholic fatty liver disease and other manifestations of insulin resistance and the metabolic syndrome.The study protocols for all animal experiments were approved by the Genentech Institutional Animal Care and Use Committee (IACUC). The approval IDs for this study are: #08-1943, #08-2004, #08-2004A, #08-2004B, #08-2004C, #08-2136, #08-2136F, #09-1001, #09-1066, #10-1818.Unless otherwise noted, recombinant human FGF21, FGF19 and variants produced in transiently transfected CHO cell and purified to homogeneity in PBS were used for experiments. For some experiments, E. coli derived FGF21  was used. All the purified proteins were tested for activity by cell based GAL-Elk1 assays prior to use in for other assays. For experiments in All the constructs also possessed a signal sequence at the N-terminal end (cleaved upon secretion) and the flag tag (DYKDDDDK) at the C-terminal end. The sequences derived from FGF21 are shown in bold.1 (hFGF19): RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEKLKPGTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK2: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK3: HPIPDSSPLLQFGGQVRQRYLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK4 (hFGF19v): HPIPDSSPLLQFGGQVRQRYLYTDDPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK5: HPIPDSSPLLQFGGQVRQRYLYTDDAQLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK6: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK7: LPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS8: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS9: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS10: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS11: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS12: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGEDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS13: RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS14: RPLAFSDAGPHVHYGWGDPIRLRHLYTLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS15: RPLAFSDAGPHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK16: HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS17 (hFGF21): \u20138 hours in serum free media containing 25 mg/L porcine heparin (Sigma) and FGF protein at a various concentration. The cells were then lysed with PLB reagent (Promega) and luciferase activity in each well was determined using Dual-Glo Luciferase Assay System (Promega) and EnVision Multilabel Reader (PerkinElmer). Firefly luciferase activity was normalized to the co-expressed Renilla luciferase activity, and is shown as an average and standard error of the mean of the three replicas.All the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37\u00b0C under 5% CO2. Rat L6 myoblasts in a 96-well plate were transiently-transfected with expression vectors encoding Renilla luciferase , human KLB, appropriate human FGFR, GAL4-Elk-1 transcriptional activator , and firefly luciferase reporter driven GAL4 binding sites , using FuGENE HD Transfection Reagent (Roche Applied Science). On the next day, the transfected cells were cultured for an additional 6A 96-well-plate was filled with 50 \u00b5L/well of 0.5% molten agarose in growth media. After the base agarose had solidified, about 670 HepG2 cells suspended in 50 \u00b5L top molten agarose solution (0.35% agarose in growth media) were added to the base agar in each well, and allowed to solidify. Following solidification, 20 \u00b5L of growth medium containing an appropriate amount of FGF protein was added to each well on designated day 0. On each of the subsequent days 2, 4, 6 and 8, a further 20 \u00b5L of growth medium with an appropriate amount of FGF protein was added to each well. A subset of the sample wells was also treated with protein synthesis inhibitor Geneticin (Invitrogen) to provide a background fluorescence signal. On day 9, 10 \u00b5L AlamarBlue reagent (Invitrogen) was added to each sample well and the plate was further incubated for 5 hrs. The resulting fluorescent intensity was measured using EnVision Multilabel Reader (PerkinElmer) and used as an indication of the total metabolic activity in each well. Each condition was tested in quintuplicate.FGFR-binding activity of FGF19 and FGF19v were measured as described in Mice were maintained in a pathogen-free animal facility at 21\u00b0C under standard 12 hr light/12 hr dark cycle with access to chow  or a high fat, high carbohydrate diet  and water ad libitum. Male mice were used for all of the experiments described. FGFR4 KO mice in C57BL/6 background were previously described Total cholesterol, triglyceride, \u03b2-hydroxybutylate (BHB), lactate (Thermo DMA) and nonesterified fatty acid (Roche) were determined by using enzymatic reactions. Serum insulin levels were determined by ELISA . BA composition was determined by liquid chromatography-mass spectrometry analysis as previously described Tissue RNAs were isolated by using QIAzol reagent (Qiagen). cDNA was synthesize with the Quantitect Reverse Transcription Kit (Qiagen). For real time qPCR, samples were run in triplicate in the ABI Prism 7900HT (Applied Biosystems) by using SYBR green universal mix (Invitrogen) or by Taqman universal mix (Roche) and normalized by levels of 36B4. Pre-designed Quantitect primers for GK, SHP, Cyp8b1, IGFBP2, and AFP were obtained from Qiagen and all other primers were designed using primer express software (Applied Biosystems). Sequences of in-house designed primers will be provided upon request.p-value <0.05 was considered statistically significant. Values were presented as means+/\u2212 standard error of the mean.Unpaired student's t-test (two-tailed) was used for statistical analyses to compare treatment groups using Prism 5 software (Graphpad) or Excel (Microsoft). A Figure S1FGF21 and 19 activates FGFR2c and FGFR3c in the presence of KLB. GAL-Elk1 luciferase assay in L6 cells. L6 cells were cotransfected with expression vectors for KLB and the indicated FGFR together with GAL-Elk1, SV40-Renilla Luciferase, and Gal-responsive luciferase reporter. Transfected cells were incubated with media containing increasing concentrations of FGF19 (\u25cb) or FGF21(\u25b4) for 6 hours before luciferase assays. Transcriptional activation was assessed by the relative luciferase activity normalized by Renilla luciferase activity and expressed as relative luciferase unit (RLU).(TIF)Click here for additional data file.Figure S2In vitro activity of FGF21, FGF19 and chimeric constructs. GAL-Elk1 luciferase assay in rat L6 cells. L6 cells were cotransfected with expression vectors for KLB and the indicated FGFR together with GAL-Elk1, SV40-renilla Luciferase, and Gal-responsive firefly luciferase reporter. Transfected L6 cells were incubated for 6 hours before luciferase assays with conditioned medium from 293 cells transiently transfected with each FGF construct indicated at the bottom. The number below each group corresponds to the number of the construct as indicated in (TIF)Click here for additional data file."}
+{"text": "Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used.Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran.Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay.This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay.This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants. Escherichia coli as insoluble inclusion bodies have been demonstrated and are commercially used. The major advantage of these approaches is that proinsulin can be produced in large scale, but the intricate process of refinement and the formation of accurate disulfide bonds during folding are critical cost factors  is one of the most important and favorable vegetable crops. It is an ideal candidate plant for the production and delivery of oral vaccines. Being a short-duration crop and having the ability to grow in greenhouses adds to its advantages for exploring the possibilities of using this crop for biopharmaceutical production  harboring the human proinsulin gene. This expression vector having a resistance gene for kanamycin (NPT II) was used for selection of the transformed bacteria, and the hygromycin resistance gene was used to create hygromycin resistance in plants to screen the transgenic line. CaMV35S (Cauliflower Mosaic Virus promoter which induces high level of transcription) promoter, NOS  terminator sequence, the reporter genes of GUS and GFP (as reporter genes) encoding beta-glucuronidase and green florescent protein was used in front of the 35S promoter, and the sequences for restriction enzymes of Bst EII and Nco I were used to replace the desired gene with the reporter genes (Staphylococcus aureus (pro A-Pins) were cloned instead of the reporter genes. Proinsulin gene was obtained from the Pasteur Institute, Iran. The coding sequence is:er genes . The humATGGCGGGATTNAACCAATTTAATAAGGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAATGAAGAACAACGCAATGGTTTCATCCAGAGCTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAAGCTGATAACAAAGGATCCCGTCGCTTTGTTAACCAACACCTGTGCGGTTCTCACCTGGTTGAAGCTCTGTACCTGGTTTGCGGTGAACGTGGTTTCTTCTACACCCCGAAGACCCGTCGTGAAGCTGAAGACCTGCAGGTTGGTCAGGTTGAACTGGGTGGTGGTCCGGGTGCTGGTAGCCTGCAACCGCTGGCTCTGGAAGGTTCTCTGCAGAAGCGTGGTATCGTTGAACAGTGCTGCACCTCTATCTGCTCTCTGTACTACCAACTGGAAAACTACTGCAACStaphylococcus aureus spa gene for immunoglobulin G binding protein A\", and black is proinsulin  , which has been deleted because there was no need for targeting recombinant protein . Proinsulin is converted into the bioactive hormone insulin by removal of its connecting peptide (C-peptide). PROTEIN A can cause high expression of recombinant proinsulin gene and high stability of recombinant proinsulin protein produced in the Escherichia coli, and this protein causes easier purification of human proinsulin bound to it. Proinsulin is converted to bioactive hormone insulin by carboxypeptidase-H (CP-H) from Arg-Arg and Lys-Arg sites in secreting vesicle  for 30 minutes at room temperature. The explants were transferred into sterile paper to remove excess Agrobacterium, and were placed into coculture medium top side down (CM) for 2 days in the dark at 28\u02daC. Finally, inoculated explants were transferred into regeneration medium (RM)  were surface sterilized by 70% ethanol for 30 seconds, and then rinsed in distilled water for 1 minute. Then seed sterilization continued by immersing in 3% NaClO and rinsed three times with sterile distilled water. Sterile seeds were germinated in the dark at 25\u02daC on Murashige and Skoog (MS)  medium fium (RM) . The traGenomic DNA of transgenic plants was extracted, using the CTAB method . A quantFor the extraction of total soluble protein (TSP), 200 mg of young tomato leaves were used. The tomato leaves were ground in liquid nitrogen to a fine powder. Proteins were extracted by using 1000 \u00b5L of extraction buffer  and 0.04% (v/v) 2- Mercaptoethanol). Cell debris was removed by two rounds of centrifugation , and the supernatant was used for dot blot analysis. Twenty nanograms of protein samples were directly spotted onto a nitrocellulose membrane. BSA 1% solution was added after dying and incubated for 1 hour at 25\u02daC. Solution was poured out and washing with PBS-T for 5 minutes was performed; this step was repeated 3 times. Then the primary antibody  was added and incubated for 1 hour. The solution was poured out; washing with PBS-T was performed for 5 minutes, and repeated 3 times. Next, nitrocellulose membrane was incubated in anti-rabbit IgG conjugated with horseradish peroxidase as a secondary antibody for an hour with 1:4000 dilutions. Color development solution was DAB and 0.01% hydrogen peroxide in 50 mM Tris (pH = 7.5).E. coli strain DH5\u03b1. The constructed vector was confirmed by colony PCR, PCR, digestion and sequencing  and mammalian cell cultures (0.1% cost reduction) . The fir"}
+{"text": "AbstractAmynthas Kinberg, 1867 species belonging in family Megascolecidae s. stricto are sketched, dissected and described. Amynthas daeari Blakemore sp. n. has spermathecae in 6/7/8 complying with an Amynthas tokioensis spp-group, whilst Amynthas jinburi Blakemore sp. n. has spermathecal pores in 5 & 6 strictly complying with Amynthas canaliculatus-group. A definitive COI gene barcode is provided for the holotype of Amynthas daeari but the age since collection or preservation of the Amynthas jinburi type in 2000 precluded its mtDNA extraction at this time.Two Korean endemic pheretimoid  Pheretima auct.) group of Oriental origin that provides approximately 970 valid species from 1,200 nominal taxa .Specimens, now in 80% Ethanol, are lodged in the NIBR facility. Small tissue samples taken for mtDNA COI barcoding as proposed 10 yrs ago by Discussion is confined to remarks after each species\u2019 description. For brevity, not all taxonomic authorities are cited in References as these may be sought elsewhere.urn:lsid:zoobank.org:act:E6B103F4-2FFE-4A05-BCD8-356A90754AC5http://species-id.net/wiki/Amynthas_daeari35.9801N, 127.2981E); collected 27th July, 2012 by Dr Hong-Yul Seo. DNA tissue sample code \u2013 w53.IV0000261261, mature specimen complete but broken in two at clitellum after being figured and dissected. Collected from small valley at Jeollabuk-do, Wanju-gun, Dongsang-myeon, Daea-ri  with GMs anterio-median and shallow clefts laterally .Female pores. Single on 14.Spermathecal pores. 6/7/8 ca 0.3 C apart at edge of puckered area and lateral to GMs.Genital markings. Paired discs just median to male and spermathecal pores as noted; composite glands on spermathecal pore GMs but none found for GMs near male pores although the body here is macerated and they may well have broken off and dissipated.Septa. Nephridial forests on septa 5 & 6; 7/8 thin, 8/9/10 aborted.Dorsal blood vessel (dbv). Single.Hearts. Last hearts in 13 (preceding vascularization unclear/damaged).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. Indeterminate as specimen macerated; caeca ventrally incised from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, seminal vesicles in 11 & 12.Ovaries. In 13 as usual.Prostates. Racemose glands in 17-19, duct short and muscular.Spermathecae. Two pairs in 7 and 8; that in 7lhs inflated, that in 8lhs deflated .Gut contents. Coarse organic debris, i.e., a litter diet suggesting superficial feeding.Amynthas daeari Holotype.>w53 PageBreakGCTCATGCATTTTTAATAATCTTCTTTCTTGTAATACCAGTATTTATTGGTGGGTTTGGAAATTGACTTCTACCTCTAATACTAGGTGCCCCAGATATAGCTTTCCCGCGACTTAACAATATAAGATTCTGATTACTGCCCCCATCACTAATTTTACTAGTATCGTCTGCAGCAGTAGAAAAAGGTGCCGGAACAGGATGGACAGTGTACCCCCCACTTGCGAGAAACATTGCACATGCCGGCCCTTCAGTAGATCTTGCAATTTTTTCTCTCCATCTAGCCGGAGCATCATCAATTCTCGGTGCCATCAACTTCATTACTACCGTAATTAATATACGATGATCTGGGCTACGCTTAGAACGAATTCCTCTATTTGTATGAGCAGTTGTAATTACTGTAATTCTTTTACTTCTATCTTTACCAGTCTTAGCCGGTGCTATTACAATATTACTAACAGACCGAAACCTAAATACATCATTTTTTGATCCAGCGGGAGGAGGTGATCCAATTCTATATCAACACTTATTTCTATATTTCATTTTAGGAATTTGAGCTGGAATAATTGGGGCAGGAATAAGACTGCTTATTCGAATTGAGCTAAGACAGCCGGGCTCTTTTCTAGGAAGGGATCAACTCTATAATACAATTGTAACAAmynthas tappensis\u201d (AB542547.1) from Japan max. identity <88% this then is a different and likely new taxon. The closest match from current Korean studies with BLASTn identity 565/653 (87%) is WO49, an immature Amynthas sp. from Jeju that itself comes closest to the Amynthas tokioensis/Metaphire hilgendorfi spp. complexes ; about twenty other species, many placed in this group after 1972, have simple intestinal caeca. Only four have simple incised caeca as here, but they all differ in characteristics of their GMs, at least, and none of these latter are known from Korea (Blakemore unpublished). The incised caeca is assumed to be a characteristic transitional or intermediate from simple to complex/manicate. The GMs in 7-8 obviously correspond to those in 18 during amphimixis but it is not known whether they interlock serially. The shape of the spermathecae and spermathecal pores are further distinguishing characteristics of Amynthas daeari that, along with its objective DNA barcode data, now serve to define this taxon.Of urn:lsid:zoobank.org:act:9BA5299B-0BA0-4E50-8EAC-C76C2D66B2EAhttp://species-id.net/wiki/Amynthas_jinburi38.2961N, 128.3546E) just north of Seoraksan Park on East coast; collected 1st \u2013 2nd June, 2000 by unknown person(s) and deposited in NIBR. DNA tissue sample w61b (unsuccessful at this time).IV0000213690, sub-mature specimen, figured and dissected. From Gangwon-do, Goseong-gun, Ganseong-eup, Jinbu-ri .Length. 210 mm.Width. ca. 10 mm at male pore level.Segments. 143 with some secondary annulation (from preservation?).Colour. Bleached pale yellow in aged alcohol, possibly darker in life.Prostomium. Open epilobous.First dorsal pore. 11/12.Setae. >100 per segment; e.g. 100+ on 11 and 112 counted on segment 12; approximately 16 setae intervene between male pore pads that are asetal on 18.Nephropores. Not found.Clitellum. Slightly darker at 14-16.Male pores: On 18 on small, rounded and flat porophores.Female pores. Single on 14.Spermathecal pores. At posterior of 5 and 6 approximately 0.3 C apart.Genital markings. None (sub-adult?).Septa. Nephridial forests on septa 5 & 6; 5/6/7/8 thick, 8/9 thin to base of gizzard, 9/10 aborted.Hearts. Seen in 11-13 (aborted in 10?).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. From 15; caeca simple elongate from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, testes small in 10 &11; seminal vesicles in 11 & 12.Ovaries. Compact in 13; ovisacs not found in 14.Prostates: Racemose glands not fully developed in 18 on short, muscular duct.Spermathecae. Two pairs in 6 & 7 exiting to anterior of 5/6 and 6/7 in 5 & 6  or denatured by pH.Amynthas serenus  from Pahang, Malaysia that also lacks GMs, and Amynthas? breviclitellatus  from Vietnam that differs, at least, in its GMs in 7, 18 and 19. From \u201cK\u00f4ry\u00f4\u201d Korea (about 30 Km from Seoul), Amynthas fibulus fibulus  is superficially similar but has spermathecal pores anteriorly in 6 & 7 (rather than posteriorly in 5 & 6) plus its caeca are incised ventrally (rather than smooth); ditto for Amynthas fibulus ranunculus  that further has slits lateral to male pores. Interestingly, Amynthas fibulus closely resembles the current specimen\u2019s gland . It appears that many of Spermathecal pores in 5/6 and 6/7...at or near leading edge of vi, vii\u201d and no useful figures are provided for the reader to decide.It should be here noted that morrisi-group\u2019s possible nearest relatives from Korea would likely be Amynthas koreanus  that, however, has manicate caeca; or Amynthas kobayashii  and Amynthas geojeinsulae  that both have male fields from 17-19 but differ in simple or incised caeca, respectively; or Amynthas assimilis Hong & Kim, 2002 that, like many of its similar cited taxa, has seminal grooves on 18.If spermathecal pores were in 5/6/7 in any of the above taxa, then the The current species has simple, superficial male pores on large disc-like pads on 18. Although not fully mature, it appears unique in the Korea fauna on its combination of this aspect of its male field, spermathecal pores in 5 & 6 and its profusion of setae that number more than 100 per segment, combined with simple elongate intestinal caeca.Fresh topotypic material is required to confirm these conclusions and to provide definitive DNA data, unless refinement of techniques allows extraction from older types."}
+{"text": "Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.We discovered that domain D of the The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in  E. coli remains an attractive host for the production of recombinant proteins, even though more complex proteins with posttranslational modifications, such as glycosylation patterns require alternative host systems ; SOD [BT008028.1]; Staphylococcal Protein A[P38507]:Domain D:GFPmut3.1 [proEDDIE [ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK NproEDDIE DNA sequence sSpAD (codon optimized):GCAGACGCACAACAGAATAAGTTTAACAAAGACCAGCAGAGCGCATTCTACGAAATTCTGAACATGCCGAATCTGAATGAGGAACAACGTAATGGCTTTATTCAGTCTTTAAAAGACGACCCATCTCAGAGCACCAACGTTCTGGGCGAAGCAAAGAAACTGAACGAATCTCAGGCACCAAAA"}
+{"text": "Exposure of neurons to 2 \u00b5M A\u03b21\u201342 resulted in significant viability loss and cell apoptosis. Accumulation of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and activation of caspase-9 and caspase-3 were also observed after A\u03b21\u201342 exposure. All these effects induced by A\u03b21\u201342 were markedly reversed by DG treatment. In addition, DG could alleviate lipid peroxidation and partially restore the mitochondrial function in A\u03b21\u201342-induced AD mice. DG also significantly increased the PGC-1\u03b1 expression in vivo and in vitro, while knocking down PGC-1\u03b1 partially blocked the protective effects, which indicated that PGC-1\u03b1 contributed to the neuroprotective effects of DG. Furthermore, DG significantly decreased the escape latency and search distance and increased the target crossing times of A\u03b21\u201342-induced AD mice in the Morris water maze test. Therefore, these results demonstrated that DG could attenuate A\u03b21\u201342-induced neuronal injury by preventing mitochondrial dysfunction and oxidative stress and improved cognitive impairment in A\u03b21\u201342-induced AD mice, indicating that DG exerted potential beneficial effects on AD.Mitochondrial dysfunction is a hallmark of beta-amyloid (A\u03b2)-induced neurotoxicity in Alzheimer's disease (AD), and is considered an early event in AD pathology. Diammonium glycyrrhizinate (DG), the salt form of Glycyrrhizin, is known for its anti-inflammatory effects, resistance to biologic oxidation and membranous protection. In the present study, the neuroprotective effects of DG on A\u03b2 Alzheimer's disease (AD), with typical pathological abnormalities including amyloid plaques, neurofibrillary tangles and neuron death, is the most prevalent neurodegenerative disease in vivo and CREB-dependent gene expression played critical roles in the neuroplasticity associated with cognitive function The peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) are a small family of transcriptional coactivators which play a critical role in the control of glucose, lipid, and energy metabolism 1\u201342-induced oxidative stress and mitochondrial dysfunction partially via induction of PGC-1\u03b1 and alleviated A\u03b21\u201342-induced cognitive impairment, suggesting DG might be developed into a promising drug for treatment of AD.Glycyrrhizin (GL), which is extracted in liquorice root, has a wide range of pharmacological actions including anti-virus, anti-allergenic and anti-immune-mediated cytotoxicity 1\u201342  was dissolved in 1% NH3\u00b7H2O at a concentration of 1 \u00b5g/\u00b5l and incubated at 37\u00b0C for 5 days to allow for fibril formation. DG was purchased from Jiangsu Chia-Tai Tianqing Pharmacy Company. The male ICR mice (weight range: 15\u201320 g) were anesthetized and A\u03b21\u201342  was injected to bilateral hippocampus by infusion cannulae. DG was co-injected intraperitoneally with A\u03b21\u201342. The mice were randomly assigned into four groups: the normal mice with saline or DG , and A\u03b21\u201342-induced AD mice with saline or DG . All animal experiments were approved by the Animal Care Committee in Nanjing University and performed according to institutional guidelines. We made every effort to minimize the number of mice used and their suffering.The A\u03b25 cells/ml on poly-D-lysine-coated plates. Cells were maintained in Neurobasal media supplemented with B27  and 25 nM glutamine at 37\u00b0C in a humidified 5% CO2 incubator. The purity of neurons was over 95%. The cells at day 8 were incubated with 2 \u00b5M A\u03b21\u201342 with DG or saline for 24 h.Primary cortical neurons were prepared from E15\u201317 mouse embryo. Cortexes were dissected and plated at 4\u00d7102 incubator.HEK293T, BV-2 and RAW264.7 cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM containing 10% of heat-inactivated fetal bovine serum (FBS), 2 mmol/L of L-glutamine, 100 U/ml of penicillin, and 100 \u00b5g/ml of streptomycin at 37\u00b0C in a humidified 5% COSmall hairpin RNAs (shRNAs) were synthesized and subsequently cloned into pCMV-U6 vector using Bbsl and BglII . Five PGC-1\u03b1 shRNAs sequences (shP1\u2013shP5) were designed to target mouse PGC-1\u03b1 gene. The plasmid expressing scrambled shRNA (sh-con) was used as a negative control. ShRNA sequences were as follows:5\u2032-TTTGGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGCTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGC-3\u2032;shP1: Forward: 5\u2032-TTTGCCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGGTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAACCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGG-3\u2032;shP2: Forward: 5\u2032-TTTGCGGAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTTTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAAAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTCCG-3\u2032;shP3: Forward: 5\u2032-TTTGCGGTAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTATTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAATAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTACCG-3\u2032;shP4: Forward: 5\u2032-TTTGTCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGATTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAATCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGA-3\u2032.shP5: Forward: 5\u2032-TTTGGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGCTTTTTG-3\u2032, Reverse: 5\u2032-GATCCAAAAAGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGC-3\u2032.sh-con: Forward: The oligonucleotides were synthesized by Biocolor BioScience and Technology Company . shRNAs were transfected into neurons using Lipofectamine 2000  according to the manufacturer's instructions. Cells were harvested for RT-PCR and western blotting at 24 h after the transfection.Apoptosis was determined by Annexin V-FITC apoptosis detection kit . After treatment, the cells were rinsed with PBS twice, centrifuged at 600 g for 10 min and resuspended in 0.5 ml binding buffer containing 5 \u00b5l Annexin V and 5 \u00b5l propidium iodide (PI), and then incubated for 15 min at 37\u00b0C in the dark. The apoptotic rate was examined by flow cytometry.Cell viability was determined using the conventional MTT assay. After treatment, primary cortical neurons were treated with 0.5 mg/ml MTT for 4 h at 37\u00b0C. The formazan crystals were dissolved in 100 ml of DMSO and the absorbance was measured at 570 nm in a plate reader. Cell survival rates were expressed as percentages of the value of normal cells.LDH is the most widely used marker in cytotoxicity study. At the end of incubation, the supernatant was collected from plates and the LDH content was determined using an LDH assay kit according to the manufacturer's instructions . LDH cytotoxicity was calculated by the following formula: LDH cytotoxicity\u200a=\u200a(sample OD\u2212blank OD)/(standard solution OD\u2212blank standard solution OD )\u00d72000.Change of the mitochondrial transmembrane potential in neurons was quantified by JC-1 . Briefly, neuronal cells were centrifuged at 600 g for 10 min, and resuspended in 0.5 ml medium containing 5 \u00b5M JC-1. After 20 min of incubation at 37\u00b0C in the dark, the cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.To monitor intracellular accumulation of ROS, flow cytometry was used with commercial kit  according to the manufacturer's instructions. After treatment, the cells were harvested, rinsed with PBS twice, centrifuged at 600 g for 10 min, and then resuspended in 10 \u00b5M DCFH-DA solutions. After 20 min of incubation at 37\u00b0C, cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.The levels of 4-HNE from hippocampus and serum were measured by the ELISA kits  according to the manufacturer's instruction. Briefly, supernatant from hippocampus or serum were added into the 96-well plate coated with purified anti-4-HNE antibody, and then HRP-labeled 4-HNE antibody was added. The absorbance was measured at 450 nm and the concentration of 4-HNE was determined by comparing the O.D. of the sample to the standard curve.For measurement of cytochrome c release, the mitochondrial and cytosol fractions were prepared according to the manufacturer's instructions . Briefly, mice hippocampus were washed twice with cold PBS, resuspended in fresh cytosolic extract buffer and incubated for 30 min on ice with frequent tube tapping. Tissues were homogenized on ice, and then nuclei, unbroken cells, and cell debris were pelleted at 600 g for 10 min at 4\u00b0C. The supernatant was spun again at 13,000 g for 20 min at 4\u00b0C. The supernatant was carefully transferred and the final pellet was used as the mitochondrial fraction. The cytochrome c levels were determined using a monoclonal antibody to cytochrome c by western blotting as described below.Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dry milk for 1 h and incubated overnight at 4\u00b0C with rabbit anti-cleaved caspase-3 , rabbit anti-caspase-3 , rabbit anti-caspase-9 , rabbit anti-PGC-1\u03b1 , mouse anti-cytochrome c , or mouse anti-GAPDH  antibody. GAPDH was used as a loading control. The proteins were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies and visualized with chemiluminescence reagents provided with the ECL kit  and exposure to film. The intensity of the blots was quantified with densitometry.Caspase-3 and caspase-9 activities of primary cortical neurons were measured by means of colorimetric assay kits , according to the manufacturer's instructions. In brief, harvested cells were incubated with 50 \u00b5l lysis buffer on ice for 30 min, followed by centrifugation at 10,000 g for 1 min at 4\u00b0C. Then, cells were suspended in 50 \u00b5l 2\u00d7reaction buffer and 5 \u00b5l caspase-3 or caspase-9 substrate incubating for 4 h at 37\u00b0C. Later, the absorbance was read in a microplate reader at 400 nm.Real-time PCR was performed as described previously 5\u2032-TGACACAACGCGGACAGAA-3\u2032, Reverse: 5\u2032-GGTAGGTGATGAAACCATAG-3\u2032;PGC-1\u03b1: Forward: 5\u2032-GCCAAGGCTGTGGGCAAGGT-3\u2032, Reverse: 5\u2032-TCTCCAGGCGGCACGTCAGA-3\u2032.GAPDH: Forward: The PCR products were analyzed on 1.5% agarose gels and visualized by ethidium bromide. The gel was visualized with UV-transilluminator and photographed.5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACTGT-3\u2032, Reverse: 5\u2032-CGCCGAGATCTAAAGCTATTAAAAAGTAGGCT-3\u2032) to facilitate directional cloning. The PCR products were cloned into the pGL3 basic in sense orientation (designated as p-PGC-3K). The truncated constructs were made using the following primers:The promoter regions of mouse PGC-1\u03b1 (\u22123000 to 0 bp) were amplified using PCR, DNAs of primary cortical neurons as templates, and specific primers with MluI and BglII restriction enzyme  cut sites engineered on the ends : Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACAC-3\u2032, Reverse: 5\u2032-GTCGAGATCTTCTACTTTCCACACAGTC-3\u2032;\u22123000\u20132000 bp (named as p-PGC-1K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACAC-3\u2032, Reverse: 5\u2032-CCGCCGAGATCTTCTGACTTTATATAGTC-3\u2032;\u22123000\u20131500 bp (named as p-PGC-1.5K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACT-3\u2032, Reverse: 5\u2032-GCCGAGATCTTCCAACCCTAGTGCCTTG-3\u2032;\u22123000\u20131000 bp (named as p-PGC-2K): Forward: 5\u2032-ATAAACGCGTAATGTGTGGCCGAACACACT-3\u2032, Reverse: 5\u2032-GCCGAGATCTGATTTTCTTTCTCTCTCTCCT-3\u2032;\u22123000\u2013500 bp (named as p-PGC-2.5K): Forward: 5\u2032-AAATAAACGCGTGGGGGTGTTGCCTTCAAAC-3\u2032, Reverse: 5\u2032-GCCCCGAGATCTAAAGCTATTAAAAAGTAGG-3\u2032.\u2212500\u20130 bp (named as p-PGC-500 bp): Forward: The sequence of \u2212100\u20130 bp in the PGC-1\u03b1 promoter (named as p-PGC-100 bp), CREB binding site mutation sequence (named as p-PGC-100 bp mutate) and deletion sequence (named as p-PGC-100 bp delete) were synthesized with MluI and BglII restriction enzyme cut sites as followers:5\u2032-CGCGTGAGGGCTGCCTTGGAGTGACGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACGTCACTCCAAGGCAGCCCTCA-3\u2032;p-PGC-100 bp: Forward: 5\u2032-CGCGTGAGGGCTGCCTTGGAGTGTGGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACCACACTCCAAGGCAGCCCTCA-3\u2032;p-PGC-100 bp mutate: Forward: 5\u2032-CGCGTGAGGGCTGCCTTGGAGGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3\u2032, Reverse: 5\u2032-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCCTCCAAGGCAGCCCTCA-3\u2032.p-PGC-100 bp delete: Forward: Renilla were cotransfected to cells followed by DG treatment for 24 h. The Luciferase activity was assayed by using the Promega Bright-N-Glo system as previously described All transfection experiments in this study were performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. pPGCs and phRL-CMV The Morris water maze test was conducted as previously described post hoc test with day and treatment as the sources of variation. Otherwise comparison between two groups was statistically evaluated by Student's t-test and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey post hoc test. Values of P<0.05 were considered statistically significant.The data were expressed as means \u00b1 SEM and analyzed by SPSS12.0 statistical analytical software . Group differences in the escape latency, searching distance and swimming speed during the Morris water maze test were analyzed using two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni 1\u201342 (2 \u00b5M) and different concentrations of DG or saline. As expected, the viability of cortical neurons exposed to A\u03b21\u201342 was reduced by 34.1% in comparison with the control group . To further investigate DG's ability to inhibit A\u03b2-induced oxidative stress, a marker of lipid peroxidation, 4-HNE was examined. As shown in 1\u201342-induced AD mice were significantly increased by 40.4% and 67.3% compared to control mice respectively (P<0.05), while 4-HNE levels were reduced 24.2% and 33.2% in the serum and hippocampus after DG treatment respectively (P<0.05).Emerging evidence suggests that mitochondrial dysfunction and oxidative stress are involved in A\u03b21\u201342 (P<0.01). However, the decrease of \u0394\u03c8 induced by A\u03b21\u201342 was greatly alleviated after DG treatment (P<0.01), indicating that DG protected mitochondrial against A\u03b21\u201342-induced injury. Meanwhile, the activities of caspase -9 and caspase-3, were assessed. As shown in 1\u201342-treated neurons, while DG-treated neurons exhibited lower caspase-9 and caspase-3 activities compared to A\u03b21\u201342-treated neurons (P<0.05).Mitochondrial membrane potential (\u0394\u03c8) is widely recognized as an indicator of mitochondrial functionality, which is measured by JC-1, a cationic lipophilic fluorescent. The results showed that there was a significant loss of \u0394\u03c8 in neurons treated with A\u03b21\u201342 , P<0.01.reatment , P<0.01,in vivo, the release of cytochrome c from the mitochondrial membrane as well as the subsequent activation of caspase-9 and caspase-3 was investigated by western blotting. The levels of cytosolic cytochrome c expression in A\u03b21\u201342-induced AD mice were significantly increased, which were significantly reversed by the treatment with DG , 6 h (1.47\u00b10.08-fold), 12 h (1.70\u00b10.11-fold), 24 h (2.11\u00b10.48-fold) and 48 h (1.24\u00b10.38-fold)  , with si38-fold) . PGC-1\u03b1 pression . To demopression  and the  neurons .P<0.01). Also DG treatment significantly up-regulated the transcriptional activity of PGC-1\u03b1 by 4.08-fold in HEK293T cells, 1.98-fold in BV-2 cells and 1.62-fold in RAW264.7 cells at a concentration of 0.001 \u00b5g/\u00b5l, indicating that induction of transcriptional activity of PGC-1\u03b1 by DG may not be cell type specific (P<0.01), which indicated that \u2212500\u20130 bp in the promoter of PGC-1\u03b1 might be involved in the protective effects of DG \u200a=\u200a3.620, P\u200a=\u200a0.016; group x day: F\u200a=\u200a0.915, P\u200a=\u200a0.516). In addition, the search distance was also significantly decreased by DG treatment compared to AD mice \u200a=\u200a41.688, P\u200a=\u200a0.014; days: F\u200a=\u200a11.536, P\u200a=\u200a0.01; group x day: F\u200a=\u200a1.942, P\u200a=\u200a0.067, P<0.05, 1\u201342-induced AD mice.To explore whether DG could improve cognitive impairment in A\u03b21\u201342-induced AD model in vitro and in vivo, this study for the first time shows: 1) DG exerts neuroprotective effects and improves cognitive impairment; 2) DG rescues mitochondrial dysfunction and inhibits oxidative stress; and 3) DG increases the expression of PGC-1\u03b1, which might contribute to the neuroprotection of DG.In the A\u03b21\u201342-induced neuronal toxicity, and is considered as an early event in AD pathology. Several evidences indicated that A\u03b2 triggered mitochondrial dysfunction through a number of pathways such as increase of ROS, interaction with ABAD, impaired mitochondrial biogenesis, and alteration of mitochondrial dynamics 1\u201342-treated neurons. In addition, DG decreased lipid peroxidation and release of cytochrome c from the mitochondria, and the activation of caspase-9 and caspase-3 in A\u03b21\u201342-induced AD mice. Furthermore, this anti-oxidation function of DG could refrain neurotoxicity mediated by A\u03b21\u201342, that is, increased cell viability, decreased apoptosis and LDH release in A\u03b21\u201342-treated neurons.Mitochondrial dysfunction is a hallmark of A\u03b21\u201342-treated neurons.Regardless of the possible mechanisms of DG restraining oxidative stress, it is clear that PGC-1\u03b1 is a major regulator of mitochondrial biogenesis and is protective against oxidative damage 1\u201342-treated neurons and CREB might play an important role in induction of the transcriptional activity of PGC-1\u03b1. PGC-1\u03b1 was a direct target of CREB induction of gluconeogenesis in vivoIt is intriguing that DG treatment increased the expression of PGC-1\u03b1 in A\u03b21\u201342-induced toxicity in vitro and in vivo. DG significantly increased the viability of A\u03b21\u201342-treated neurons by inhibiting oxidative stress and reversing mitochondrial dysfunction. Furthermore, PGC-1\u03b1 upregulated by DG treatment might play an important role against A\u03b21\u201342-induced neurotoxicity. Findings of current study revealed new function and mechanism of DG on neurotoxicity induced by A\u03b21\u201342, suggesting that DG may be developed into a new drug for treatment of AD.Taken together, DG exerted neuroprotective effects against A\u03b2"}
+{"text": "Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by  Methanocaldococcus jannaschii (M. jannaschii) tyrosyl-tRNA synthetase (TyrRS) to charge an amber codon-suppressing tRNA with an UAA Transfer ribonucleic acids (tRNAs) serve as an adaptor molecule, bridging between genetic information and amino acid sequences during protein biosynthesis. Aminoacyl-tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to a corresponding tRNA and have evolved active sites that are highly specific for a given substrate M. jannaschii TyrRS can only be used in prokaryotic cells; they are not orthogonal in eukaryotic systems M. jannaschii TyrRS can thus be used in a eukaryotic system if they are manipulated into ignoring endogenous tRNAs.All of the existing unnatural aaRSs derived from M. jannaschii TyrRS can be made orthogonal in mammalian cells by using peptide transplantation involving the connective polypeptide 1 (CP1) domain of E. coli TyrRS M. jannaschii that is not orthogonal in mammalian cells can be made orthogonal by switching C1:G72 to G1:C72 thus generating 1bp-tRNACUAM. jannaschii TyrRS (aaRS) specific to an unnatural amino acid in E. coli allowing it to charge 1bp-tRNACUA with the same unnatural amino acid in mammalian cells We have previously shown that M. jannaschii in mammalian cells, the tRNA was previously flanked with sequences taken from a human tyrosyl-tRNA gene. Six tandem repeats of the resulting genes were cloned into plasmid pZeoSV2 (+) to create 6x_wt-tRNACUA  of green fluorescent protein (GFP) gene in vector p-EGFP-N1 to afford p-GFP_39TAG. Mammalian HEK 293T cells were co-transfected with p-GFP_39TAG and the plasmid expressing the amber-suppressor tRNA. Suppression of the 39th amber codon TAG led to the expression of a full-length GFP in vivo  region in recognizing its corresponding tRNA acceptor stem M. jannaschii TyrRSs (E. coli (pdb.org 1X8X) and M. jannaschii TyrRS (pdb.org 1J1U) were used to design six E. coli CP1 swapped mutants: TyrRS_36CP1, TyrRS_39CP1, TyrRS_39CP1_2, TyrRS_42CP1, TyrRS_44CP1, and TyrRS_fullCP1. Amino acid sequence alignment  of the two TyrRS was used to design another three CP1-transplanted mutants: TyrRS_29RED, TyrRS_34RED, and TyrRS_38RED. The N-terminal regions of these swaps are similar to the structural homology designs, although the C-terminal region is based on a RED sequence that is shared by both TyrRSs. The crystal structure of Thermus thermophilus (T. thermophilus) TyrRS is also available, and it is known to charge a G1:C72-containing tyrosyl-tRNA T. thermophilus TyrRS and the M. jannaschii TyrRS, two additional CP1-substituted mutants were designed: TyrRS_39tt and TyrRS_45tt. All CP1-swapped TyrRSs also contained an Arg286 mutation that has previously been shown to increase recognition of the anticodon region of a suppressor tRNA by M. jannaschii TyrRSs Our next focus was manipulating the i TyrRSs Figure 4CUA, and with a plasmid containing a gene for a CP1-swapped TyrRS, harvested 72 hours later, and analyzed by FACS analysis. Since the tRNA is not recognized by any endogenous aaRS, full-length GFP could only be detected when the 1bp-tRNACUA was charged by the CP1-swapped TyrRS. Wild type E. coli TyrRS was used as a positive control and offered a 13.5-fold increase in fluorescence over that seen when cells were transfected with a plasmid harboring 1bp-tRNACUA only  orthogonality in the mammalian host system; b) stop codon-suppression efficiency; c) expression of a protein, reaching yields up to five-fold higher than realized with previously used systems that rely on the amber suppression approach in mammalian cells. These improvements pave the way for efficient and specific incorporation of UAA into recombinant mammalian proteins.E. coli TOP10 cells and isolated using a Qiagen Miniprep Kit. HEK293T cells (ATCC\u00ae CRL-11268TM) were cultured in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% (v/v) fetal bovine serum  in a 37\u00b0C humidified incubator containing 5% CO2. Transfections were performed using FuGENE HD according to product manual . Full-length GFP was excited at 460-500 nm and detected using a Nikon Eclipse TE2000-S microscope equipped with a FITC HyQ filter (Chroma).All plasmids were amplified in The commercially available plasmid p-EGFP-N1 (Clontech) was used to express a gene for EGFP using a CMV promoter. The 39th codon in the EGFP gene was mutated from TAC to TAG by site-directed mutagenesis (SDM) using primers designed by the Stratagene online primer design program5\u2032-GCGAGGGCGAGGGCGATTAGACCTACGGCAAGC-3\u20325\u2032-GCTTGCCGTAGGTCTAATCGCCCTCGCCCTCGC-3\u2032A QuikChange II Site-Directed Mutagenesis Kit was purchased from Stratagene , and site-directed mutagenesis was completed according to the provided protocol. Plasmids were sequenced by the University of Texas at Austin ICMB DNA Sequencing Facility. The new plasmid created was termed plasmid GFP_39TAG.The MCS-1 region from a commercially available plasmid, pTRE-TIGHT-BI (Clontech) was removed by digesting the plasmid with the KpnI and EcoRI restriction enzymes . Oligonucleotides, containing a sequence for the T7 promoter:5\u2032-GGGGTACCCCTAATACGACTCACTATAGGGGGAATTCC-3\u20325\u2032-GGAATT-CCCCCTATAGTGAGTCGTATTAGGGGTACCCC-3\u2032were annealed and digested with KpnI and EcoRI. The digested insert was then ligated using DNA T4 Ligase into the pre-digested p-TRE-TIGHT-BI plasmid to form plasmid p-TRE-TIGHT-T7. Next, oligonucleotides containing the sequence for the human H1 promoter were annealed and extended using a Klenow enzyme. The oligonucleotide sequences were:5\u2032-CAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAAC ACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAA-3\u20325\u2032-GAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTG ATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCC-3\u2032The sequence was then amplified by PCR using the primers:5\u2032-GGAATTCCAATTCGAACGCTGACGTCATCAACCCGCTCCAAGG AATC-3\u20325\u2032-GAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCA-3\u2032The resulting product was digested with the KpnI and BglII restriction enzymes and ligated into a pre-digested p-TRE-TIGHT-T7 plasmid to generate plasmid pT7-H1.M. jannaschii TyrRS (amino acids 110-148) with the CP1 region of E. coli TyrRS (amino acids 129-172). The 5\u2032 end of the gene (containing sequences for amino acids 1-109) was amplified using the following primers:TyrRS_44CP1 was created by replacing the CP1 region of 5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3\u2032The 3\u2032 end (containing amino acids 149-306) was amplified by PCR using primers:5\u2032-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3\u20325\u2032-CCCGAATTCTAATCTCTTTCTAATTGGCT-3\u2032E. coli CP1 region (amino acids 129-172) was amplified from a wild type E. coli TyrRS gene by PCR using the following primers:Finally, the 5\u2032-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032All three PCR fragments were combined, denatured for 15 minutes at 85\u00b0C and elongated with a Klenow enzyme for 30 minutes at room temperature. The Klenow product was again amplified by PCR, purified, and digested with the HindIII and EcoRI restriction enzymes. Finally, TyrRS_44CP1 was created by ligating the digested product into a pre-digested pEF6-V5-His6-TOPO plasmid  using T4 DNA ligase (NEB). All other CP1-transplanted mutants were created by PCR in a process similar to that described in the previous section. The chimeric gene was divided into three segments, and each was amplified by PCR. Finally, a joint gene was also amplified by PCR, digested, and ligated into plasmid pEF6-V5-His6-TOPO. Primers are listed for each of the segments, based on the size and sequence of the CP1 swap.TyrRS_36CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-CGCAGGAAGGTCAGCACATTCATCTGGAATTCACTTCCATAAACAT-3\u20325\u2032-ATGTTTATGGAAGTGAATTCCAGATGAATGTGCTGACCTTCCTGCG-3\u20325\u2032-TGGATAGATAACTTCAGCAACAATCCCCTGATCTTCACGGTTGAGA-3\u20325\u2032-TCTCAACCGTGAAGATCAGGGGATTGTTGCTGAAGTTATCTATCCA-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_39CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3\u20325\u2032-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_39CP1_2:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3\u20325\u2032-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_42CP1:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3\u20325\u2032-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3\u20325\u2032-TTCAGCAACCTTTGGATTACGGTTGAGACGCTGCTTAACC-3\u20325\u2032-GGTTAAGCAGCGTCTCAACCGTAATCCAAAGGTTGCTGAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_44CP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3\u20325\u2032-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3\u20325\u2032-CCCGAATTCTAATCTCTTTCTAATTGGCT-3\u20325\u2032-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_fullCP1:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-CCAACTGCAACATCAACGCCACCGTACTGTTTGTTCAGAC-3\u20325\u2032-GTCTGAACAAACAGTACGGTGGCGTTGATGTTGCAGTTGG-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_29RED:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_34RED:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3\u20325\u2032-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_38RED:5\u2032-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3\u20325\u2032-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3\u20325\u2032-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3\u20325\u2032-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3\u20325\u2032-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3\u20325\u2032-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3\u2032TyrRS_39tt:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-GTGAGGCCCTCCAGCCACTCGGAATAAACATATTTTGCCTTTAAC-3\u20325\u2032-GTTAAAGGCAAAATATGTTTATTCCGAGTGGCTGGAGGGCCTCAC-3\u20325\u2032-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3\u20325\u2032-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032TyrRS_45tt:5\u2032-AAGGATCCACCATGGACGAATTTGAAATGAT-3\u20325\u2032-CTCGGAGTTGTAGCGGAGCTCAAATAACCCCATTGCTTCAAAAAC-3\u20325\u2032-GTTTTTGAAGCAATGGGGTTATTTGAGCTCCGCTACAACTCCGAG-3\u20325\u2032-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3\u20325\u2032-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3\u20325\u2032-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3\u2032.CUA gene were annealed, extended using a Klenow enzyme, and amplified by PCR. The sequences of oligonucleotides were:Two oligonucleotides for the 1bp-tRNA5\u2032-GAAGATCTGCGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCT AAATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCGCAGACAAGTGCGGTTTTTTT-35\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGCGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3\u2032CUA plasmid.The PCR product was then digested with the BglII and PstI restriction enzymes and ligated into a pre-digested pT7-H1 (described above) plasmid to create the 1bp-tRNACUA was created in a similar fashion using the following oligonucleotides to create the tRNA gene:H1_2bp-tRNA5\u2032-GAAGATCTGGGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCCCAGACAAGTGCGGTTTTTTT-3\u20325\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGGGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3CUA was generated using the following oligonucleotides:Finally, H1_3bp-tRNA5\u2032-GAAGATCTGGTGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCACCAGACAAGTGCGGTTTTTTT-3\u20325\u2032-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTG TCTGGTGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3\u2032.E. coli TyrRS was amplified from a plasmid by PCR using the following primers:A gene for 5\u2032-GCTCTAGATTATTTCCAGCAAATCAGACAGTA-3\u20325\u2032-CGGGATCCATGGCAAGCAGTAACTTGATTAAA-3\u2032E. coli TyrRS in HEK293T cells.The PCR product was digested with the XbaI and BamHI restriction enzymes  and ligated using T4 ligase into a pre-digested pEF6-V5 plasmid to express All new sequences of TyrRSs have been deposited in GenBank with the following accession numbers:TyrRS_36CP1: KF050826TyrRS_39CP1: KF050827TyrRS_39CP1_2: KF050828TyrRS_42CP1: KF050829TyrRS_44CP1: KF050830TyrRS_fullCP1: KF050831TyrRS_29RED: KF050832TyrRS_34RED: KF050833TyrRS_38RED: KF050834TyrRS_39tt: KF050835TyrRS_45tt: KF050836A BD FACSCalibur flow cytometer was used to gate approximately 10,000 cells, based on forward and side scatter. Each cell was then excited at a wavelength of 488 nm and the resulting emission was detected with an FL-1 filter (515\u2013545 nm). Positive fluorescent populations were gated based on negative or positive controls and analyzed using Cyflogic v.1.2.1 computer software ."}
+{"text": "The aim of this study was to investigate the presence of nerve growth factor (NGF) and its receptors tyrosine kinase A (TrkA) and p75 in the ovaries of the wild ground squirrels during the breeding and nonbreeding seasons. In the breeding period, NGF, TrkA and p75 were immunolocalized in granulosa cells, thecal cells, interstitial cells and luteal cells whereas in the nonbreeding period, both of them were detected only in granulosa cells, thecal cells and interstitial cells. Stronger immunostaining of NGF, TrkA and p75 were observed in granulosa cells, thecal cells and interstitial cells in the breeding season compared to the nonbreeding season. Corresponding for the immunohistochemical results, immunoreactivities of NGF and its two receptors were greater in the ovaries of the breeding season then decreased to a relatively low level in the nonbreeding season. The mean mRNA levels of NGF, TrkA and p75 were significantly higher in the breeding season than in the nonbreeding season. In addition, plasma gonadotropins, estradiol-17\u03b2 and progesterone concentrations were significantly higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, and TrkA and p75 were correlated with changes in plasma gonadotropins, estradiol-17\u03b2 and progesterone concentrations. These results indicated that NGF and its receptors, TrkA and p75 may be involved in the regulation of seasonal changes in the ovarian functions of the wild ground squirrel. The nerve growth factor (NGF) belongs to a family of related proteins required for the survival, maintenance, and development of discrete neuronal populations in the central and peripheral nervous systems -3. It isIt is now well known that NGF and its receptors are expressed in the mammalian ovary, including women -14, ratsCitellus dauricus Brandt) is a typical seasonal breeder which has a strict and extremely compressed breeding period  from April to May and a long period of sexual dormancy from June to the following March including a 6-month hibernation period [The wild ground squirrel . Wild female ground squirrels that were regarded as adults according to their body weights (242\u2013412\u00a0g) were captured on April 13 (10.2 hours of daylight) after emergence from hibernation in the breeding period (n\u2009=\u200910) and on June 9 (12.6 hours of daylight) in the nonbreeding period (n\u2009=\u20098) of 2009 in Hebei Province, PR China.Animals were anesthetized with 4% isoflurane and blood samples were rapidly collected from leg vein. Plasma samples were frozen and stored at -20C, after the blood samples were added heparin sodium and centrifuged . An overdose of pentobarbital was applied afterwards for euthanasia. Ovary and brain were quickly removed and dissected. Length, width and weight of each ovary were measured. A part of the tissues were fixed in 0.05\u00a0M phosphate-buffered saline  containing 4% paraformaldehyde for histological and immunohistochemical observation, while the rest were immediately frozen in liquid nitrogen and stored at -80C for RNA isolation and protein extraction.Ovarian samples were dehydrated in ethanol series and embedded in paraffin wax. Serial sections (4\u00a0\u03bcm) were mounted on slides coated with poly-L-lysine . Sections were stained with hematoxylin-eosin (HE) for observations of general histology. The sections were screened using an Olympus photomicroscope with a\u2009\u00d7\u200920 objective lens and imaged with software Image-Pro Plus 4.5 . Every one in ten serial sections, and altogether 50 and 30 sections for the breeding and nonbreeding season ovary respectively were selected for follicle identification [2O2. Finally, the sections were counterstained with hematoxylin  and NGF, TrkA and p75 were detected, respectively. The immunostained slides were scanned using the software Image-Pro Plus 4.5  at 20\u00d7 magnification. The specificity of NGF and its receptors, TrkA and p75 antibodies have been described previously [Ovarian sections were blocked with 10% normal goat serum to prevent the non-specific binding of the second antibody. The sections were then incubated with polyclonal primary antibody against NGF , TrkA  or p75   for 12\u00a0h at 4C, and incubated with the second antibody, goat anti-rabbit IgG conjugated with biotin and peroxidase with avidin for 1\u00a0h at room temperature. The sections were visualized using a rabbit ExtrAvidin\u2122 staining kit  in 150\u00a0ml of 0.05\u00a0M Tris\u2013HCl buffer containing 30\u00a0mg 3,3-diaminobenzidine  plus 30\u00a0\u03bcl Heviously . The imm2O2 and visualized with Odyssey infrared imaging system. Brain tissue of wild ground squirrel was used as a positive control and water, instead of primary antisera, was used as a negative control. \u03b2-actin was selected as the endogenous control. The intensities of the bands were quantified using Quantity One software  and expression ratios were calculated.Ovarian tissues were weighed and dissected into small pieces using a clean razor blade. The tissues were homogenized in a tissue homogenizer containing 300\u00a0\u03bcl of 10\u00a0mg/ml PMSF and incubated for 30\u00a0min on ice. Homogenates were centrifuged at 12,000\u00a0g for 10\u00a0min at 4C. Protein extracts (25\u00a0\u03bcg) were mixed with equal volumes of 2\u2009\u00d7\u2009Laemmli sample buffer. Equal amounts of proteins from each sample were loaded onto a 12% SDS-PAGE gel and electrophoretically separated at 18\u00a0V/cm and transferred to nitrocellulose membrane using a wet transblotting apparatus . The membrane was blocked in 3% BSA for 1\u00a0h at room temperature. Primary incubation of the membrane was carried out using NGF, TrkA or p75 antibody (1:1000 dilution) for 1\u00a0h at room temperature. Secondary incubation of the membrane was then carried out using an IRDye  for 1\u00a0h at room temperature. Finally, the membrane was washed in 25\u00a0ml Tris-Buffered Saline with Tween-20  plus 3\u00a0\u03bcl HTotal RNA from each sample was extracted using ISOGEN . Approximately 1\u00a0g of ovarian tissues were thawed and immediately homogenized in 10\u00a0ml of ISOGEN\u2122. The homogenate was incubated for 5\u00a0min at room temperature to allow the complete dissociation of nucleoprotein complexes. After the addition of 2\u00a0ml of chloroform, the mixture was vigorously shaken for 3\u00a0min at room temperature and centrifuged at 12,000\u00a0g for 10\u00a0min at 4C. The aqueous phase was then transferred to a fresh tube and washed with an equal volume of chloroform. An equal volume of isopropanol was added, and the sample was kept for 10\u00a0min at room temperature. RNA was precipitated by centrifugation at 12,000\u00a0g for 10\u00a0min at 4C. The RNA pellet was washed twice with 75% ethanol, briefly dried under air, and dissolved in 100\u00a0\u03bcl of diethylprocarbonate-treated water.12\u201318 according to the manufacturer\u2019s protocol. The 20\u00a0\u03bcl of reaction mixture contained 4\u00a0\u03bcg of total RNA, 0.5\u00a0\u03bcg of oligo (dT)12\u201318, 2.5\u00a0mM MgCl2, 0.5\u00a0mM dNTP, 10\u00a0mM dithiothreitol, 20\u00a0mM Tris\u2013HCl (pH\u00a08.4) and 200 U of Superscript II enzyme. The first-strand cDNA was used for PCR amplification with the appropriate primers previously proved  and 2.5 U of Taq polymerase . The amplification was under the following condition: 94C for 5\u00a0min for the initial denaturation of the RNA/cDNA hybrid, 30 cycles of 94C for 1\u00a0min, 52C for 1\u00a0min, and 72C for 2\u00a0min for amplification. The PCR product was electrophoresed in the 2% agarose gel and individual bands were visualized by ethidium bromide staining. Brain tissue of wild ground squirrel was used as positive control and water, instead of cDNA, was used as negative control. The housekeeping gene, RpL7, was selected as the endogenous control as it is an estrogen-independent gene. The bands were quantified using Quantity One software  and expression ratios were calculated.The first-strand cDNA from total RNA was synthesized using Superscript II Reverse Transcriptase  and oligo (dT)E. coli using TOPO TA Cloning Kit . Plasmids were extracted from the bacteria and positive clones containing the proper insert were sequenced in both directions using Thermo Sequenase II Dye Terminator Cycle Sequencing Premix Kit  with an automatic sequencing system . After obtaining the sequence of each PCR product, we blasted with the known mRNA sequences of mouse , rat , bovine  and human , find the homologous sequence fragments in each species and compare for homology using DNAman.The purified PCR products were ligated into pCR 2.1-TOPO  and the ligation products were used to transform the competent 125I-labeled radioligands as described previously [Plasma concentrations of estradiol-17\u03b2 and progesterone were determined by double-antibody RIA systems using eviously . Antisereviously  and progeviously  was kindMeans and standard deviations were calculated. Data were analyzed using a one-way ANOVA and the means were compared for significance using Duncan\u2019s Multiple Range Test (P\u2009=\u20090.05) using the SPSS computer software package.Ovaries of breeding and nonbreeding seasons were observed morphologically and histologically Figure\u00a0. In lineImmunohistochemistry was performed to detect the localization pattern of NGF and its receptors in the wild ground squirrel ovary and representative stainings were shown in Figure\u00a0We then moved on to detect the protein and mRNA expression levels of NGF, TrkA and p75 using Western blot and PCR, and representative bands were shown in Figure\u00a0To further confirm the nature of the PCR signals, cDNA fragments of NGF, TrkA and p75 in ovarian tissues were sequenced and compared to the corresponding fragments in mouse, rat, bovine, and human. The partial mRNA sequences in the wild ground squirrel are as below:NGF#GGGGGACTCAGTGTGTGTGCTGGTGTCAGTGTGTGGGTTGGAGATAAGACCACAGCCACAGACATCAAGGGCAAGGAGGTGACAGTGCTGGCCGAGGTGAACATTAACAACAGTGTATTCAGACAGTACTTTTTTGAGACCAAGTGCCGAGCTrkA#GGAAGTGACATCTCTACCGCAGTTCAGCACCGAGAGCGATGTGTGGAGCTTTGGGGTGGTGCTCTGGGAGATCTTCACCTATGGAAAGCAGCCCTGGTACCAGCTCTCTAACACTGAGGCGATCGAGTGTATCACGCAGGGCCGGGAGCTGGAGCGGCCGCGCGCCTGCCCTCCTGATGTCTACGCCATCATGCGAGGCTGCTGGCAGCGAGAACCGAATCAACGCCTP75#CCAGTAGGGCAGTGTGGCGGAGCCTTGCGGAGCCATCCAGACCGTGTGTGAACCCTGCCTGGACAGTGTTACGTTCTCTGACGTGGTGAGCGCCACCGAGCCGTGCAAGCCGTGCACCGAGTGCCTGGGCCTGCAGAGTATGTCCGCTCCCTGTGTGGAGGCAGACGATGCCGTGTGCCGATGCTCCTATGGCTACTACCAGGACGAGGAGACTGGCCGCTGCGAGGCTTGCAGCGTGTGCGGGGTGGGCTCAGGATCGGTGTCCTCCCRpL7#AAGGATCTGCTGCTGCTTCTGTTCCAGATCTCAATGGCACCTTTGTTAAGCTCAACAAGGCTTCAATTAACATGCTGCGGATTGTGGAGCCATACATTGCATGGGGGTACCCCAACCTGAAGTCAGTAAACGAGCTCATCTACAAGCGAGGCTACGGCAAAATCAACAAGAAGCGGATTGCCTTGACAGATAATTCCTTGAATGCACGGTCTCTTThe 176-bp NGF cDNA nucleotide sequence identity was 92.11%, 90.20%, 81.70% and 86.18%, respectively; the 261-bp TrkA cDNA nucleotide sequence identity was 94.32%, 90.83%, 84.72% and 83.41%, respectively; the 295-bp p75 cDNA nucleotide sequence identity was 94.42%, 88.10%, 84.01% and 83.64%, respectively; the 207-bp RpL7 cDNA nucleotide sequence identity was 94.55%, 84.65%, 74.23% and 76.87%, respectively  that involves both sex steroids and growth factors.The authors declare that they have no competing interests.BL participated in performing the experiments, analyzing the data and drafting the manuscript. XS, LB, SH, QL and YL assisted with sample collection, all experiments and helped revising the manuscript. YH and QW designed, supervised the study, and revised manuscript. GW and KT provided some reagents and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA.Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition. Regions were defined by using SICER (v.1.03). Only regions that were called by using both input and 'no antibody' as a background control with Benjamini corrected false discovery rate < 0.05 were kept. Finally, only regions called in both antibody hmeDIP-seq experiments were kept and analyzed. Gene ontology analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT) . PublishFully hydroxymethylated DNA was produced by endpoint PCR using Phusion polymerase  and hm-dCTP  followed by PCR purification . Unmethylated and fully methylated control DNAs were produced in the same manner with dCTP and m-dCTP , respectively. Various amounts of DNA were denatured, snap cooled and dotted onto positively charged nylon membranes . Membranes were crosslinked, blocked with 5% milk, and incubated with Active Motif  5-hmC antibody for 1 hour. Membranes were washed and then incubated with anti-rabbit secondary horseradish peroxidase-linked antibody  for 1 hour, washed, and developed with ECL reagent (CST) and Biomax MS film (Kodak). DNA sequences were (primer sequences in bold):TACTCTATACTCTACTCATCATTACACGCGCGATATCGTTAACGATAATTCGCGCGATTACGATCGATAACGCGTTAATATGAGATATGAGATGTGTATG; 6 CG-TACTCTATACTCTACTCATCATTACAATATATATATCGTTAACGATAATTCGCGCGATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 3 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTCGCGAAATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 1 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTAACGAAATTATAATTTATAATTAATTAATATGAGATATGAGATGTGTATG.12 CG-Human stem cell genomic DNA 5 to 10 \u03bcg) was treated with the EpiMark 5-hmC and 5-mC Analysis Kit as per the included protocol (NEB). Briefly, DNA was either glucosylated with beta-glucosyltransferase and UDP-Glc or mock treated with beta-glucosyltransferase and no UDP-Glc for 12 to 18 hours. These reactions were then split into three and mock digested, digested with MspI, or with HpaII for at least 4 hours. Samples were treated with proteinase K that was then heat inactivated. All DNA were diluted to a final concentration of 16 ng/\u03bcl to be used for PCR analysis. Quantitative PCR was completed with iQ SYBR Green Supermix  using a CFX384 Real-Time PCR Detection System (Biorad). Primers used for quantitative PCR are listed in Table S1 in Additional file 0 \u03bcg was 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; bp: base pair; ESC: embryonic stem cell; H3K4me1: histone H3 mono-methylated at lysine 4; H3K27ac: histone H3 acetylated at lysine 27; hESC: human embryonic stem cell; hmeDIP: hydroxymethyl-DNA immunoprecipitation; RPKM: reads per kilobase per million mapped reads; TFBS: transcription factor binding site.HS, SP and SEJ designed the study. SF and SMK performed the experiments. HS and SEJ analyzed the data. HS wrote the paper.Supplementary figures and table. Figure S1: 5hmC dot blots on oligos with varying amounts of 5hmC. Figure S2: characterization of defined 5hmC peaks. Figure S3: correlation of 5hmC and gene expression. Figure S4: 5hmC and different classes of putative enhancers [nhancers . Figure nhancers . Figure nhancers ,14,15. FClick here for file"}
+{"text": "L26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.The human cytomegalovirus (HCMV) U Human cytomegalovirus (HCMV), a betaherpesvirus, is a widespread opportunistic pathogen. HCMV causes severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients L32 and UL99 HCMV is a relatively large virus, with a \u223c240-kb DNA genome that encodes >200 open reading frames. The viral particle is enveloped and its genome is encased within a protein capsid. Packaged in between the capsid and the viral envelope is a protein layer called the tegument, a structural feature unique to herpes viruses L26 gene, which has been found to be critical for high-titer viral replication L26 protein is expressed with early kinetics, and synthesis of the protein initiates at one of two start codons resulting in 21- or 27-kDa products L26 deletion grow to lower final titers, with slower growth kinetics, and exhibit a small plaque phenotype L26 has been implicated in transcriptional activation of the immediate early promoter L26 also impacts the structural characteristics of virions. These mutants are less stable than wildtype virions and contain hypophosphorylated tegument constituents L26 protein localizes to the nucleus at early times post infection, and to viral assembly compartments at late time points While many HCMV tegument proteins are known to be important for HCMV replication, the mechanisms through which many of these proteins contribute to the infectious cycle are unclear. One such tegument protein is encoded by the UL26 that contribute to UL26-dependent phenotypes through the creation of a panel of mutant UL26 viruses. Site-directed mutagenesis was employed to target both of UL26\u2019s initiation methionines and to introduce stop codons throughout the UL26 ORF. Analysis of these mutant viruses indicates that the UL26 short isoform is dispensable in vitro when in the presence of the UL26 long isoform. In contrast, the extra N-terminal 34 amino acids of the long UL26 isoform was found to be important for UL26-dependent phenotypes, exhibiting intermediate defects in plaque size and virion stability in comparison to wildtype and UL26-null viruses. Lastly, the carboxy terminal 38 amino acids were found to be critical for wildtype replication as deletion of this domain phenocopied UL26-deletion viruses. Deletion of these of 38 amino acids reduced the nuclear localization and tegumentation of the resulting UL26 protein product. These studies indicate that distinct domains of UL26 contribute to different UL26-dependent phenotypes and shed light on how these domains contribute to HCMV replication.Here, we analyzed specific domains of UBAdwt, a bacterial artificial chromosome (BAC) clone of Ad169 4 cells per cm2. Prior to infection, cells were serum starved for 24 hours. In all infections, viral innocula were added to cells for a 2 hr adsorption period and then aspirated. For experiments involving measurement of viral titers via plaque assay, unbound virus was inactivated through a sodium citrate wash  followed by a DMEM wash immediately following viral adsorption.MRC5 fibroblasts (passages 23\u201329) were cultured in Dulbecco\u2019s modified Eagle medium  supplemented with 10% fetal bovine serum. The wild type HCMV strain used in this study was L26 mutants were derived from the BAdwt clone of Ad169 (Genebank accession number: FJ527563) L26 mutants constructed are: BAdUL26 double methionine deletion (referred to as DBmet\u0394 in the text); BAdUL26 1st Methionine deletion (referred to as 1stmet\u0394 in the text); BAdUL26 2nd Methionine deletion (referred to as 2ndmet\u0394 in the text); BAdUL26 double methionine deletion rescue (referred to as DBrescue in the text); BAdUL26 #68 stop codon mutant (referred to as #68stop in the text); BAdUL26 #107 stop codon mutant (referred to as #107stop in the text); BAdUL26 #146 stop codon mutant (referred to as #146stop in the text); BAdUL26 #185 stop codon mutant (referred to as #185stop in the text). Wild type BAdwt is referred as WT in the text and BAdUL26 transposon insertion virus L26 flanking sequences was recombined into BADwt through electroporation into E. coli (strain SW105) containing BAdwt. Recombination was screened by growth in kanamycin. The Kan/Isce I cassette containing BAC was then electroporated into GS1783 cells, which contain an arabinose-inducible I-Sce 1 restriction site used for negative selection L26 gene. The UL26 DBmet\u0394 mutant was created by deletion of the 2nd Methionine from the UL26 1stmet\u0394 mutant. The primers for generating mutant viruses via two sequential PCR reactions (first reaction to introduce Kan/Isce I cassette and second reaction to insert the point mutation) were as follows (5\u2032 to 3\u2032): UL26 1stmet\u0394 insertion:F-GGCCCTCGGTGCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCAll UTTCATAGGATGACGACGATAAGTAGGG; R-GCGGCTTCATGTGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACAACCAATTAACCAATTCTGATTAG;stmet\u0394 Met to ATCUL26 1: F -GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATCTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA;R\u2013TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTAGATGAAGACGCTCAAACTTTTGAATGTGGGCCC GGTAGCGC; stmet to ATC negative controlUL26 1:F-GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATGTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA; R- TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACATGAAGACGCTCAAACTTTTGAATGTGGGCCCGGTAGCGC;ndmet\u0394 insertionUL26 2:F-GCGGCGCGTTATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATAGGATGACGACGATAAGTAGGG;R- CGCATAAAATCGTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCAACCAATTAACCAATTCTGATTAG; ndmet\u0394 Met to ATCUL26 2: F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATCACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTGATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; ndmet to ATC negative controlUL26 2:F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATGACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; UL26 DBmet\u0394: F-CGCCGTTTTCGGCCTCACGAG; R-GGTGCCGATGACGCGCAACTG. The single step PCR process employs only a single set of primers that contain homology sufficient for both recombination events UL26#185stop:F-CACGGTGACGTAGCAGCACGCGGCTCACGTAGCAGGCCGATTAGCGGATGACCTGGCCGTCGGAGGATGACGACGATAAGTAGGG;R-CTCGGGCCTGCGACGCGACGCCGACGGCCAGGTCATCCGCTAATCGGCCTGCTACGTGAGCCGCAACCAATTAACCAATTCTGATTAG; UL26#146stop:F-GCTCCACGTCTTCAAAGTAGCTGTGTAGCAGGCCGCGCTCTTACAGCTGCGGCAGCGAGTCGGAGGATGACGACGATAAGTAGGG; R-GAACTTTGTAGTGCGCGCCGCCGACTCGCTGCCGCAGCTGTAAGAGCGCGGCCTGCTACACAGCAACCAATTAACCAATTCTGATTAG; UL26#107stop:F \u2013CGGCCGCCACGCCGGCCACGCTGCGGTCCCAACTGAAAAGTTAGGCGAGTCCGATGGTGCCGAAGGATGACGACGATAAGTAGGG; R-CCAGGGTCAGTTGCGCGTCATCGGCACCATCGGACTCGCCTAACTTTTCAGTTGGGACCGCAGCAACCAATTAACCAATTCTGATTAG; UL26#68stop:F-GCGGGGTGAGGATGGTCTCCTCCACGTCGCAGACAAACAATTAGTAGCCGCGCGGATAGGGCAAGGATGACGACGATAAGTAGGG; R-GCGCGGTCGCCACCTGGATCTGCCCTATCCGCGCGGCTACTAATTGTTTGTCTGCGACGTGGACAACCAATTAACCAATTCTGATTAGL26 [7H19] , PP28 [10B4\u201329], UL83-coded pp65 (8F5) and pUL44  and cellular protein tubulin [Epitomics]. A rabbit UL26 N-terminal specific antibody was generated by Biomatik (http://www.biomatik.com/) using the following underlined sequence: MTSRRAPDGGLNLDD. The methionine preceding this sequence is the 2nd UL26 initiation methionine. The secondary antibodies were rabbit polyclonal  and mouse monoclonal [Abcam].Protein accumulation was assayed by Western blotting. Protein from cell lysates was solubilized in disruption buffer , separated by either 10% or 15% SDS-PAGE, and transferred to nitrocellulose in Tris-glycine transfer buffer. Blots were then stained with Ponceau S to visualize protein bands and ensure equal protein loading. The membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 (TBST), followed by incubation in primary antibody. After subsequent washes, blots were treated with secondary antibody and protein bands were visualized using the enhanced chemiluminescence (ECL) system (Pierce). The primary antibodies were specific for viral proteins UL123-coded IE1 [1B12], UViral DNA accumulation was monitored by real-time PCR. At various times post infection, medium was aspirated from cells and viral DNA was harvested in lysis buffer . The extracted nucleic acid was quantified and checked for purity through 260\u2236280 absorbance by NanoDrop. Quantitative PCR (qPCR) was performed using Fast SYBR green master mix, a model 7500 Fast real-time PCR system, and Fast 7500 software (Applied Biosystems) according to manufacturer\u2019s instructions. Viral DNA was quantified with specific primer pairs targeting UL83 (pp65), 5\u2032-CAG-GAA-GAT-TTG-CTG-CCC-GTT-CAT-3\u2032 (forward) and (5\u2032-GGC-TTT-ACG-GTG-TTG-TGT-CCC-AAA-3\u2032 (reverse).For immunofluorescence, MRC5 fibroblasts were grown on glass coverslips. At various time points post infection, cells were washed once with PBS, fixed with 2% paraformaldehyde in PBS for 20 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 and 0.1% SDS for 15 min, then washed twice with PBS containing 0.05% Tween-20. Cells were subsequently blocked by overnight incubation in PBS containing 2% bovine serum albumin (BSA), 5% goat serum, 5% human serum, and 0.3% Triton X-100. Cells were incubated with anti-UL26 sera [7H19] that had been diluted 1\u22362 in PBS containing 0.05% Tween-20 for 1 hr. Slides were subsequently washed with PBS containing 0.01% Tween-20 three times, incubated with fluorochrome-conjugated anti-mouse secondary antibody for 1 hr, and washed three times in the same buffer lacking antibody. Coverslips were mounted in slow-fade Gold antifade reagent (Molecular Probes) and DAPI . Confocal images were captured with FV1000 Olympus laser scanning confocal microscope. All images were captured under identical confocal settings.L26 and anti-pp65-specific western to ensure the linearity of UL26 and pp65 detection. Quantitation of UL26 and pp65-specific protein bands was performed using ImageLab software tools from BioRad.To produce partially purified virions for the analysis of their constituent proteins, WT and #185stop virus stocks were first clarified by low speed centrifugation and then centrifuged through a sorbitol cushion at 26K rpm for 1 hr. The virion pellet was then resuspended in T.N. buffer containing 20 mM Tris-HCl, PH 7.4, 100 mM NaCl, and 1.5% BSA and purified by centrifugation through a glycerol tartrate gradient as previously described Replicate cultures of MRC5 fibroblasts were infected with 25 PFU of the indicated recombinant virus. Representative plaques at day 15 post infection for each virus are shown. Areas of representative plaques for each virus were quantified by Image J and normalized to the WT plaque size. To investigate the stability of virion infectivity, an equivalent number of plaque forming units from freshly thawed viral stocks were incubated at 37\u00b0C for 0, 4, 8, or 20 hours. After the indicated incubation period, confluent MRC5 fibroblasts were infected. The percentage of plaques remaining relative to the 0 h control was plotted.To investigate virion stability after trypsin exposure, infected MRC5 cells (MOI\u200a=\u200a3.0) were harvested when the CPE reached 80%. The media containing infectious virus was reserved, and cells were scraped in a small volume of media and sonicated. The sonicated cells and reserved culture media were combined and centrifuged at 6,000 rpm for 30 min. The supernatants were then sedimented at 38,000\u00d7g for 60 min. The pellets containing virus were resuspended in serum-free minimal essential medium. Two hundred \u00b5l of either 2.5% trypsin (Invitrogen) or media was mixed with 1.8 ml of the resuspended virus, and incubated at 37\u00b0C for 30 or 60 minutes for the trypsin treated, or 0 minutes for the media control. To inactivate the trypsin, at the end of the prescribed intervals, calf serum was added to a final concentration of 10%. The suspension was then tittered by plaque assay. The percentage of plaques remaining relative to the media control was plotted.t-test unless otherwise indicated. A probability of value (p) <0.05 was considered statistically significant. For comparison of the viral growth between wildtype and the #185stop mutant from 48\u2013120 hpi a homoscedastic paired two-tailed ttest of viral titers at each time point was performed. Averages are plotted with either standard deviation (SD), or standard error of the mean (SE) as indicated.Statistical significance was assessed by a non-paired two tailed homoscedastic student\u2019s L26 gene have been shown to be growth attenuated L26 protein is expressed from a spliced mRNA transcript that is also responsible for the expression of the UL29, UL28, UL27, and UL29/28 open reading frames L26-null viruses contain large deletions within the UL26 open reading frame which could impact the expression of the other open reading frames that are expressed from this mRNA transcript. Further, it is unclear how the two separate isoforms of the UL26 protein contribute to HCMV infection. To address these issues, and to map the domains of UL26 that impact viral replication, we employed BAC-mediated recombineering to create a panel of viruses containing site-directed UL26 mutations. This panel included viruses containing a mutation ablating one or both initiating methionines as well as viruses containing stop codon insertions throughout the UL26 open frame.Viruses containing deletions in the UL26 protein is illustrated in L26 reading frames that could be stably expressed, stop insertions were made at locations that approximated transitions between these predicted domains , nor did the previously described transposon-deleted UL26 virus (UL26TI)  and were not analyzed in further detail. The specific mutations of the remaining recombinant viruses, and the UL26 ORFs they produce, are illustrated in A plot of manavalan hydrophobicity  domains . To veri isoform . Further(UL26TI) . The repisoforms . The stoL26 protein impacts production of viral progeny, we wanted to elucidate how specific domains of the UL26 protein contribute to viral replication. We first wanted to assess whether the double methionine deletion mutant (DBmet\u0394), grew with similar kinetics as the transposon insertion mutant (UL26TI). As shown in L26 ORF does not substantially impact the in vitro viral growth over and above what is observed with less disruptive targeting of the UL26 initiation methionines.As it has previously been found that deletion of the UL26 protein contribute to viral growth, we analyzed the replication of the individual methionine mutants and the #185 stop insertion mutant. At high MOI, the 2ndmet\u0394 virus, which only expresses the long isoform of UL26   . The #18(DBmet\u0394) . Similarl titers . Interesl titers . The comL26 is important for wildtype levels of IE1 accumulation L26 domains on viral gene expression, we analyzed the accumulation of viral proteins during infection. Upon infection at an MOI\u200a=\u200a3.0, both UL26-null viruses, UL26TI and DBmet\u0394, accumulated less IE1 at 4 hpi, but recovered by 24 hpi , viral DNA accumulated similarly between the WT and 2ndmet\u0394  from our panel of mutants, overlaid with agarose and incubated at 37\u00b0C for 15 days. Images of the resulting plaques were captured with subsequent analysis of the area of each plaque. Images of representative plaques are shown in L26-null viruses. The plaques produced by the 1stmet\u0394 virus were intermediate in size between the UL26-null viruses and WT HCMV, whereas the 2ndmet\u0394 virus produced plaques of WT size. These results indicate that the C-terminal 38 amino acids of the UL26 protein, and to a lesser extent, the N-terminal 34 amino acids are important for WT plaque size.We and others have observed that deletion of the UL26 protein is important for virion stability, inasmuch as prolonged incubation at 20\u00b0C causes UL26-defective virions to lose their ability to initiate infection faster than WT virions do. L26 mutations impact virion stability, viral supernatants containing equivalent PFUs were incubated at 37\u00b0C for various times and then plated. The percentage of plaques remaining after incubation in comparison to control was plotted in L26-null viruses demonstrated a \u223c50% drop in infectivity, a statistically significant difference . This suggests that large differences in virion protein constituents are likely not responsible for the decreased stability of mutant UL26 viruses. Another major question is the function of UL26 in the nucleus at early times. It seems likely that this nuclear UL26 is responsible for impacting IE1 gene expression at early times, although the potential mechanism involved still needs to be elucidated. Our identification of the important UL26 N-terminal and C-terminal domains will facilitate addressing these questions. The C-terminal 38-amino acids of UL26 are important for proper UL26 tegumentation, nuclear localization, and viral replication. Our studies highlight the importance of these C-terminal 38-amino acids for future study. Further functional analysis will distinguish how the specific residues within this domain contribute to UL26 nuclear localization and proper tegumentation, and subsequently to HCMV replication. Given its importance to HCMV infection, elucidating the mechanisms through which UL26 domains contribute to high-titer replication may shed light on possibilities for therapeutic intervention.A number of questions remain about U"}
+{"text": "Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools\u2014referred to as siPools\u2014containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects. RNA interference (RNAi) is not only a potent cellular pathway to silence endogenous or exogenous genes but is also a widely used tool for sequence-specific gene knockdown . The triThe core protein component of RISC and direct interaction partner of siRNAs is a member of the Argonaute (Ago) protein family. Ago proteins are characterized by PAZ, MID and PIWI domains . The PAZAfter an initial hype and the hope that RNAi develops into a potent novel strategy for therapy, it became clear that several problems associated with RNAi experiments have not been solved. Besides delivery issues in RNAi-based therapeutic approaches, one of the major problems of siRNAs are off-target effects ,21. MostHere, we report the development of a new generation of siRNA tools without off-target effects. Using an enzymatic approach, we generate complex pools of accurately defined siRNAs. While synergistically silencing one single on-target gene, each individual siRNA is present at very low concentrations, effectively diluting off-target effects below detection limits. The novel enzymatic strategy allows for the free combination of individually selected siRNA sequences to obtain optimal siRNA pools. We refer to our novel siRNA pools as siPools and overcome off-target effects, the major shortcoming of classical siRNA reagents.in vitro transcribed using an integrated T7 promoter sequence followed by RNA precipitation with 2.5 M LiCl. Sense and antisense strands were annealed by incubation at 95\u00b0C for 5 min followed by slow cooling to room temperature. Annealed dsRNAs were subsequently digested with 10 U/\u03bcg RNase T1 to obtain ds siRNAs with 2 nt 3\u2032 overhangs. RNase T1 digested siRNAs were purified by native 20% polyacrylamide gel electrophoresis and eluted from the gel overnight at 4\u00b0C in elution buffer  followed by ethanol precipitation. To obtain siPools with 30 siRNAs, siPool #1 was mixed with siPool #2, and to generate siPool 45 with 45 different siRNAs, siPool #1 was mixed with siPool #2 and #3. siPool 60 containing 60 different siRNAs was mixed by the use of all four different siPools #1, #2, #3 and #4. TNRC6A, TNRC6B, TNRC6C and unspecific control siPools (neg. pools) each comprised of 30 individual siRNA sequences. For all template and siRNA sequences, see Supplementary Material. Ready-to-use siPools against various genes were purchased from siTools Biotech, Munich, Germany.DNA templates were synthesized by GeneArt (Life Technologies). SiPool templates were Antisense strands of siRNA sequences were depicted: PolG siRNA off-T: 5\u2032-AAUAUCCAGCGCUUCACCC-3\u2032, Scyl1 siRNA off-T: 5\u2032-ACAUUGUUGUGGAUGAGGC-3\u2032; Mad2 siRNA: 5\u2032-CCAAUCUUUCAGUUGUUCC3\u2032; neg. ctrl. siRNA: 5\u2032-UUGUCUUGCAUUCGACUAA-3\u2032. esiRNAs (Sigma) were delivered as RNase III digested double-stranded products derived from the following sequences, PolG esiRNA: 5\u2032-GGAAGAAGTGGGAGGTGGTTGCTGAACGGGCATGGAAGGGGGGCACAGAGTCAGAAATGTTCAATAAGCTTGAGAGCATTGCTACGTCTGACATACCACGTACCCCGGTGCTGGGCTGCTGCATCAGCCGAGCCCTGGAGCCCTCGGCTGTCCAGGAAGAGTTTATGACCAGCCGTGTGAATTGGGTGGTACAGAGCTCTGCTGTTGACTACTTACACCTCATGCTTGTGGCCATGAAGTGGCTGTTTGAAGAGT-3\u2032, Scyl1 esiRNA: 5\u2032-CAGCCGAGAAGCAAAAATTCTTCCAGGAGCTGAGCAAGAGCCTGGACGCATTCCCTGAGGATTTCTGTCGGCACAAGGTGCTGCCCCAGCTGCTGACCGCCTTCGAGTTCGGCAATGCTGGGGCCGTTGTCCTCACGCCCCTCTTCAAGGTGGGCAAGTTCCTGAGCGCTGAGGAGTATCAGCAGAAGATCATCCCTGTGGTGGTCAAGATGTTCTCATCCACTGACCGGGCCATGCGCATCCGCCTCCTGCAGCAGATGGAGCAGTTCATCCAGTACCTTGACGAGCCAACAGTCAACACCCAGATCTTCCCCCACGTCGTACATGGCTTCCTGGACACCAACCCTGCCATCCGGGAGCAGACGGTCAAGTCCATGCTGCTCCTGGCCCCAAAGCTGAACGAGGCCAACCTCAATGTGGAGCTGA-3\u2032.To generate low-complexity pools, the following siRNAs were used: PolG siRNA 1: 5\u2032-UCAUCCGACAGCCGAUACC-3\u2032, PolG siRNA 2: 5\u2032-AAUUCUUGCAGGUCCCACU-3\u2032, PolG siRNA 3: 5\u2032-GCUAUUACCAUCCUUGUGA-3\u2032, PolG siRNA 4: 5\u2032-UUAAACUGCAUUAGUAAGC-3\u2032. Scyl1 siRNA 1: 5\u2032-UUUCUCAGGAUCUACAGUGAG-3\u2032, Scyl1 siRNA 2: 5\u2032-UUGAGGUAUAUUCCCAACGGG-3\u2032, Scyl1 siRNA 3: 5\u2032-UUGGUUUCUACAAAGCGGUUG-3\u2032, Scyl1 siRNA 4: 5\u2032-UUGUACAAUAAAUACAUCUGU-3\u2032. Three siRNAs were mixed in different combinations with the corresponding siRNA off-T to obtain four different siRNA-containing low-complexity pools with possible Mad2 off-target effects. For pool #1, siRNA 1, 2, 3; for pool #2, siRNA 1, 2, 4; and for pool #3, siRNA 1, 3 and 4 were mixed with off-T siRNAs. Low-complexity pools consisting of siRNA 1\u20134 were used as non-Mad2 off-target controls (pool #4).HeLa cells were cultivated in Dulbecco's modified Eagle's medium substituted with 10% FCS  and penicillin/streptomycin. siRNA transfections were done using Lipofectamine RNAiMax (Life Technologies) according to the manufacturer's protocol. Cells were harvested 24 h or 48 h after transfection.RNA was isolated 24 h after transfection followed by cDNA synthesis and quantitative polymerase chain reaction (qPCR). Following primers were used: PolG for: 5\u2032-TTCCAGGACCTGATGCAGTA-3\u2032, PolG rev: 5\u2032- ACAGGCAGGTAGGAGACACC-3\u2032, Scyl1 for: 5\u2032-CTGGAGGAAGTGGAGAAGGA-3\u2032, Scyl1 rev: 5\u2032-TCAGCTTGGAGGTGAGTGAG-3\u2032, Mad2 for: 5\u2032-AGATGACAGTGCACCCAGAG-3\u2032, Mad2 rev: 5\u2032-TCCAACAGTGGCAGAAATGT-3\u2032 GAPDH for: 5\u2032-ATGGGTGTGAACCATGAGAA-3\u2032, GAPDH rev: 5\u2032-GTGCTAAGCAGTTGGTGGTG-3\u2032, TNRC6A for: 5\u2032-CCCTCAGGTTCCAGTTTCAT-3\u2032, TNRC6A rev: 5\u2032-GCTGGTTTAGCTGGGATAGC-3\u2032, TNRC6B for: 5\u2032-ATGGTTCTGGCTTCAGCTCT-3\u2032, TNRC6B rev: 5\u2032-CATATTGGCTTCCTGTGTGG-3\u2032, TNRC6C for: 5\u2032-TAGCAAGCATGGTGCTATCC-3\u2032, TNRC6C rev: 5\u2032-GTACCGGCCATAGGAGTCAT-3\u2032, OAS1 for: 5\u2032-TGCCATTGACATCATCTGTG-3\u2032, OAS1 rev: 5\u2032-GAGCCACCCTTTACCACCT-3\u2032, INFB1 for: 5\u2032-AGTAGGCGACACTGTTCGTG-3\u2032, IFNB1 rev: 5\u2032-GAAGCACAACAGGAGAGCAA-3\u2032, IL6 for: 5\u2032-AATGAGGAGACTTGCCTGGT-3\u2032, IL6 rev: 5\u2032-GCAGGAACTGGATCAGGACT-3\u2032, STAT1 for: 5\u2032-AATTGACCTCGAGACGACCT-3\u2032, STAT1 rev: 5\u2032-CACATGGTGGAGTCAGGAAG-3\u2032.For western blot analysis cells were harvested and lysed in NET buffer  48 h after transfection. Proteins were separated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by semi-dry electroblotting. Following antibodies were used: polyclonal anti-MAD2 (Bethyl Laboratories) at a dilution of 1:5000 and a monoclonal mouse anti beta actin antibody (clone AC15 from Abcam) at a dilution of 1:5000 in TBS-Tween with 5% milk powder. Fluorescently labeled IRDye 800 CW antibodies were used as secondary antibodies (Li-COR). Western blots were imaged with an Odyssey Fluorescence scanner (Li-COR).To generate an off-target reporter construct, a modified pMIR dual luciferase reporter plasmid  was usedHeLa cells were transfected with 10 nM siPools and lysed in NET buffer  48 h after transfection. Lysates were used for Ago2-siRNA co-immunoprecipitation. Protein-G sepharose beads (GE) were pre-incubated with monoclonal anti-Ago2 (11A9) antibody . LysatesHeLa cells were transfected with 3 nM Scyl1 siRNA, Scyl1 siPool 15, Scyl1 siPool 60, or mock transfected in three biological replicates. RNA was isolated 48 h after transfection and further processed for Affymetrix Human Gene 1.0 ST array analysis. The Human Gene 1.0 ST array platform from Affymetrix  was used to assess transcriptome-wide expression profiles. Normalization of raw intensity values from CEL files was performed using variance stabilization , and thelimma , or mock transfected in three biological replicates. RNA was isolated 48 h after transfection. Transcription profiles were analyzed using the Human Gene 2.0 ST array platform (Affymetrix). Raw intensity values (from CEL files) were normalized using RMA (oligo ). The nsanalyzeR . All logAnalyses were performed within the statistical programming environment R and using Bioconductor  packagesP-value for the number of repressed transcripts with a seed match for the Scyl1 siRNA to belong to the same distribution. For the pool 15 and pool 60, three additional siRNA seed sequences were drawn from the pools and tested analogously.Human 3\u2032-UTR sequences were retrieved from Ensembl version 68 for the transcripts represented on the cartridge microarray. The reverse compliment of the siRNA seed sequences (7 nt) was searched for in the 3\u2032-UTRs of the transcripts and 2292 transcripts with a seed sequence match were found. To analyze whether there is significant enrichment of transcripts with a seed-binding site among the repressed transcripts after siRNA transfection, the number of repressed transcripts with a seed match was compared to the mean number of transcripts with a random seed match. Seven thousand five hundred random seed sequences were drawn and from these, 2000 with similar numbers of transcripts with a seed match in the 3\u2032-UTR (range 1692\u20132892 transcripts per sequence) were drawn and the number of repressed transcripts with a seed match were recorded for each random seed sequence. The distribution of these values was used to calculate the We hypothesized that siRNA pools containing up to 60 individual siRNAs against one target possess very low off-target effects because of the very low concentration of each individual siRNA. However, chemical synthesis of high numbers of siRNAs is cost-intensive and therefore not practicable. Therefore, we set out to generate complex siRNA pools enzymatically. For our experiments, we chose the human target genes PolG and Scyl1. siRNAs with strong off-target effects against the gene MAD2 had been reported for both genes . These sFor siRNA pool production, we selected siRNA sequences using standard siRNA prediction tools, which are connected by non-complementary linker sequences Figure . The DNAThe two strands were transcribed, annealed and analyzed on a native polyacrylamide (PAA) gel , but not in the siPool transfections   or siRNASince siPools and esiRNAs derive from longer dsRNA precursors and such precursors might cause interferon response, we tested the expression of interferon response genes after siRNA transfection Figure . siPoolsOff-target effects are a severe but often ignored error source in RNAi experiments. This is particularly important for genome-wide screening approaches, in which only a number of hits can be validated individually . In factO-methylation of the guide strand leads to a reduction of miRNA-like off-target effects presumably due to less efficient binding to the seed sequence of the off-target mRNA. For siRNA on-target activity, this modification seems to be tolerated  and theobserved  highlighEscherichia coli RNase III generates more discrete size products thus improving RNase III generated siRNAs  have been discovered in various organisms and tissues ,59. Theshttp://www.ncbi.nlm.nih.gov/geo/) under accession GSE57674.Raw intensities and normalized expression data of the microarrays are publicly available at the NCBI Gene Expression Omnibus (GEO, Supplementary Data are available at NAR Online."}
+{"text": "CCND1) and a 3\u2032UTR consisting of sequences from both the CCND1 3\u2032UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3\u2032UTR with siRNA results in decreased CCND1 levels.The t translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3\u2032UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (The online version of this article (doi:10.1186/s13045-016-0260-7) contains supplementary material, which is available to authorized users. CCND1) transcript [CCND1 mRNA has a long 3\u2032UTR (~3.1\u00a0Kb) that contains numerous destabilizing elements [CCND1 transcripts with truncated 3\u2032UTRs correlating with reduced survival [Mantle cell lymphoma (MCL) is considered incurable upon relapse . The halanscript . The CCNelements , 5. MCL survival . In somesurvival . In othesurvival , has beesurvival . Aside fsurvival .CCND1 mRNA. We observed 3\u2032rapid amplification of cDNA end (3\u2032RACE) products in all the MCL lines that would indicate that 3\u2032UTR shortening has occurred  gene  from MRCK, which triggers the subsequent addition of the poly(A) tail, creating a chimeric 3\u2032UTR. The observed CCND1/MRCK fusion gene likely is formed by a second translocation event between chromosomes 11 and 14, which positions the full open reading frame of CCND1 and a truncated 3\u2032UTR within intron one of MRCK  or the truncated CCND1 3\u2032UTR from Jeko-1 (Tr-CCND1) and co-transfected them with three miRNA mimics known to repress CCND1. The FL-CCND1 reporter was downregulated in response to each mimic tested  and 5% penicillin/streptomycin. All three MCL cell lines: Jeko-1, Granta-519, and SP-53 contain the t11;14) translocation [;14 per the manufacturer\u2019s protocol. HF Phusion buffer was used together with MgCl2. Primers were designed to provide the largest DNA PCR product possible, and were validated to work with genomic DNA. The primer sequences used were CCND1 forward 5\u00b4TCCGGAGCATTTTGATACCAG and MRCK reverse 5\u00b4TCCAATTCTGCTAGACCTTTGTGATA.Total mRNA was extracted using TRIzol reagent (Life Technologies). After DNase (Promega) treatment, cDNA synthesis was carried out using M-MLV Reverse Transcriptase (Life Technologies). For 3\u00b4RACE, oligo(dT25)T7 primer was used in cDNA synthesis, and the first round of PCR was performed using the CCND1 primer 5\u00b4 TGGTGAACAAGCTCAAGTGG and oligo(dT25)T7. The second round of PCR was performed using a nested CCND1 primer 5\u00b4TGGCATTTTGGAGAGGAAGTG and a T7 primer. All PCR was performed using pfu polymerase. The resulting PCR product was cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Life Technologies) and sequenced . The qRT-PCR protocol used, as well as the amplicons used to measure 7SK and CCND1, were previously described . The priThree different CCND1 3\u00b4UTR constructs were cloned downstream of the Renilla luciferase gene of psicheck2 (Promega) between Xho1 and Not1. To clone the gene fusion, the CCND1-MRCK sequence: CTCGAGGGGCGCCAGGACGGCGGGCGCCACCGCCACCCGCAGCGAGGGCGGAGCCGGCCCCAGGTGCTCCCCTGACAGTCCCTCCTCTCCGGAGCATTTTGATACCAGAAGGGAAAGCTTCATTCTCCTTGTTGTTGGTTGTTTTTTCCTTTGCTCTTTCCCCTAAAAAAGCATAATAGGTTAGCTGACCAATGATTAAAACATTATAGATCCGGAACGAACTAGAGAAGGCATGCAAAATTCAGATGAGAAAGTTTCTAAGTGATAGCTCCAGCTACCCCTCCAAATATCACAAAGGTCTAGCAGAATTGGAATTAAAAAAAAAAAGTCCTCTTGCACGGTTTATTTAAAATAAAGCCTTAACCTTAGGTGGTGCACCAAGTTGAACCTGACAGTGGAACTGTGTGGGTTTCAAGATCGAGTGATCAGAAAGGAACGGTAAACAAGCTGGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACCCGAGGTCAGGAGTACAAGACCAGCCTGGCCAACACTGTGGCGGCCGC was synthesized by GenScript and ligated into pUC57-Kan. After Xho1 and Not1 Restriction enzyme digestion, the sequence was directly cloned into the psicheck2 plasmid. The Jeko-1 specific truncated 3\u00b4UTR was cloned from Jeko-1 cDNA using the following primers: forward 5\u00b4GGCCCTCGAGGGGCGCCAGGCAGGCGGGCGC and reverse 5\u00b4GGCCGCGGCCGCTGCCTAGAACCCCACTACAGCTGTGC. Full length CCND1 3\u00b4UTR was cloned from the CCND1-pLightSwitch 3\u00b4UTR plasmid (S813994) from SwitchGear Genomics. The primers used were: forward 5\u00b4 GGCCGTCGACGGGCGCCAGGCAGGCGGGCGC and reverse 5\u00b4 GGCCGCGGCCGC CGTCTTTTTGTCTTCTGCTGGA.Either control siRNA , CCND1-sCells were lysed using RIPA buffer and 20ug of each protein sample was resolved on a 12% SDS-PAGE gel. After transfer to a PVDF membrane, blocking was done for 1hr in 5% non-fat milk resuspended in PBS (+0.001 % Tween 20). The membranes were probed with the monoclonal cyclin D1 antibody  and polyclonal alpha tubulin antibody (Abcam). After probing with HRP-conjugated secondary antibody, proteins were detected by luminol.We used predicted CCND1-miRNA interactions using starBase software, which consolidates TargetScan, PicTar, PITA, Miranda and RNA22 miRNA prediction softwares, and overlaps the data with CLIP-Sequencing (CLIP-Seq.) data. We used a high stringency cutoff, where only miRNAs supported by >/=3 CLIP-Seq. experiments were selected, to reduce false positives ."}
+{"text": "We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol \u03b5 is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol \u03b4 is not present. Conversely, Pol \u03b4-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols \u03b5 and \u03b1. We have shown earlier that Pol \u03b4-PCNA is suppressed on the leading strand with CMG . We propDOI:http://dx.doi.org/10.7554/eLife.04988.001 Cells must replicate their DNA before they divide so that the newly formed cells can each receive a copy of the same genetic material. DNA replication requires complex molecular machinery called a replisome, which comprises multiple proteins, enzymes, and other molecules. First, an enzyme called a helicase starts to unwind the double-stranded DNA into two single strands. This process continues while other enzymes, called polymerases, use the exposed single strands as templates to make complementary new strands of DNA. One of these new strands is built continuously and called the \u2018leading strand\u2019. The other newly forming strand\u2014the \u2018lagging strand\u2019\u2014is made in the opposite direction, as a series of short fragments that are later joined together.The replisomes in bacterial cells have been well studied, but many researchers are investigating the composition of the replisome in animals, plants, and fungi . Now, Georgescu et al. have essentially rebuilt a eukaryotic replisome from 31 different proteins in a test tube and confirmed that it can make both leading and lagging DNA strands\u2014just like in a normal cell. Further experiments revealed that the polymerase enzyme that operates on the leading strand cannot work on the lagging strand and vice versa. This exclusivity is unique to eukaryotic DNA replication, as bacterial polymerases can use either DNA strand as a template.Georgescu et al. then found that the eukaryotic polymerases are actively prevented from copying the \u2018wrong\u2019 strand of DNA and suggest that the helicase enzyme that unwinds the DNA might be behind this activity. Important future studies must now address how the replisome deals with obstacles created by certain DNA-binding proteins and damaged DNA and how it interfaces with the molecules that control cell division and DNA repair.DOI:http://dx.doi.org/10.7554/eLife.04988.002 Composition of the eukaryotic replisome and the function of its various proteins is an area of active investigation. Cellular studies reveal that eukaryotes use two different DNA polymerases for the leading and lagging strands, Pols \u03b5 and \u03b4, respectively . PrimingWhile in vitro synthesis of the leading strand replisome has been accomplished with the purified CMG complex from budding yeast , the disStudy of the eukaryotic replisome identifies a new process that has no precedent in bacterial systems. Bacteria use simple recruitment processes to attract and hold polymerases to the fork. These are typically mediated by polymerase interactions with other proteins at the replication fork, such as the helicase and sliding clamps . However32P-dCTP (leading) or 32P-dGTP (lagging).We expressed and purified yeast CMG helicase in our previous studies and demonstrated its function in leading strand synthesis with Pol \u03b5 . The disEscherichia coli. A mixture of cells containing the 4 subunits were lysed, and Pol \u03b1 was purified as an intact 4-subunit holoenzyme . The analysis also confirms that Pol \u03b1 cannot perform priming and extension in the absence of CMG (lanes 1\u20133) and that \u03d529 Pol cannot perform lagging strand synthesis (lanes 13\u201315).To determine if Okazaki fragments are distributed over the length of the DNA template, we used tive gel . The anaPol \u03b5 is the leading strand enzyme and presumably takes over the leading strand from Pol \u03b1 after this distributive enzyme dissociates from DNA. To determine if the Pol \u03b1/\u03b5 switch occurs as expected, we preloaded CMG on the forked DNA, then added increasing amounts of Pol \u03b1 either with or without Pol \u03b5, and stopped the reactions after 20 min. If Pol \u03b5 takes over the leading strand from Pol \u03b1, full-length products will be observed sooner in reactions that contain Pol \u03b5 because in the presence of CMG, this enzyme synthesizes DNA faster than Pol \u03b1. The results show full-length product in all the lanes containing Pol \u03b5 with Pol \u03b1 .In order to compare total leading and lagging strand synthesis rates, we setup standard replication reactions by loading CMG onto the nucleotide-biased forked DNA, followed by the addition of all three DNA polymerases in the presence of RFC and PCNA; the reactions were divided, and replication was initiated by the addition of either 32P-dCTP (leading) or 32P-dGTP (lagging) in the three polymerase replisome system shows similar amounts of leading and lagging strand synthesis . Indeed, polymerase recruitment by binding clamps and the helicase underlies attraction of polymerases to replication forks of bacteria, its phages, and the SV40 virus. Recruitment is also partly responsible for polymerase placement in eukaryotes. For example, Pol \u03b5 binds directly to CMG helicase, stabilizing it on the leading strand . HoweverWe were surprised to find that Pol \u03b1 polymerase activity is highly functional with CMG on both leading and lagging strands in the absence of other polymerases. Pol \u03b1 lacks the high fidelity of Pols \u03b5 and \u03b4 and does not provide bulk leading or lagging strand synthesis in cells, and thus processes must exist that suppress the polymerase activity of Pol \u03b1. In fact, we find many ways that Pol \u03b1 polymerase is suppressed. One mechanism is by Pol \u03b5 positioning on the lagging strand. Interestingly, Pol \u03b5 is also suppressed on the lagging strand; perhaps, Pol \u03b5 is suppressed from function on the lagging strand by the relative orientation in which CMG holds Pol \u03b5. On the leading strand, Pol \u03b5 simply prevents Pol \u03b1 polymerase extension by switching with it and becoming processive with CMG. This can be likened to the switch of Pol \u03b4 with Pol \u03b1 that prevents leading strand synthesis by Pol \u03b1 in the SV40 system . Pol \u03b1 pThe exclusion processes that underlie eukaryotic fork leading/lagging strand function are summarized in E. coli Pol III replicase  complex, directing the single Pol \u03b5 molecule to the leading strand. Suppression of Pol \u03b1 by Pol \u03b5 on the lagging strand could perhaps be explained by competition of these polymerases for CMG, where Pol \u03b5 is suppressed from extending the primer. In this connection, the Pol2 gene encoding the catalytic polymerase actually contains two polymerase structures: the active polymerase/exonuclease on the N-terminal half and B-family polymerase in the C-terminal half of Pol2 that is presumed to be inactive . One posNumerous proteins travel with the replication fork, and it is not possible to know a priori how many are needed for functional leading and lagging strand replication. This is the first study to reconstitute leading/lagging strand replication with three pure polymerases in a eukaryotic system. It reveals that many of the proteins that move with forks are not required to recapitulate leading/lagging strand synthesis in vitro. For example, Ctf4/AND-1 is essential in most cells (not in budding yeast), and yeast Ctf4 helps recruit Pol \u03b1 bind to RPCs , yet theThe current report also reveals that Pol \u03b1 can prime the leading strand directly. To our knowledge, before this report, all proposed models of leading strand initiation in bacteria and SV40 show priming on the lagging strand of a bidirectional origin, which becomes the leading strand of one fork, rather than directly priming the leading strands . We showCellular studies indicate that nucleosomes are involved in determining Okazaki fragment size , but datReconstitution of cellular replisomes in vitro should provide a framework to explore the effects of other proteins that move with forks and of post-translational modifications that control eukaryotic forks in response to the cell cycle and DNA checkpoint mechanisms. Reconstituted systems should also enable detailed study of factors that maintain the epigenetic state of a cell during replication.2, 5 mM imidazole, 20 mM KOAc, and 350 mM KCl. Buffer D is 25 mM Tris-OAc pH 7.6, 40 mM K-OAc, 40 mM K glutamate, 2 mM Mg-OAc2, 1 mM DTT, 20% glycerol, and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA. Buffer H is 20 mM Hepes pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT, 350 mM KCl, 1 mM ATP, and 4 mM MgCl2.Radioactive nucleotides were from Perkin Elmer. Unlabeled nucleotides were from GE Healthcare. DNA modification enzymes and \u03d529 DNA polymerase were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Bradford Protein stain and bovine serum albumin as a standard. Buffer A is 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 4% glycerol. Buffer B is the same as buffer A except 20 mM Tris-acetate, pH 7.5 was used in place of 20 nM Tris-HCl. Buffer C is 25 mM Tris-Cl pH 7.9, 10% glycerol, 1 mM DTT, 1 mM MgClE. coli SSB , a strain constructed from W303  . Pol12 was transformed into E. coli BL21(DE3)codon plus RIL , then induced with IPTG for 8 hr at 15\u00b0C. Pri1 and Pri2 were co-expressed in E. coli BL21(DE3) cells by IPTG induction for 8 hr at 15\u00b0C. A 12 l culture of induced yeast cells for Pol1 and 1 l of each induced E. coli cultures for Pol12 and Pri1 and Pri2 were co-crushed in a cryogenic mill as described for CMG (g for 1 hr at 4\u00b0C. Anti-Flag agarose (1.2 ml) was added to the supernatant (80 ml) and incubated with slow rotation for 1.5 hr at 12\u00b0C. Beads were collected by centrifugation at 1500\u00d7g, washed twice with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, then loaded into a gravity column. The column was washed with 15 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol, and Pol \u03b1 was eluted with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol containing 20 \u03bcg/ml Flag peptide. The eluent was diluted to a conductivity equal to 150 mM NaCl using 25 mM Hepes pH 7.4, 1 mM EDTA, and 10% glycerol, then applied to a 1 ml Heparin agarose column. Pol \u03b1 was eluted with a gradient of 100 mM to 1 M NaCl in 25 mM Hepes pH 7.4, 1 mM EDTA, 10% glycerol. Peak fractions were pooled, aliquoted, and stored at \u221280\u00b0C. Typical yield of Pol \u03b1 was about 3 mg.Proteins were purified as described: RPA , E. colicoli SSB , PCNA  . The Pol for CMG . Frozen To make the linear fork DNA substrate, a 3.2 kb sequence of DNA was designed such that one strand lacked dC residues and thus the other strand lacked dG. The G-rich strand was examined to eliminate runs of four G residues. The resulting 3260 bp sequence was synthesized by Biomatik . The first four and last two bases represent overhangs generated by BsaI and BtsC1, respectively. Both of these enzymes cut outside of their recognition sequences, and their recognition sequences are excluded from the template.CGGTATTCTAACCACATTAATCTACACCTCTCACACACTACTCATATCATCTTCCAAAACCCACCTTTAAAAAACCCTTTATCCACACTCATCACCATTTCACCAACCTTTTTCTTAATTCTACACAAATCCAATTAACCTATCTCCAATTTTAACTCCATCACCTCTTATATTAACCCACCTACTACTCCAACAATACCCTATCAAATCTACTTCTATCTCAAAACTATCACCTACTCCTTCCATCATAATCCACTCTTATCAATTAAACAATTATCCTTCTTTCCCACCATCACTCACCATCTTTTCTTAACACCCTTAACATTTCCTTTTATAAAACACTTCCAATCCTATTTTCTCACTATCCCACCCACCATAAAAACTATCTCACCCTAACTCAACCCTTTCCCTCTCACCAACACTCCTTTATCACACACACTTTACCACACAAATCCCTCCATCATACACCTTTACCCTCAAATCCTAAACCACCTAACTATTCCACACAATATATCTACAAAAATTTACTTTTCCACATCTCCAACCCTTCCAACACCTTAATCCCAAACCTTAACTAACCTCTCTTTAATACTTCCTCCCATTCCCACCACATACACCAAATTATCTTCAACTCAAAAACCTAAACTCTCCTTTTTATTCCCTATAAAAACTCTTAACCCTCCAATATACAACTCTAACTAACTTCATTATCAACCAATCTTCCTCTACTTCCTCATCTTATAATTTATCCATTCAAAATAACCTACCTACCACCTCTCCTCTTCACTTCCTACCCTAAAATCACCACCTTATCCCTAATTTACCTCTTTCAACTTTCCTTAACCCAACTTCTCAATCCTACTTCACTTACTTCTTATAAAACCATCATTATCACACTACACATTACTCTATCTATCCAATCATCACCTTCTACAATCCAAACTATCCCACTACCCTCTCATTCTACCTTTTCATCTATCTCAAACTATCCACCAATCCATACCTCAAACTTTAACCACCCACTCCAAATCTACAACATAAAATTAACCTATCACATTCCAAACTAATCACCCTAACCCTAACACCCTTTTATCCTCACCAAAATTACCATTTTCCTCTTTACTCATAAACAAACATTCTCACCCATTTATAAAACACTTAATACCCACTTAATTCACTTCCTTTTTCTACCTCACCATCATCAACTCCTAATATCAACAACCCAAAATCACCACCTATATCCTCATCTCCTATATAAAAAACTTCACATCTCAACCTCAAACCACCTATTCCACTTAATCCCAATCAACCTATCAACTCTACAACCTACTCTTCAAATACATCTCCTATCACTTTCCCACCTCCTTCAATCAATTATAACTTTATCACCTAAACATTCTAAAATCTCCTATCCACTACATCACATAAACCTAAACCTACTACCAACCTACCATTATCCTTATCAAACATCATCAATTCCATCTTTCTTTATACCCTCTCCATATCTCTCTCATTAAAAAAACCAAAATCTAACAACTTCTTATTTCTAATCAAAAAAACAATCAACCTAACTCATAAAATCTTCACCTTAAAATTCCTTTACATTTAACAAATCCACTCTCCCTATCTTTTTCATATCAAACTTATCTAAACCACTATCCTCATTTATCCTAATACTCCATATACAACACCAAATTTCTTATATCTCATCTAAAATCCTCCACCAATATAAACTCCTCTTTACCATTTCCACTCAACACACCAAATCTTATTATTCCATCAAATCTAATCCATTACCATCATCAACCCTAATAAACCTACTTCCCAACTTTTATCTCTCCACTACCACACCAAAAATTAACCTCCTCTAAAAACTATCATTCCCTTTACCTCTTCCACATTCCACCTATAACTCCTCATCTTAAAACCAATCAACACCAAACAACTATCTCACCATATTTCCTCTCCAAACCAACAAATTAACAATCCTACCCACTCCAACCTCCACATTACTAATAACTAAACTTACCTTACCTACCACACCCTATCAACCATATTTAAAAAATTACTTATTCACTAACTAAAACATCACCCACAAACTTAAATCATCACCTCCTCTTTTCCACCTTATTCACCAACCCAATCTATCTATCTCACCTATACCTTTCCCTAATATCTTTTACTAACCCTATAAATACCACAATTCTAAAAAACCCATACTTATCTCACACATCACTTTATACTTCACTCTTAAAATACCCTCCAATATATATTACAACCCAAAAATATCTCCCTTCTATCTCCTACACACAAATTATACCACTTTTAAACCACTCCTCATCTCTAACCCAACCCTCTACAATTCCATACATCTCTACTATCAACATCACTCCTTCTTTTCCACTCTTCTCTCCACATCTTTATTAAACATCTCCTCCTCATTTTCACATAACTATTTACTAAATAAATTTACCTAAACTACATTTATTAAAACCCTACAACATACTCCTTATTCTCCTACCTACCATTCTCTAATCTCTTTACATTCTACTACTTTCCTACCTACTATCTCAATAAATTCACTTTCCTTTCACCACACACCAACACACCTTCCTCCAAATTCTTTATATCTCCTTCTCCTAACCAAATTCCTCACTAATAACATCTTACCTCCCTACCTTTTTCCTTCTACCCTCCACCATTTCCCAACCTCATACTCAATAATCAATTTACCCTCCCACAACATAACTCTATTAACACCCATTTTCTATCCATCAACTTCCTATTACTTAAATTATCTTTTAAACCAATAAAACTCCACCTCAACCACCCACCTCTCTCTTTCAATCCAATTTCAATCTTTCCAACCATTCTATCTACCCTAAACTATTAACTATCTATCTCACCCACAATCCCTCCTACAATTCACAACAACATTCCACCACTTCACTTTATCTTCACCTCCAAACTTATTCCTTCCCATTATCACCTTCTCCAAAACCCACAAACATCTAACTCTCATCTCTCAACACTTTCTACCCATTCTCTCTAACAAAATTCCCTTACTCTTTATTCACAAAACCACTAAAATCACCACAACAACCCAACAAAAACAAAATCCTCACTTACCTATACTCAATAAATCCTTCAACTCATTATTCTATTTCTAACCCTAAATCAAAACTCCCATATCTACCATTCTTTCCACTCAATTAAATCCCACCAACCCTTATTTTCCTCCAATAACTTAACC.The synthetic 3.2 kb DNA was cut from the plasmid using BsaI-HF and BtsCI and purified from low-melting agarose gel. 35 pmol of the 3.2 kb linear DNA template was ligated to a fivefold excess of forked junction on the BsaI end and to a 10-fold molar excess of a short blocking duplex on the BtsCI end. To make the forked junction, 1050 pmol 1T oligonucleotide was annealed to 175 pmol 160mer oligonucleotide, as described . The 16032P-dCTP, while for lagging strand replications, we used 20 \u03bcM dGTP and 10 \u03bcCi 32P-dGTP. Exceptions to this protocol are noted in the figure legends. Timed aliquots were removed and quenched upon adding SDS and EDTA to final concentrations of 0.5% and 20 mM, respectively. Quenched reactions were analyzed in 0.7% or 2% alkaline agarose gels and imaged in a Typhoon 9400 PhosphorImager .Replication assays contained 1.5 nM linear forked DNA, 24 nM CMG, 400 nM RPA (unless noted otherwise), 20 nM PCNA (unless noted otherwise), 6 nM RFC (unless noted otherwise), and Pol \u03b1, Pol \u03b5, and Pol \u03b4 as indicated in the figure legends, in 25 mM Tris-acetate pH 7.5, 10 mM Mg-acetate, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 \u03bcg/ml BSA, 0.1 mM AMP\u2013PNP, 5 mM ATP, 200 \u03bcM each rCTP, rUTP, rGTP, 60 \u03bcM of each unlabeled dNTP, and 20 \u03bcM of the labeled dNTP. Reactions were staged as follows. CMG was added first and pre-incubated with the DNA and 0.1 mM AMP-PNP for 10 min at 30\u00b0C, then the noted polymerases together with the RFC and PCNA (where indicated) were added, along with dATP, dCTP for an additional 2 min. Replication was then initiated upon the addition of a solution containing the RPA, ATP, dTTP, and dGTP. It is important to note that for leading strand replication reactions, we used 20 \u03bc\u039c dCTP and 10 \u03bcCi 32P-dCTP (leading) or 32P-dGTP (lagging) was added. Timed reactions were stopped with an equal volume of 2\u00d7 STOP solution (40 mM EDTA and 1% SDS) and spotted on DE81 filter papers, then analyzed using a Perkin Elmer Liquid Scintillation Analyzer . Separately, we performed control replication reactions using \u03d5X174 ssDNA coated with RPA, containing a known amount of Gs and Cs, confirm that 32P-dCTP and 32P-dGTP are equally incorporated by each of the DNA polymerases in our experimental conditions.In order to compare total DNA synthesis rates on leading and lagging strand, we performed standard replication reactions containing all three DNA polymerases at 10 nM, CMG (30 nM), RFC (10 nM), PCNA (20 nM), and RPA (400 nM) in presence of 1 mM ATP, rNTPs , and 30 \u03bcM dNTPs; reactions were divided, and either 32P-dGTP to label the lagging strand; the second reaction utilized 1.5 nM unprimed forked DNA and 10 nM each Pol \u03b5 and Pol \u03b1 along with 20 \u03bcM dGTP and 10 \u03bcCi 32P-dGTP to label the lagging strand, and the third reaction contained DNA-primed forked DNA and 1 U of \u03d529 DNA polymerase along with 20 \u03bcM dCTP and 10 \u03bcCi 32P-dCTP to label the leading strand. Each reaction was quenched upon heating to 65\u00b0C for 10 min to inactivate the CMG and polymerases. Then, reactions were divided into three tubes, one was untreated, the second was treated with EarI, and the third was treated with PsiI, adjusting the reaction buffer for each enzyme with the commercial provided buffer. Reactions were analyzed in a 2% native agarose gel followed by autoradiography in a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Three replication reactions were performed as described above with the following differences. The first reaction used 1.5 nM unprimed forked DNA and 10 nM Pol \u03b1 with 20 \u03bcM dGTP as well as 10 \u03bcCi 32P) dTTP and incubated at 30\u00b0C. At the times indicated, 25-\u03bcl aliquots were removed and quenched by addition of an equal volume of 1% SDS/40 mm EDTA. Products were analyzed in 0.7% alkaline agarose gels. Gels were dried, exposed to PhosphorImager screens, and imaged using a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Reactions contained 1.5 nM \u03d5X174 circular ssDNA (as circles) primed with a DNA 30-mer and pre-incubated for 10 min with 420 nM RPA (as heterotrimer) in 20 mM Tris-Cl (pH 7.5), 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 \u03bcg/ml BSA, 8 mM MgOAc, 0.5 mm ATP, 5% glycerol, and 60 \u03bcM, each of dGTP and dATP. Reactions also contained the indicated amounts of RFC, PCNA, and Pol \u03b1 and were pre-incubated for 5 min at 30\u00b0C. DNA synthesis was initiated by adding 15 \u03bcl of 60 \u03bcM dCTP, 20 \u03bcM dTTP, 15 \u03bcCi of . Proteins were brought to a final volume of 200 \u03bcl in 100 mM sodium phosphate, 150 mM NaCl pH 8.0, and incubated with 50 \u03bcl (as a 10% slurry) Strep-Tactin magnetic beads  for 1 hr at 4\u00b0C with end-over-end mixing. Beads were collected in a magnetic separator and washed twice in 200 \u03bcl 100 mM sodium phosphate, 150 mM NaCl pH 8.0, then eluted in 75 \u03bcl 10 mM biotin in 100 mM sodium phosphate, 150 mM NaCl pH 8.0 for 20 min on ice. Samples were analyzed in 8% SDS-PAGE followed by staining with Coomassie Blue Denville stain."}
+{"text": "AbstractSymmerista H\u00fcbner  is reviewed for Costa Rica, based on 49 wild-caught specimens. Four species are newly described: Symmerista luisdiegogomezi Chac\u00f3n, Symmerista inbioi Chac\u00f3n, Symmerista minaei Chac\u00f3n and Symmerista aura Chac\u00f3n. All are from the cloud forests of the Talamanca moutain range, southern Costa Rica. Photographs of the adults, male and female genitalia, and barcodes are also provided. The species Symmerista tlotzin Schaus (1892) is removed from Symmerista and assigned to the genus Elymiotis Walker as a new combination.The genus  Notodontidae includes about 3500 described species worldwide , from the cloud forests at altitudes between 1000 and 2600 meters.H\u00fcbner (1821) established the genus  species . SpeciesSymmerista tlotzin  male genitalia morphology and barcodes , Santo Domingo de Heredia, Costa Rica were examined, sexed and identified. Of those, 12 were DNA barcoded and dissected. All holotypes and paratypes are deposited in the collections at INBio. Fifty-six specimens of reared and wild-caught Elymiotis tlotzin, previously known as Symmerista tlotzin, were also examined.Forty-nine specimens of  Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio National Museum of Natural History, Smithsonian Institution, Washington DC, USAUSNM Adterminal lineAD Corpus bursaeCB Ductus bursaeDB ForewingFW HindwingHW Medial lineM Postmedial linePM Sternum 8ST8 Tergum 8T8 Wing lengthWLPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeH\u00fcbnerSymmerista H\u00fcbner, 1821. Verz. bekannt. Schmett. : 248.Noctua albicosta H\u00fcbner, 1809, Samml. eur. Schmett. 4: pl. 93, fig. 440. By subsequent designation by, Kirby, 1892, Syn. Cat. Lepid. Het. Het. 1: 572.Noctua albicosta occurs in Canada and the US.Type(s), [North America]: Mistakenly included by H\u00fcbner as a European species; Symmerista was originally proposed in the Noctuidae.Nye (1975) stated: Noctua albicosta is a form of Phalaena albifrons Smith, 1797, in Smith & Abbot, Nat. Hist. rarer lepid. Insects Georgia 2: 159, pl. 80.Watson et al. (1980) stated: Adults \u2013 Medium-sized notodontid moth, FW = 16\u201322 mm, females larger than males; male antenna pectinate nearly to apex, terminal 10\u201312 annulations simple or antenna bipectinate, six terminal flagellomeres without rami; antennae of female simple; labial palpus porrect; haustellum reduced to two small lobes, completely hidden by labial palpi; ocelli absent; eye smooth, round; thorax generally dark brown with beige tegula; all scales of thorax long and forked; scales of patagium and prothorax beige and cream colored. FW with the costa straight, apex marked, the outer margin evenly rounded; accessory cell present, narrow; R2-5, stalked, R5 arising beyond R2, M1 from apex of accessory cell or nearly so. Male genitalia \u2013 ST8 with a wide, deep emargination; valva membranous, finely pubescent, costulae absent; tegumen narrowed dorsally; uncus concave, the socii subquadrate and lobed or long, wide, pubescent at bases, narrow and flattened at apices; vinculum slightly sclerotized; manica membranous, fused to juxta; ventral process of phallus long and forked, its tip well beyond the tip of the medial, dorsal process; vesica bulbous with a scobinate patch.Female genitalia \u2013 Papillae anales membranous, covered with short, scattered setae; posterior apophyses long and slender; DB sclerotized for approximately two-thirds of its length; CB rounded; anterior vaginal plate asymmetrical, slightly swollen and inflated in middle.Symmerista is characterized by: haustellum reduced to two small lobes, completely hidden by labial palpi; valva membranous; costulae absent; uncus concave; manica membranous, fused to juxta and ductus bursae sclerotized for approximately two-thirds of its length. Symmerista differs of the genera Elasmia and Elymiotis by the following characteristics: Elasmia has a well-developed haustellum, sacculus pleated and saccular scent organ present, manica sclerotized, deciduous cornuti, costulae present, costal margin sclerotized, ST8 with long and curvy lateral sclerotized projections like forceps, midplate with tiny pleats and membranous; uncus elongate, setose, with two dorsal protuberances and two tiny spines at the apex. Elymiotis has the haustellum well developed, sacculus pleated, deciduous cornuti, costal margin sclerotized, St8 posterior margin irregularly sclerotized and convex with tiny lateral proyections, uncus thin and long with apex acute and setose. All of these characterisctics are absent in Symmerista.The genus PageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/81A8E740-7368-45A7-9BBE-6113BBC43D3E16 specimens Holotype male: INB0003536238 , Costa Rica, Prov. Cartago, P.N. Tapanti, Macizo de La Muerte, Est. La Esperanza 9.69129-83.87683, 2600 m, September 2002, R. Delgado (INBio).Paratypes: 13 males, 2 females. 2 males: INB0003756237, INB0003756364 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y el Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio). 4 males: INB0003316532, INB0003316533, INB0003316534, INB0003316535 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003352709 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho. El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio). 2 males: INB0003387641 (dissected), INB0003387642, Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). 2 males: INB0003536234 (COI barcoded), INB0003536235 (COI Barcoded), INB0003536237 (COI barcoded), Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). 2 males: INB0003545221 , INB0003545222 (COI barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). Female: INB0003339209  Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Est. La Esperanza. 9.686771-83.87775, 2600 m. July 2001. R. Delgado (INBio). Female: INB0003756274 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio).This species is named in honor of the late Professor Luis Diego G\u00f3mez Pignataro of San Jose, Costa Rica, for his outstanding contribution to our knowledge of Costa Rican biodiversity, his support of the Museo Nacional de Costa Rica, and for inspiring me to become a naturalist and taxonomist.Male genitalia: St8 posterior margin concave with a pair of short postero-lateral projections, projections robust and heavily sclerotized with blunt apices; valva membranous, finely pubescent, sacculus smooth, its margin uniform, costa straight with a distal protuberance near apex; valva with two triangular spine-like processes, one at base, the other at juxta near anellus; uncus slightly concave, somewhat helmet shaped, dorsal surface PageBreakrough, pubescent, ventral surface smooth with sparse pubescence; socii long, wide, pubescent at bases, narrow and flattened at apices, shape as in the figure; length of the phallus 3.1 mm, proximal part of phallus tube wide at the base, narrow in the middle, distal part of phallus tube robust, sclerotized, with a tubular lateral projection and rounded apex; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: posterior apophyses longer and more slender than anterior apophyses; CB rounded and membranous, lacking a signum; DB wide and sclerotized; posterior margin of postvaginal plate sclerotized and emarginate.Dorsal FW ground color dark brown with costal margin black; an irregular, long thin cream-colored mark from the reniform spot to the apex; fringe dark brown with beige scales where veins touch termen; dorsal HW dark brown. Male , southern Costa Rica .Holotype male: INB0003487050 , Costa Rica, Prov. Cartago, El Guarco, Reserva Forestal Rio Macho, Macizo de la Muerte, Sector La Esperanza 9.686771-83.87775, 2600 m, May 2002, R. Delgado (INBio).Paratypes: Male: INB0003153991 Costa Rica, Prov. Cartago, El Guarco, San Isidro, Est. La Esperanza 9.687685-83.884582, 2450 m, March 2001, R. Delgado (INBio). 2 males: INB0003316531, INB0003316537 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003320716 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, El Guarco, San Isidro, Est. La Esperanza, 9.683922-83.876688, 2600 m, May 2001, R. Delgado (INBio). Male: INB0003334468 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza, 9.686771-83.87775, 2600-2700 m, April 2001, R. Delgado (INBio). 3 males: INB0003339195, INB0003339197, INB0003339198 Costa Rica, Prov. Cartago, El Guarco, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza 9.686771-83.87775, 2600 m, July 2001, R. Delgado (INBio). Male: INB0003387640 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). Male: INB0003478225 , Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69397-83.854504, 2700 m, 13\u201314 May 2002, J. Montero (INBio). 2 males: INB0003536236 , INB0003536239 (COI Barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). Male: INB0003545219  Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). 3 males: INB0003756229, INB0003756321, INB0003756418 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22\u201327 September 2003, D. Rubi, R. Gonzalez, R. Delgado(INBio). Male: INBIOCRI001359567 Costa Rica, Prov. Cartago, Quebrada Segunda, Parque Nacional Tapanti, 9.762583-83.788328, 1250 m, February 1993, G. Mora. Male: INBIOCRI002210601 Costa Rica, Prov. Cartago, La Represa, Tapanti, 9.695643-83.768399, 1800 m, July 1995, R. Delgado (INBio). Male: INBIOCRI002253344 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, March 1995, R. Delgado (INBio). Male: INBIOCRI002423774 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1800 m, February 1995, R. Delgado (INBio). Male, INBIOCRI002427627 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, 22 August\u201315 September 1995, R. Delgado (INBio).PageBreakThis species is dedicated to the Instituto Nacional de Biodiversidad (INBio) in recognition of its 25 years of support for developing an understanding of the biodiversity of Costa Rica and exporting that understanding to the nation and the world.Symmerista inbioi differs from Symmerista luisdiegogomezi on: dorsal FW ground color light brown; from the reniform spot to the apex there is an uniform long, thin, white cream-colored band; fringe reddish brown; dorsal HW dirty beige. Male genitalia: St8 wide at base, anterior margin convex, posterior margin slightly irregular with a pair of very short projections, these sclerotized with blunt apices; valva membranous, saccular margin slightly jagged, costal margin smooth, with a distal protuberance near the apex; elongate finger-shaped process on the saccular margin at the internal base of the valve; uncus plate slightly concave, with papillae and setae on the dorsal and ventral edge; socii elongated, broad at base, narrow and flattened at the apex, with papillae and setae in the dorsal surface; length of the phallus 3.9 mm, proximal part of phallus curved at the base, slightly narrow in the middle, distal part of phallus tube sclerotized, narrow at the base, robust, irregular and wide at the end, with a tubular lateral projection with the distal nipple; proximal part of the vesica with a dorsal scobinate patch, distal part of the vesica bulbous. The genital armature of Symmerista inbioi is more robust and larger than Symmerista luisdiegogomezi.Male  (n Range) .DNA barcode of holotype male INB0003487050Symmerista inbioi | COI-5P:MHMXP003-08 | INB0003487050 | AACATTATATTTTATTTTTGGGATTTGAGCAGGTATAGTAGGAACTTCTTTAAGTCTATTAATTCGAGCTGAATTAGGAAACCCCGGATCACTTATTGGGGATGATCAAATTTATAATACAATTGTTACAGCCCATGCCTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCTCTTATACTAGGAGCCCCAGATATAGCATTCCCCCGCATAAATAATATAAGTTTTTGACTTTTGCCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATCGTAGAAAATGGAGCAGGAACTGGATGGACAGTGTACCCCCCACTATCCGCCAACATTGCCCATAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCTGGAATTTCCTCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGCCTCAATAATATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTTTAGCGGGAGCTATTACAATACTACTAACTGACCGTAATTTAAATACATCCTTTTTTGACCCTGCTGGGGGAGGAGATCCAATTTTATACCAACATTTATTTTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/71A2F18E-A8EC-4BF6-9991-ACEC39D207994 specimens Holotype female: INB0003339208 , Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Estacion La Esperanza, 9.68677-83.87775, 2600 m, July 2001, R. Delgado (INBio). Paratypes: Female: INB0003155283 (COI barcoded), Costa Rica, Prov. Limon, Bratsi, Valle del Silencio, 9.107197-82.961749, 2472 m, 11\u201312 October 2000, R. Delgado (INBio). Female: INB0003352700 (COI barcoded), Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio).Other material examined: 1 Male, INB00033387642 (dissected) Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.68677-83.87775, 2600 m, October 2001. R. Delgado (INBio).This species is dedicated to the Ministerio del Ambiente y Energ\u00eda (MINAE) of the government of Costa Rica in recognition of its 28 years of continuous and widespread support for the survival and conservation of the wild biodiversity of Costa Rica.PageBreakSymmerista minaei differs from Symmerista luisdiegogomezi on: dorsal FW ground color beige and light brown, square mark creamy near the reniform spot; beige mark in the apex; fringe beige yellow; dorsal HW beige. Male genitalia: T8 anterior margin slightly concave, posterior margin finely serrated with a window in the center; St8 lateral margins wide at the base, narrow to posterior margin, anterior margin concave, slightly sclerotized with a short projection in the center, posterior margin with robust projections, highly sclerotized on each side, with blunt apices, a little dome in the middle of the posterior margin; length of the phallus 3.3 mm, proximal part of phallus tube wide at base, distal part of phallus tube robust, sclerotized, with a tubular lateral projection rounded apex with the distal nipple; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: Anterior and posterior apophyses the same size, long an slender; DB sclerotized; CB rounded, membranous and pleated; posterior margin of postvaginal plate sclerotized, slightly irregular, inverted V-shape.Male  (n Range) .DNA barcode paratype female INB0003155283.Symmerista minaei | COI-5P:MHMXP006-08 | INB0003155283 | PageBreakTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCCCTTATGCTAGGAGCCCCAGATATAGCATTCCCACGTATAAATAATATAAGTTTTTGACTTTTACCCCCCTCCTTAACCCTTTTAATTTCAAGAAGAATCGTCGAAAATGGGGCAGGAACCGGATGGACAGTGTACCCCCCACTATCCTCCAATATTGCCCACAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTACATTTAGCTGGAATTTCATCAATTTTAGGGGCCATTAATTTTATCACAACAATTATTAATATACGTCTCAATAACATATCTTTTGATCAAATACCCTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTCTAGGGAGCTATTACAATACTACTAACGGATCGTAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTAACATTATATTTCATTTTTGGAATTTGAGCAGGTATAGTTGGAACTTCATAAGCCTATTAATTCGAGCTGAATTAGGAAATCCCGGATCCCTTATTGGAGATGATCAAATTTATAACACAATTGTTACAGCCCATGCCTTTaxon classificationAnimaliaLepidopteraNotodontidaeChac\u00f3nsp. n.http://zoobank.org/31DCFCFA-B792-406E-A667-CAAA17B5111F4 specimens Holotype male: INB0003116415 , Costa Rica, Prov. Cartago, Paraiso, P.N. Tapanti, Macizo de La Muerte, Estacion Quebrada Segunda, 9.762583-83.788328, 1300 m, November 2000, R. Delgado (INBio).Paratypes: 1 males, 2 females. Male: INBIOCRI002442681 (COI barcoded), Costa Rica, Prov. Puntarenas, Buenos Aires, Potrero Grande, Estacion Altamira, 1 Km. S del Cerro Biolley, 9.032987-83.010887, 1450 m, 13\u201326 May 1996, R. Villalobos (INBio). Female: INBIOCRI002549508 (COI barcoded), Costa Rica, Prov. Puntarenas, Coto Brus, Sabalito, Send. El ripario a 3 Km NE. de Progreso, 8.917676-82.78469, 1300 m, 6\u20139 April 1997, A. Picado (INBio). Female: INBIOCRI002754030 Costa Rica, Prov. Cartago, Turrialba, Tayutic, Moravia de Chirripo Shipiri, 9.837781-83.453639, 1000 m, 10 May 1983, D. H. Janzen & W. Hallwachs (INBio).This species is dedicated to Isidro Chac\u00f3n\u2019s daughter, Aura Chac\u00f3n, for 25 years of understanding an absent father obsessed with his work.Male genitalia: St8 anterior margin with a sclerotized triangular projection in the middle, posterior margin sclerotized, irregular, with a rectangular projection at the center bearing two highly sclerotic and sharply serrated structures; proximal part of phallus tube wide and short at the base, with a blade-like lateral projection, sharp at the distal apex; a small rounded projection and curve off the previous, distal part of phallus tube robust, sclerotized, with a small bulge on the ventral side; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: papillae anales ovoid-triangular, setose; posterior apophyses longer and more slender than anterior apophyses; DB sclerotized; CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex.Dorsal FW ground color light gray; a white cream mark from the discal cell to the apex. PageBreakSymmerista aura differs from female of Symmerista meridionalis Thiaucourt, 2007 in the shape of the antivaginalis plate; the CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex. This is the description of the female genitalia of Symmerista meridionalis published by Thiaucourt in 2007 which mentiones the differences, \u201cFemale terminalia: distal edge of lamella postvaginalis slightly incurved; lamella antevaginalis rectangular, almost square; its distal margin strongly sclerotized; mouth of the ostium bursae oval, densely sclerotized under the margin; ductus bursae beyond the constriction under the ostium, forming a short funnel; bursa membranous inserted on the dorsal surface of the extremity of the duct; signum distinct.\u201d The male of Symmerista meridionalis is unknown.The female of Male   .DNA barcode paratype male INB0003116415.Symmerista aura | COI-5P:MHMXP017-08 | INB0003116415 | ACATTATATTTTATTTTTGGGGTTTGAGCTGGGATAGTTGGAACTTCCCTAAGTTTACTAATTCGAGCTGAATTGGGTAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACAATTGTAACAGCCCATGCTTTTATTATAATTTTTTTTATAGTTATACCCACCATAATCGGGGGATTTGGTAATTGACTAGTTCCTCTTATATTAGGGGCACCGGATATAGCATTTCCACGTATAAATAACATAAGTTTTTGACTTCTACCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATTGTCGAAAATGGAGCTGGAACAGGATGAACAGTGTACCCCCCATTGTCATCTAATATTGCTCATGGTGGTAGTTCCGTAGATTTAGCTATTTTTTCACTTCATTTAGTGGAATTTCTTCAATTTTAGGGGCTATTAATTTTATTACAACAATCATTAATATACGTCTTAATAATATATCTTTTGACCAAATACCTTTATTTGTGTGAGCTGTAGGGATTACAGCATTTTTACTTTTACTTTCTTTACCTGTATTAGCTGGAGCTATTACAATATTATTAACTGATCGTAATCTAAACACATCTTTTTTTGATCCCGCTGGAGGAGGAGATCCTATTTTATACPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaecomb. n.11 males 12 females.PageBreakINB0003319696 Costa Rica, Prov. Guanacaste, Nicoya, P.N. Barra Honda, Sector Barra Honda, 10.169826-85.379137, 50 m, 25\u201330 December 2000, H. Mendez (INBio). Female: INBIOCRI001184551 Costa Rica, Prov. Guanacaste, Bagaces, P. N. Palo Verde, Estacion Palo Verde, 10.349119-85.352345, 10 m, 20 June 1993, U. Chavarria (INBio). Female: INB0003300310 Costa Rica, Prov. Guanacaste, Hojancha, Z.P. Nosara, Hojancha, R.F. Monte Alto, 10.011248-85.402778, 400 a 500 m, 27 July \u2013 3 August 2000, H. Mendez (INBio). Female: INBIOCRI000674401 Costa Rica, Prov. Guanacaste, Liberia, P.N.Sta. Rosa, Playa Naranjo, 10.802713 \u2013 85.67479, 0\u201310 m, March 1991, E. Alcazar (INBio). Female: INB0003956448 Costa Rica, Prov. Guanacaste, Nicoya, San Antonio, Humedal Mata Redonda, 10.328094-85.42111, 8 m, 6 July 2005, B. Gamboa, J. Azofeifa, J. Gutierrez, M. Moraga, Y. Cardenas (INBio).2 Males: INBIOCRI000584965 Costa Rica, Prov. Guanacaste, Bagaces, Ref. Nac. Fauna Silv. R. L. Rodriguez, Estacion Palo Verde, 10.349119-85.352345, 10 m, May 1991, U. Chavarria (INBio). Male: INB0003072431 Costa Rica, Prov. Guanacaste, Bagaces, Pque. Nal. Palo Verde, Sector Palo Verde, 10.366668-85.383266, 0\u201350 m, 3 May 2000, H. Mendez (INBio). Male: INBIOCRI000386810 Costa Rica, Prov. Guanacaste, Liberia, P. N. Sta. Rosa, Playa Naranjo, 10.80275-85.666572, 0\u201310 m, May 1991, E. Alcazar (INBio). Male: INBIOCRI002426620 Costa Rica, Prov. Guanacaste, Liberia, Sector Las Pailas, 4.5 Km. SW del Volcan Rincon de la Vieja, 10.776784-85.351913, 800 m, 24 June 1995, K. Taylor (INBio). Male: INB0004065577 Costa Rica, Prov. Guanacaste, Liberia, Santa Rosa Nat. Pk., 10.83641-85.615491, 300 m, 4\u20136 December 1979, D. H. Janzen (INBio). Male: Male: 94-SRNP-2964 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 9 June 1994, Gusaneros. Male: 98-SRNP-9137 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa, 10.83764-85.61871, 295 m, 14 August 1998, Manuel Pereira. Male: 01-SRNP-17295 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 2 November 2001, Gusaneros. Male: 06-SRNP-13290 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 31 May 2006, Eilyn Camacho.PageBreakGuanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 1 June 2011, Daniel H. JanzenFemale: 92-SRNP-736 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Vado Nisperal, 10.80212-85.65372, 10 m, 20 May 1992, Gusaneros. Female: 96-SRNP-1331 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 31 May 1996, Gusaneros. Female: 96-SRNP-1332 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 2 June 1996, Gusaneros. Female: 98-SRNP-9134 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 2 August 1998, Guillermo Pereira. Female: 98-SRNP-9137 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 14 August 1998, Guillermo Pereira. Female: 01-SRNP-17336 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 01-SRNP-17336 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 07-SRNP-112736 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero los Patos (adult at light), 10.82097-85.63323, 251 m, 8 December 2007, H. Cambronero & S. Rios. Female: 11-SRNP-12732 (COI Barcoded), Costa Rica, Area Conservacion PageBreakAdults \u2013  in this genus, the species should be now in another genus.\u201d KIRBY (1892) lists Edema  as a junior synonym of Symmerista Hubner, [1821].Nystaleini, but it differs from that of Symmerista .Eulophidae:Euplectrus (n=1).Elymiotis tlotzin have been collected in the dry forest ecosystem of Peninsula de Nicoya, and in the dry forests of Sector Santa Rosa and Sector Pailas of ACG, at elevations of 0 to 800 m. (Adult o 800 m. .DNA barcode female 11-SRNP-12732.Elymiotis tlotzin | COI-5P:MHMYM2073-11 | 11-SRNP-12732 | PageBreakAACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCTTTAATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATGCCTATTATAATTGGAGGATTTGGAAATTGACTAGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAATATAAGATTTTGACTACTTCCACCCTCACTAACTTTATTGATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTTTATCCCCCCCTTTCATCTAA0TATTGCACATAGAGGAAGATCTGTAGATTTAGCAATTTTTTCACTTCATTTAGCTGGTATTTCATCGATTTTAGGAGCTATTAATTTTATTACAACGATTATTAATATACGACTTAATAACATAACTTTTGATCAAATACCTTTATTTGTTTGAGCAGTAGGAATTACAGCTTTTTTATTATTATTATCTTTACCTGTTTTAGCCGGAGCGATTACTATATTATTAACAGACCGTAATTTAAATACTTCATTTTTCGACCCTGCTGGTGGAGGAGATCCAATTCTTTATCAACATTTATTT"}
+{"text": "Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres  and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(\u2212) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2  expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells  Telomerase plays a central role both in the biology of normal aging, as well as in the development of human cancers , 2. HoweRecently, Clarke and colleagues have taken advantage of the properties of human telomerase (hTERT), to enrich for a population of osteosarcoma cells with stem-like properties . For thiHere, we have adapted this approach to the study of breast cancer stem-like cells, with a focus on proteomics analysis, biomarker discovery and cell metabolism. Importantly, we demonstrate that hTERT-high-activity breast cancer cells form mammospheres with higher efficiency, and show a proteomics profile consistent with an anabolic cancer stem cell phenotype. In support of this notion, we also show that hTERT-high-activity breast cancer cells have increased mitochondrial mass and activity, consistent with an increase in mitochondrial biogenesis.To enrich for a population of immortal CSCs, we exploited a sensitive eGFP reporter system for the fluorescent detection of high telomerase transcriptional activity. Briefly, MCF7 cells were transduced with a lentiviral vector driving eGFP protein expression, under the control of a 1.5-kB fragment of the hTERT promoter. This DNA construct also contains a puromycin-resistance cassette for antibiotic-resistance selection. Schematic diagrams illustrating this overall experimental strategy and the construction of the hTERT-promoter-vector are shown in Figures After selection with puromycin, MCF7-hTERT-eGFP cells were subjected to FACS analysis to visualize the broad distribution of eGFP expression, which serves as a surrogate marker of telomerase activity. Importantly, fewer than 1 in a 100 cells in MCF7-hTERT-eGFP cell monolayers visually showed high GFP fluorescence. In striking contrast, there was a dramatic enrichment of GFP(+) cells in MCF7 cell mammospheres (> 50 \u03bcm), each containing usually 1-to-2 GFP-high cells  and GFP-low (negative) groups. Then, five thousands cells from each group were seeded per well in 6-well low-attachment plates. These two groups were compared to the total unfractionated cell population.Remarkably, Figure In order to dissect the mechanism(s) by which high telomerase activity drives the survival and clonal expansion of CSCs, we next employed an unbiased label-free proteomics approach. The proteome of fractionated MCF7-hTERT-eGFP cells was directly determined, after FACS separation into GFP-high and GFP-low populations. For simplicity, we focused on the proteins that were over-expressed in GFP-high cells by > 1.5-fold. Our results are summarized in Tables Importantly, >30 mitochondrial-related proteins were over-expressed in GFP-high cells Tables . Most ofCSCs undergo an EMT, which facilitates cell migration, invasion and metastatic dissemination . Thus, wElevated protein synthesis is another important feature of the anabolic CSC phenotype . As predTaken together, hTERT-eGFP-high cells over-express >100 proteins related to an anabolic CSC phenotype.in vivo. For this purpose, we exploited a clinical data set of tumor samples from N = 28 breast cancer patients. These tumor samples were subjected to laser-capture micro-dissection, to separate epithelial cancer cells from adjacent tumor stroma [in vivo. Tables To determine the clinical relevance of our findings, we next assessed whether the hTERT proteomic targets that we identified in GFP-high cells were transcriptionally over-expressed, in human breast cancer cells r stroma . OverallTo directly validate the mitochondrial phenotype of hTERT-eGFP-high cells top 5%), we used two well-established fluorescent probes to quantitate mitochondrial membrane potential and mass, by FACS analysis. More specifically, we used MitoTracker Orange (561-nm) as a reporter for mitochondrial membrane potential and MitoTracker Deep-Red 640-nm) as a marker of mitochondrial mass. Importantly, Figures 0-nm as a%, we usein vivo [Previous studies using mouse mammary epithelial cells have demonstrated that stem-like cells can be enriched solely based on cell size . For exain vivo .larger cells  and ii) smaller cells   ) Figure . Interes) Figure .As such, larger cell size in MCF7 cells directly correlates with telomerase activity  and mitochondrial mass/activity, which would be consistent with an anabolic CSC phenotype. These results provide independent validation for the idea that high hTERT activity (\u201cstemness\u201d) is functionally associated with increased mitochondrial mass and activity in breast cancer cells, and co-segregates with large cell size. Importantly, large cell size is determined by increased PI3K/AKT/mTOR-signaling, which drives significant increases in overall protein synthesis \u201314. ThisHere, we have used an hTERT-promoter-eGFP-reporter system to identify and purify a sub-population of MCF7 cells, with high hTERT transcriptional activity, by FACS analysis. These hTERT-eGFP-high cells formed mammospheres with greater efficiency, as predicted, consistent with the idea that this sub-population of cells is enriched in cancer stem-like cells. Importantly, proteomics analysis of these hTERT-eGFP-high MCF7 cells revealed the upregulation of mitochondrial proteins, glycolytic enzymes and EMT markers, as well as components of the protein synthesis machinery, such as ribosome-related proteins and chaperones for protein folding. Interestingly, MT-CO2  expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-eGFP-high MCF7 cells. We then functionally validated that hTERT-eGFP-high MCF7 cells show increases in mitochondrial mass and activity, using two distinct MitoTracker probes. Complementary results were obtained using cell size to fractionate MCF7 cells. Larger stem-like cells showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, these two independent approaches for the enrichment of immortal anabolic CSCs should allow the development of new prognostic biomarkers and related novel anti-cancer therapies.Interestingly, recent studies in aging and cancer have both directly linked telomerase activity to mitochondrial function, via the hTERT-p53-PGC1 signaling axis \u201318. Moreprotein synthesis machinery  and ii) are associated with large cell size , which share several properties with CSCs or TICs \u201326. For We recently directly compared the proteome of MCF7 cell monolayers to MCF7-derived mammospheres, using label-free unbiased proteomics analysis. Consistent with our current findings, this analysis demonstrated that MCF7-derived mammospheres show the over-expression of >60 mitochondrial-related proteins and >80 components of the protein synthesis machinery , 29. ComImportantly, functional validation studies revealed that mammosphere formation could be efficiently blocked with well-established inhibitors of mitochondrial function and/or inhibitors of protein synthesis, such as oligomycin A and puromycin, respectively , 29. Morin vivo. Thus, the new hTERT metabolic targets that we identified here may be important for improving human breast cancer diagnosis and therapy.In summary, we show here that high telomerase activity metabolically defines a sub-population of anabolic CSCs, with increased mitochondrial mass and large cell size. Overall, greater than seventy hTERT targets  that we identified in hTERT-eGFP-high cells were also transcriptionally elevated in human breast cancer cells MCF7 cells were purchased from the ATCC. Gibco-brand cell culture media (DMEM and DMEM/F12) was purchased from Life Technologies. The lentiviral vector encoding the hTERT promoter linked to eGFP was custom-made by Genecopoeia (USA). MitoTracker probes (Deep Red and Orange) were purchased from Molecular Probes/Invitrogen, via Life Technologies. The telomerase inhibitor IX  was obtained commercially from Santa Cruz Biotech (USA).R cassette, and cloned into a lentiviral vector, custom-made by Genecopoeia: 5\u2032-TAAAATTGTGTTTTCTATGTTGGCTTCTCTGCAGAGAACCAGTGTAAGCTACAACTTAACTTTTGTTGGAACAAATTTTCCAAACCGCCCCTTTGCCCTAGTGCAGAGACAATTCACAAACACAGCCCTTTAAAAAGGCTTAGGGATCACTAAGGGGATTTCTAGAAGAGCGACCTGTAATCCTAAGTATTTACAAGACGAGGCTAACCTCCAGGAGCGTGACAGCCCAGGGAGGGTGCGAGGCCTGTTCAAATGCTAGCTCCATAAATAAAGCAATTTCCTCC GGCAGTTTCTGAAAGTAGGAAAGGTTACATTTAAGGTTGCGTTTGTTAGCATTTCAGTGTTTGCCGACCTCAGCTACAGCATCCCTGCAAGGCCTCGGGAGACCCAGAAGTTTCTCGCCCCTTAGATCCAAACTTGAGC AACCCGGAGTCTGGATTCCTGGGAAGTCCTAGCTGTCCTGCGGTTGTGCCGGGGCCCCAGGTCTGGAGGGGACCAGTGGCCGTGTGGCTTCTACTGCTGGGCTGGAAGTCGGGCCTCCTAGCTCTGCAGTCCGAGGCTTGGAGCCAGGTGCCTGGACCCCGAGGTTGCCCTCCACCCTGTGCGGGCGGGATGTGACCAGATGTTGGCCTCATCTGCCAGACAGAGTGCCG GGGCCCAGGGTCAAGGCCGTTGTGGCTGGTGTGAGGCGCCCGGTGCGCGGCCAGCAGGAGCGCCTGGCTCCATTTCCCACCCTTTCTCGACGGGACCGCCCCGGTGGGTGATTAACAGATTTGGGGTGG TTTGCTCATGGTGGGGACCCCTCGCCGCCTGAGAACCTGCAAAGAGAAATGACGGGCCTGTGTCAAGGAGCCCAAGTCGCGGGGAAGTGTTGCAGGGAGGCACTCCGGGAGGTCCCGCGTGCCCGTCC AGGGAGCAATGCGTCCTCGGGTTCGTCCCCAGCCGCGTCTACGCGCCTCCGTCCTCCCCTTCACGTCCGGCATTCGTGGTGCCCGGAGCCCGACGCCCCGCGTCCGGACCTGGAGGCAGCCCTGGGTCTCCGGATCAGGCCAGCGGCCAAAGGGTCGCCGCACGCACCTGTTCCCAGGGCCTCCACATCATGGCCCCTCCCTCGGGTTACCCCACAGCCTAGGCCGATTCGACCTCTCTCCGCTGGGGCCCTCGCTGGCGTCCCTGCACCCTGGGAGCGCGAGCGGCGCGCGGGCGGGGAAGCGCGGCCCAGACCCCCGGGTCCGCCCGGAGCAGCTGCG CTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTT-3\u2032.The following 1.5 kB sequence was used as the hTERT-promoter to generate the hTERT-eGFP-PuroR cassette were prepared and used to stably transduce MCF7 cells, according to the manufacturer's protocol (in the presence of 5 \u03bcg/ml polybrene). Twenty-four hours post-infection, media containing the virus was removed and replaced with standard media. Cells were then selected with puromycin (2 \u03bcg/ml), for up to 10 days. Please note that for most of the experiments described in this paper, we compared the GFP-high (top 5%) fraction to the GFP-low/negative (bottom 5%) fraction, unless stated otherwise. However, for label-free proteomics analysis, we compared the GFP-high (top 10%) population to the GFP-low (bottom 10%) population, to insure that enough material was collected for sample processing. For these experiments, singlet FACS gating of live cells was utilized.Lentiviral particles harboring the hTERT-eGFP-Puro2 in mammosphere medium (DMEM-F12/B27/20-ng/ml EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) . Cells were grown for 5 days and maintained in a humidified incubator at 37\u00b0C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres >50 \u03bcm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation, and was normalized to one (1 = 100% MSE). Mammosphere assays were performed in triplicate and repeated three times independently.A single cell suspension of MCF7-hTERT-eGFP cells was prepared using enzymatic , and manual disaggregation (25 gauge needle) . Cells wCells were treated on day 0 with MST-312 at a concentration of 10 \u03bcM and compared to vehicle alone controls, processed in parallel, after 5 days of mammosphere culture. A dose-response analysis of the effects of MST-312 on parental MCF7 cells was first performed to establish an effective concentration for the inhibition of mammosphere formation; our results showed that 1 \u03bcM had little or no effect, while 10 \u03bcM inhibited mammosphere formation by approximately 70% (data not shown). Therefore, experiments with eGFP fractionated MCF7 cells were carried out at 10 \u03bcM.Fluorescent imaging of MCF7 cells in adherent conditions and non-adherent spheroid culture was performed using a Leica SP8 multi-photon DM6000 microscope to detect GFP expression and bright field images.Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS . Then, tN = 28 human breast cancer patients) [To firmly establish the clinical relevance of our results from the quantitative proteomics, we re-analyzed the transcriptional profiles of epithelial breast cancer cells and adjacent tumor stromal cells that were physically separated by laser-capture microdissection (from atients) .For live cell sorting experiments, hTERT-eGFP transfected MCF7 cells were resuspended in PBS and sorted according to eGFP expression using the BD influx. Untransfected MCF7 cells were used as a negative control to determine positive GFP expression. Cells were sorted into two groups, those with the highest GFP expression and those with the lowest GFP expression. For FACs analysis experiments, hTERT-eGFP cells were labeled with MitoTracker Deep Red  and MitoTracker Orange . Cells were kept of ice until analysis using the FACS Calibur. Results were analyzed using FlowJo software version 10. Cells were gated into two populations, those with the highest 5% of GFP expression, and those with the lowest 5% and negative GFP expression. The median fluorescent intensity of MitoTracker Deep Red (640nm) and MitoTracker Orange (561nm) was then determined within the two distinct GFP cellular populations. Additionally, cells were discriminated by cell size into two populations, small and large using SSC (side scatter) and FSC (forward scatter). The median intensity of eGFP, MitoTracker Deep Red, and MitoTracker Orange, within these populations, was then determined.To measure mitochondrial activity, cells were stained with MitoTracker Orange , whose accumulation in mitochondria is dependent upon membrane potential. To measure mitochondrial mass, cells were stained with MitoTracker Deep Red , localizing to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution  for 30-60 min at 37 \u00b0C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, and re-suspended in 300 \u03bcL of PBS. Cells were then analyzed by flow cytometry. Data analysis was performed using FlowJo software.hTERT-eGFP-MCF7 cells, co-labeled with MitoTracker dyes, were analyzed by cell size using FlowJo software. Cells were separated by gating of forward scatter (FSC) and side scatter (SSC) plots into two populations for analysis, large cells  and small cells . The median fluorescent intensity of eGFP and MitoTracker dyes were determined in each population of cells. Very similar results were obtained, with either singlet FACS gating or all live cell FACS gating.Statistical significance was determined using the Student's t-test or ANOVA, where appropriate. Values of less than 0.05 were considered significant. Data are shown as the mean \u00b1 SEM, unless stated otherwise."}
+{"text": "Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the  The orphan ligand Dilp8 has been shown to coordinate growth and developmental timing in Drosophila. Here, using Gal4 drivers and CRISPR/Cas9 approaches, Garelli et al. identify a role for relaxin-like receptor Lgr3 in regulating the Dilp8 developmental delay pathway. Drosophila, the insulin/relaxin-like peptide (Dilp8), which ensures organ and body size coordination3dilp8 uncouples the endocrine communication between imaginal discs and the prothoracic gland, making dilp8 mutants susceptible to uncoordinated disc growth when intrinsic errors of development or noxious environmental stimuli affect the growth status of one or more discs. This results in an increase in random deviations from bilateral symmetry (fluctuating asymmetry (FA)), measurable at the population levelDrosophila3456How different organs in the body sense growth perturbations in distant tissues to coordinate their size and differentiation status during development is poorly understood1bona fide relaxin peptide homologues89Drosophila genome encodes two orphan receptors, Lgr3 and Lgr4, with clear homologies to vertebrate relaxin receptors  neurons.Type C1 Leucine-rich repeat-containing G protein-coupled receptors (Lgrs) are a conserved protein family in metazoans that act as receptors for insulin-like peptides of the relaxin subfamily in vertebrates, where they play diverse roles in tissue homeostasis and remodelling, behaviour and reproductionLgr3, we remobilized an MB Minos elementLgr3+/+), which served as genetic background controls, and one imprecise excision allele, Lgr3ag1, which consists of a 3.8-kb deletion that completely removes exon 8 and partially removes exon 9 (Lgr3ag1 produces a transcript with a premature termination codon (PTC) that truncates the Lgr3 protein one amino acid after D326  are increased by an order of \u223c3 when compared with their Lgr3+/+ controls  under control of the wing-pouch Beadex-Gal4 (Bx>) driver3Lgr3+/+ animals ; To generate a mutant for s exon 9 . Lgr3ag1ter D326 . We concarea FAi ). This p animals . This deLgr3 should be necessary for the developmental delay produced by ectopic Dilp8 expression in the absence of tissue growth abnormalities3UASP-dilp8::3xFLAG (UAS-dilp8a) transgene under the control of armadillo-Gal4 (arm>) was suppressed in larvae homozygous for Lgr3ag1 or trans-heterozygous for Lgr3ag1 and a deficiency that completely uncovers the Lgr3 locus (Lgr3Df(3)BSC321) )tubulin-Gal4 (tub>), and reduced Lgr3 activity by concomitant RNA intereference (RNAi) knockdown using a short hairpin (TRiP VALIUM22 (Lgr3-IR-V22)) lineLgr3 mRNA levels by \u223c85%  (Lgr3 completely suppressed the dilp8-dependent delay (Lgr3 (TRiP VALIUM10 (Lgr3-IR-V10))dilp8-dependent delay, albeit to a lesser extent than Lgr3-IR-V22 (Lgr3 mRNA levels (\u223c50%) than Lgr3-IR-V22  . Coherennt delay . A secon3-IR-V22 . This pa3-IR-V22 , suggestLgr3 at the protein level, we used CRISPR/Cas9-mediated homologous repair15Lgr3ag5, hereafter named sfGFP::Lgr3, which contained an intact Lgr3-coding sequence downstream of the sfGFP insertion  indels in the Lgr3-coding region , which induces apoptosis, tissue damage/regeneration, an imaginal-disc-specific Dilp8 upregulation and consequentially a robust delay in the onset of metamorphosissfGFP::Lgr3 ag5 allele encodes a functional receptor. Together, these results strongly suggest that the Lgr3 protein acts in a subpopulation of CNS neurons.To gain insight into the tissue and cellular expression pattern of nsertion . sfGFP::nsertion . No otheof Dilp8 . The effg region  or by usWe were unable to detect sfGFP::Lgr3 expression in neurons directly innervating the ring gland , suggestsfGFP::Lgr3 neurons in more detail. sfGFP::Lgr3 expression was not homogeneous in the \u223c180 CNS cell bodies. It was most strongly expressed in a single pair of neurons in the very tip of the VNC, in a single dorso-ventral pair of midline neurons located deep in the thoracic segment of the VNC ), and in a pair of bilateral neurons localized in the anterior part of the pars intercerebralis ) -Gal4 driving a UAS-myr::tdTomato reporter indicates that many Lgr3-positive neurons are cholinergic, including all three major neuronal populations -Gal4 and Vesicular glutamate transporter promoter (VGlut)-Gal4, which drive expression in GABAergic and glutamatergic neurons, respectively, label very faintly the PIL neurons and the pair of distal VNC neurons and show no detectable staining in MIL neurons ) . All thrL1 stage . Co-stai neurons ). Co-sta neurons . MIL neu neurons . PIL neu neurons . The lat neurons . PIL neu neurons . These rLgr3-IR-V22 RNAi line to the panneuronal driver elav-Gal4 (elav>) and performed EMS assays -Gal4 did not rescue the EMS-induced delay  and GMR19B09-Gal4 (GMR19B09>)  throughout embryonic and larval development was compatible with developmental progression and led to a significant suppression of the EMS-dependent delay  , selecteMS-delay , the GMRhoc test . These rGMR19B09> driver labels \u223c270 neurons, \u223c30 of which populate each brain hemisphereGMR19B09> neurons with myr::tdTomato in a sfGFP::Lgr3 background, we detect overlapping expression in \u223c10 neurons per hemisphere, 2 of which are the bright PIL neurons  line drives expression in similar type of neurons, named #5 neurons25. MZ699>myr::tdTomato expression analysis in a sfGFP::Lgr3 background demonstrates that PIL neurons are a subset of #5 neurons -(FRTmCherry) luciferase reporterCRE-luciferase and the sfGFP::Lgr3 reporters  in heterologous studies in human cell lines ery cell . We concDrosophila SL2/DL2 cells. In this assay, Dilp8 is covalently attached to TriCEPS2727Drosophila insulin-like receptor (InR)Drosophila cells  assays27la cells . An endoCTLH2) -like factor derailed (Drl), neuroglian (Nrg), the laminin receptor integrin \u03b1-PS3 (ITA3) and the choline transporter (CTL)-like protein 2 (CTLH2) . Any of CTLH2) . While tDrosophila. Our study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations , and relayed through one or more steps before reaching the Lgr3-positive cells , it is tempting to propose a direct ligand\u2013receptor interaction between them. This possibility is supported by the strong genetic interaction between in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique we used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell . However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissueThor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signallingThor levels are higher in imaginal discs in the same animalsSecond, we have failed to identify Lgr3 among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl37Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons , indicatsfGFP::Lgr3 or GMR19B09>myr::tdTomato expression in the ring gland or in neurons innervating the ring gland , UAS-rpr , Lgr3-IR-V10 , Lgr3-IR-V22 , elav-Gal4 (P{w[+mW.hs]=GawB}elavC155), elav-Gal4 (P{w[+mC]=GAL4-elav.L}2/CyO), w1118;Mi{ET1}Lgr3MB06848, w1118; snaSco/SM6a, P{w[+mC]=hsILMiT}2.4, tub-Gal4 , w1118; Lgr3Df(3)BSC321/TM6C Sb1 cu1, Gad1-Gal4 (P{w[+mC]=Gad1-GAL4.3.098}2/CyO), Cha-Gal4 , GMR19B09-Gal4 , GMR17G11-Gal4 , y1 w* P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::RFP}attP18 P{y[+t7.7] w[+mC]=13XLexAop2-mCD8::GFP}su(Hw)attP8, and w1118; P{y[+t7.7] w[+mC]=GMR19B09-lexA}attP40w were obtained from the Bloomington Drosophila Stock Center at Indiana University. The stock 1 M{w[+mC]=Act5C-Cas9.P}ZH-2A w*y (reference #16) was obtained from Bestgene. The stock arm-gal4  was a gift from P. Domingos. The stocks UAS-dilp8b::3XFLAG and UAS-dilp8c::3XFLAG (reference #3) were a gift from M. Dominguez. The stock w*; P{GawB}Mz699 and UAS-myr::tdTomato/CyO; TM2/TM6B were a gift from M.L. Vasconcelos. Balanced lines were generated by crosses to the stock w1118; If/CyO; MKRS/TM6B, which was a gift from A. Jacinto. The stock w; CRE-F-luc (II) was a gift from J.C. Yin. The stock y w; ptth-HA was a gift from M. O'Connor. Stocks are maintained at low densities at 18\u2009\u00b0C in a 12-h light/dark cycle.The Drosophila Lgr3 locus by using the MiET1 transposase to remobilize the MB Minos element Lgr3MB06848 (reference #11), which is inserted in the seventh Lgr3 intron, <100\u2009bp from exon 7  and transferred to new vials every dayCy-Minos/+; Lgr3MB06848/MKRS or TM6B male adults were selected and individually crossed to the balancer strain w1118; If/CyO; MKRS/TM6B. A single eGFP-negative (lacking the Mi{ET1} element) male was selected from each cross and mated with w1118; If/CyO; MKRS/TM6B females. The putative Lgr3 excisions were balanced over TM6B to obtain the following genotypes w1118; +/+; Lgr3MB06848excision/TM6B. We obtained one imprecise excision that generated the Lgr3ag1 mutant allele and three precise excisions named Lgr3ag2, Lgr3ag3 and Lgr3ag4 all of which behaved the same way. In this study, the Lgr3ag2 line was used as the genetic background control for the imprecise excision allele Lgr3ag1.We generated a mutation in the m exon 7 . Virgin Lgr3ag1 deletion, we performed a series of PCR assays with primer pairs located around the Lgr3MB06848 insertion . This suggested that while the Lgr3ag1 deletion was very large, its breakpoints were confined within the Lgr3 locus. We then tried to amplify a 5.3-kb PCR product with a primer upstream of the Lgr3MB06848 position  are lacking in the Lgr3ag1 leucine-rich repeat domains, which are critical for relaxin ligand binding in vertebrate relaxin receptorsTo molecularly characterize the nsertion . Lgr3ag1r3 locus . This alLgr3ag1 and controls Lgr3+/+ and the original Lgr3MB06848 stock. Lgr3ag1 generated a smear with a major band that was \u223c200\u2009bp smaller than the other control genotypes \u2013PCR analyses with mRNA isolated from o exon 9 . The resn 9 (326 . We concterminus .Drosophila and Homo sapiens codon-optimized complementary DNA (cDNA) corresponding to full-length Lgr3 was synthetized de novo and cloned into pUASPpUASP-Lgr3. This plasmid was injected into w1118 and two independent insertions were tested, pUASP-Lgr3a  and pUASP-Lgr3b . A similar protocol was used to place the dilp8::3xFLAG construct described in reference #3 into pUASP, making pUASP-dilp8a.A http://tools.flycrispr.molbio.wisc.edu/targetFinder/)We used a CRISPR/Cas9-mediated homologous repair strategy15gRNA 1Fw: 5\u2032-CTTCGGAGCACTCAATTCCCACTC (CGG)-3\u2032Rv: 5\u2032-AAACGAGTGGGAATTGAGTGCTCC-3\u2032gRNA 2Fw: 5\u2032-CTTCGCAAACTCAAGTAGAATATCA (CGG)-3\u2032Rv: 5\u2032-AAACTGATATTCTACTTGAGTTTGC-3\u2032The PAM regions (CGG) are located right after the forward primers of both gRNAs 1 and 2, as shown above.As a repair cassette we designed the following sequence, where each colour represents the following:Lgr3 ATG siteOrange: Homology region up to Lgr3 ATGRed: Green: sfGFPBlue: Spacer (GSGSGS)Lgr3 ATG siteViolet: Homology region after ATGCGTAAGGGCGAGGAGTTGTTCACGGGAGTTGTGCCCATATTGGTTGAGCTGGATGGAGATGTGAATGGCCACAAGTTCAGTGTGCGGGGTGAGGGAGAAGGAGACGCAACAAACGGTAAGCTGACACTGAAGTTCATTTGTACTACGGGCAAGCTCCCGGTGCCATGGCCCACATTGGTCACCACCCTGACCTATGGCGTGCAATGCTTCGCCCGATATCCAGATCATATGAAGCAGCATGATTTCTTTAAGTCGGCCATGCCCGAGGGTTACGTACAAGAGCGCACTATTAGCTTTAAGGACGACGGTACGTATAAAACCAGGGCTGAGGTGAAGTTTGAGGGTGATACCCTGGTGAACCGCATTGAATTGAAGGGCATCGATTTTAAGGAGGACGGCAACATCCTGGGCCACAAGCTCGAATATAATTTTAATAGCCATAATGTTTACATTACCGCGGACAAGCAGAAGAATGGAATTAAGGCTAATTTCAAGATCCGACATAATGTGGAGGACGGATCCGTTCAGTTGGCCGATCACTACCAGCAAAACACCCCCATCGGAGATGGCCCCGTCCTGCTGCCCGATAACCACTACCTGAGTACCCAGTCCGTCCTGTCGAAGGATCCTAATGAGAAGCGGGATCATATGGTGCTGCTGGAGTTTGTGACTGCCGCCGGCATAACGCATGGAATGGACGAGCTGTATAAAGGCTCCGGTAGTGGTTCCGTCTACGGCAGGAGCATCGCCGTAGGCTTCTGTCTGATGACCGTCGTCCTTCTGCTGGCCGCCGTGATATTCTACTTGAGTTTGGGTGAGTCCTTAGAGTGATGTCCTTTCAAAATTCCATCATTCGCAAACCTAAATAATTTCTGAATCAAGAATGTTCAAAATCTTAGCAATTATTATACGCATAATTTGTGAAACTACTTAAAGTTCTTTTAAAACTTGAGCTGCTGTAAATTTCTATATATACTTTCGTATCCTTAAAGGGTTCCTTCGCTTGAAGCAAAAACCAAAATCAAATTCCAAACTGCAAA-3\u20325\u2032-CACTTAAAACTCTTCTCCGCGAGCTGTGAACATTAGCCAAATGAAGTGACAAGAAATTAACGCAAAAATAAAACAAGAAGACGGAGCGGTATAAGAAATAATAATATAAAAACTCAATGAGTCAGCACCGCATCAGCTCCTGCTGCTGTTGTTCTTCTTATTGCTGTTGTTTGTGGGGGCGTGGCCGGAGTGGGAATTGAGTGCTCCTAATGATGAACTCGGTCAAGGAGCCAGTGCAGCCATGGTGGCCAAGTAATTAGATAAGCGAGCGTGCAAAACAGGAGCAAACCGATAAATCGCCde novo into a pUC57 plasmid and co-injected with the two gRNAs into the stock y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*], which strongly and ubiquitously expresses a human codon-optimized cas9 . Ten negative hits were also sequenced and two were retained as background controls (ag10 and ag11). The y1M{w[+mC]=Act5C-Cas9.P}ZH-2A w* cassette was removed by selecting against eye colour and stocks were kept as homozygous stocks, except for allele ag8, which was kept balanced over TM6B.These primers were designed to indicate the correct insertion of the sfGFP repair cassette from both sides in the genome. Positive hits were sequenced . The next morning, the flies were transferred to a fresh plate to lay eggs for 3\u20136\u2009h, depending on the experiment. To control for overcrowding, between 10 and 30 larvae were transferred to vials containing normal Egg collections were performed on normal food plates and larvae were reared at controlled densities without additional yeast . Newly molted third instar larvae were collected every 2\u2009h as described previously48Larvae were collected as described above and transferred 72\u2009h after egg laying to fresh food with 10\u2009mM of EMS (Sigma) or PBS as control. Developmental time was measured as indicated above. EMS food was prepared as follows: food was melted and cooled to 55\u2009\u00b0C, an appropriate volume of freshly made EMS stock solution in PBS was added and thoroughly mixed and 3\u2009ml per tube were dispensed. EMS and PBS tubes were kept in the dark as much as possible throughout the experiments.Brains of wandering third instar larvae or first instar larvae were dissected in cold PBS, fixed for 30\u2009min in 4% paraformaldehyde, rinsed with PBS with Triton (0.3%) (PBST), incubated with primary antibody overnight and with fluorescently labelled secondary antibody for 2\u201324\u2009h in PBST with 1% bovine serum albumin. Nuclei were counterstained with DAPI (Sigma) and tissues were mounted in Fluoromount-G (Southern Biotech). Antibodies used were: mouse anti-GFP 1:200 , mouse anti-nc82 1:250 (DSHB), mouse anti-HA 1:50 , rabbit anti-GFP 1:200 , rabbit anti-Dilp7 1:5000 (gift from I. Miguel-AliagaAleft-Aright/[(Aleft+Aright)/2]. Results were compared statistically using the F-test for the significance of the difference between the FAi of the samples, an appropriate test for dispersion3\u03b1/n) for multiple comparisons was applied to \u03b1=0.05.Pairs of the left and right wings of male individuals reared at 29\u2009\u00b0C and rinsed with ethanol were dissected in a glycerol/ethanol solution and mounted in glycerol. Photos were obtained in a Zeiss Axiovert 40 CFL microscope. The wing areas and wing lengths were calculated as previously described3Lgr3+/+ by a factor of \u223c23 .To control for measurement errors, we measured the area of the same wing three times. Values obtained were 18310.2\u00b130.5\u2009a.u., which gives a coefficient of variation (CV) of 0.17%, which is smaller than the CV of wing areas of control \u22121 (Roche), and incubated the mixture at 37\u2009\u00b0C for 1\u2009h, followed by 95\u2009\u00b0C for 5\u2009min, to inactivate the protease.gDNA was extracted from a group of flies or single fliesg for 1\u2009min, to lower tissue debris. An extra DNAse treatment  was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT\u2013quantitative PCR (Thermo Scientific), following manufacturer's instructions.RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research), following manufacturer's instructions. The material used for the RT\u2013PCR experiments described in For this study, PCR and RT\u2013PCR primers were designed and their specificity tested using Primer BLAST or Primer3. A T100 Thermal Cycler (Bio-Rad) was used for performing the PCR steps. The following primers were used for PCR analyses described in Lgr3_salto_fw (expected product size 866\u2009bp)Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-TTTATGGAGCGGGCGTGGTC-3\u2032Lgr3_exonshort Lgr3 (expected product size 331\u2009bp)Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-GTGCGTTATGAGGTTGTGCTG-3\u2032Lgr3_exon3p Lgr3 (expected product size 240\u2009bp)Fw: 5\u2032-CGCCTTGTCGGTAATCCCAT-3\u2032Rv: 5\u2032-GTGGCTCCATTAAACTGCTGC-3\u2032Lgr3_exons Fw: 5\u2032-CCGACGCCTTGCTGCTAACT-3\u2032Rv: 5\u2032-CAAAGACCACCAACCAGGCGTA-3\u2032rp49 (control)Fw: 5\u2032-TTGAGAACGCAGGCGACCGT-3\u2032Rv: 5\u2032-CGTCTCCTCCAAGAAGCGCAAG-3\u2032qRT\u2013PCR experiments were performed using Lightcycler 96 (Roche) using the FastStart Essential DNA Green Master dye and polymerase (Roche). The final volume for each reaction was 10\u2009\u03bcl, consisting of 5\u2009\u03bcl of dye and polymerase (master mix), 2\u2009\u03bcl of 10 \u00d7 diluted cDNA sample and 3\u2009\u03bcl of the specific primer pairs. The following primers were used:rp49 (control)Fw: 5\u2032-TTGAGAACGCAGGCGACCGT-3\u2032Rv: 5\u2032-CGTCTCCTCCAAGAAGCGCAAG-3\u2032Lgr3Fw: 5\u2032-GCTGGGTGCCCATCATCGTTAT-3\u2032Rv: 5\u2032-CAAAGACCACCAACCAGGCGTA-3\u2032InRFw: 5\u2032-TGTCAGCTGCACAATAATAGGC-3\u2032Rv: 5\u2032-TGCACTTTTCAGGGCATTT-3\u2032DrlFw: 5\u2032-CGGAGTTCCATACCCAGATTAC-3\u2032Rv: 5\u2032-GCCTCTTGTTATATTTACAGGTCTTGG-3\u2032rp49 according to the formula: %rp49=(2\u0302\u2212(\u0394CqLgr3\u2212\u0394Cqrp49)) \u00d7 100. The geometric mean\u00b1s.e.m. of three biological repeats was used and data were analysed by one-tailed unpaired Student's t-test using Bonferroni corrections (\u03b1/n) for multiple comparisons, using \u03b1=0.05. This test assumes the independent samples have equal variances.Primer efficiency for qRT\u2013PCR was tested by serial dilution. Data were expressed as %9Drosophila SL2/DL2 cells (ATCC) cultured in Schneider medium supplemented with 10% foetal bovine serum. The cells were gently oxidized with NaIO4 and TriCEPS\u2013ligand complexes were incubated with the cells for 1.5\u2009h to allow capture of oxidized glycomoieties. Following cell lysis, trypsinization and biotin-mediated affinity purification of ligand\u2013receptor peptides, the glycosylated peptides were selectively released using PNGase F, and samples were analysed by Dualsystems Biotech AG on an LTQ Orbitrap XL (Thermo Scientific) spectrometer fitted with an electrospray ion source. The samples were measured in data-dependent acquisition mode in a 40-min gradient using a 10-cm C18 column. Peptide identifications were filtered to a false-discovery rate (FDR) of \u22641%   and qua014 ref. . For stav2) ref. . The adjIn all experiments reported in this work, no data point was excluded. All data points, including outliers, are represented in the figures and were used in the statistical analyses. No blinding was done and no particular randomization method was used to attribute individuals to experimental groups.Accession codes: DNA sequence data has been deposited in GenBank under accession codes KT321103-KT321113.How to cite this article: Garelli, A. et al. Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat. Commun. 6:8732 doi: 10.1038/ncomms9732 (2015).Supplementary Figures 1-15 and Supplementary Tables 1-2"}
+{"text": "Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.DOI:http://dx.doi.org/10.7554/eLife.08828.001 Mitochondria are structures within cells that provide the energy to power many biological processes that are essential for complex life. These structures are also highly dynamic and go through cycles of fission (in which a single mitochondrion splits in two) and fusion (in which two mitochondria merge into one). These processes both maintain the correct number of mitochondria in a cell and remove damaged ones, and defects in either can result in many diseases.Previous research had shown that mitochondria are in close contact with another cellular structure called the endoplasmic reticulum. The points of contact mark the sites where mitochondria undergo fission, as small tubes of the endoplasmic reticulum wrap around, and then constrict, to split a mitochondrion.Other recent work revealed that a protein called INF2 is anchored on the endoplasmic reticulum where it promotes mitochondrial constriction. This protein builds actin subunits into long filaments that provide the force for constriction. However, it was not clear how INF2 became active, and whether there are proteins on mitochondria that interact with INF2 or actin.Manor, Bartholomew et al. have now used a combination of microscopy-based methods and biochemical analysis to discover that a mitochondrial protein called Spire1C performs all of these roles. Spire1C is found on the outer membrane of mitochondria; it interacts with INF2 to drive the formation of actin filaments that constrict mitochondria. These results suggest that Spire1C bridges the endoplasmic reticulum with the network of actin filaments. Further experiments then showed that increasing Spire1C levels in cells resulted in the mitochondria becoming fragmented due to increased constriction. On the other hand, depleting Spire1C had the opposite effect and caused mitochondria to become unusually elongated. Following on from this work, the next challenge is to see if Spire1C is used differently or similarly in the different processes that involve mitochondrial fission.DOI:http://dx.doi.org/10.7554/eLife.08828.002 Mitochondrial division is a complex process that is essential for survival in mammals  and is fInverted formin 2 (INF2) is a formin family protein that promotes actin filament polymerization in a regulated fashion , 2014. ASpire proteins are membrane-binding actin-nucleators that interact with and regulate formin proteins . SynergiVertebrates have two known Spire genes, Spire1 and Spire2. Each Spire protein contains highly conserved domains with specific capabilities, including: four actin-monomer binding WH2 domains necessary for nucleating actin filaments; an mFYVE domain that binds to intracellular membranes and facilitates oligomerization ; and an We identified and characterized a novel alternate splice-isoform of Spire1 that contains KIND and WH2 domains common to all Spire proteins, as well as a previously uncharacterized unique 58 amino acid alternate exon sequence (ExonC) . After dWhen cells were transfected with a myc-tagged Spire1C construct, the protein showed extensive co-distribution with the mitochondrial marker mitoRFP  also shoTo examine whether Spire1C distributes on the surface or interior of mitochondria we compared the distribution of GFP-ExonC (marking Spire1C) with mitoRFP  using structured illumination microscopy (SIM), which gives a twofold resolution improvement over conventional confocal imaging . GFP-ExoTo confirm Spire1C's mitochondrial outer membrane localization, we employed a fluorescence protease protection (FPP) assay , which cIn support of this notion, transmembrane domain prediction software  indicateWe next performed photobleaching experiments to investigate the dynamics of Spire1C's association with mitochondrial membranes. Photobleaching of a portion of a mitochondrial element that expressed GFP-Spire1C resulted in rapid recovery of fluorescence, with replenishment arising first in regions close to the bleach site and later at regions further away , similarGiven that Spire proteins can nucleate actin via their highly conserved WH2-repeat domain , we nextGiven that actin assembly has been shown to play an important role in regulating mitochondrial fission , the obsSince ER tubules have been implicated in mitochondrial constriction and division , we inveOne way the KIND domain of Spire1C could affect ER-mediated mitochondrial division is by binding to ER-anchored INF2. To test this possibility, we performed in vitro GST pull-down assays. We found that the C-terminal half of INF2 (INF2-CT), but not the N-terminal half (INF2-NT), associated with a GST-tagged Spire1C KIND domain, but not GST alone . AdditioWe next tested whether disrupting Spire1C's interaction with INF2 inhibits mitochondrial fission. To test this hypothesis, we first asked whether removing the KIND domain from Spire1C blocks the increase in mitochondrial division associated with overexpressing a constitutively active INF2 mutant, INF2 A149 , 2014. CMitochondrial fission would be co-dependent on Spire1C and INF2 if Spire1C's interaction with INF2 drives ER-mediated mitochondrial constriction. To test this hypothesis, we employed confocal fluorescence imaging of ER and mitochondria to examine mitochondrial constriction sites in cells co-expressing the ER marker Ii33-mCherry and different variants of Spire1C. Mitochondria constriction was visible at sites of ER-mitochondria crossover slightly more frequently in cells overexpressing Spire1C compared to cells not overexpressing the construct . U2OS cells stably expressing GFP-INF2 was described in Ii33-mCherry was a generous gift from P Satpute . MitoEmerald and mitoRFP were gifts from A Rambold . Spire1C was amplified from mouse brain cDNA using the sequence of NM_194355 as a reference. We expected to obtain a sequence yielding protein corresponding to GI 37595748, however, it contained an additional 58 residues (ExonC). Thorough examination of all available mammalian Spire1 isoforms revealed at least 3 alternatively-spliced exons, which we refer to as exons A (majority of KIND domain), B (protein sequence AVRPLSMSHSFDLS), and C (protein sequence VPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSK).To obtain a full-length Spire1C construct, we amplified the mouse Spire1 gene AK129296, which contains the full KIND domain through the first 3 WH2 domains, along with NM_194355, which contains a partial KIND domain and ExonC without Exon B. A series of amplifications of partial gene sequences was then performed to obtain versions of mouse Spire1 that were \u00b1 each of exons A, B, and C . Each vaGGCGCGCCATGGAACTGCATACATTTCTGACCAAAATTAAGAGPrimer 1 GCGCTTAATTAATCAGATCTCGTTGATAGTCCGTTCTGAAGPrimer 2 GCGCGGCGCGCCATGGCCAATACCGTGGAGGCTGPrimer 3 GAGCATTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 4 GGCGGGCGCGCCATGGCGCAGCCCTCCAGPrimer 5 GACTTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 6 GGCGPrimer 7 CCATGTGCTCCAGGAAGAAGCCPrimer 8 CTGCCTTCCAAGCCATACTCTACTCTACPrimers for spire1 gene amplification and plasmid constructionunderlined.All primers are 5\u2032 to 3\u2032. AscI or PacI restriction sites are CGAGGCTGCAGATGAAGGCCCGGAAGATGAAGACGGAGAGAAGAGAAGCATCTCAGCCATCCGGTCCTATCAGGACGTTATGAAGATCTGTGCTGCTCACCTCCCAACTGAGTCGGAGGCACCCAATCATTATCAGGCAGTATGTCGGGCCCTGTTCGCAGAAACCATGGAACTGCATACATTTCTGACCAAAATTAAGAGTGCAAAGGAGAACCTTAAGAAGATTCAAGAAATGGAAAAGGGTGATGAATCTAGCACAGATCTGGAGGACCTGAAAAATGCAGACTGGGCCCGGTTCTGGGTACAAGCGGCGAGGGATTTGCGAAATGGGGTAAAAGCTAAGAAAGTCCAGCAGCGGCAGTACAACCCTCTGCCCATTGAGTACCAACTGACCCCTTATGAGATGGCCGCGGACGACATTCGGTGCAAAAGATACACCGCGAGAAAAGTAATGGTAAATGGTGACGTCCCCCCTCGGTTGAAAAAGAGTGCTCATGAGGTCGCCGCTGACTTTATCAGATCAAGACCCCCTGCAAATCCAGTTTCAGCCAGAAAACTGAAACCAACCCCACCACGGCCACGGAGCCTCCATGAAAGAGCAGCAGAAGAAATTAAAGCAGAAAGAAAGGCTCGGCCTGTGTCACCAGAAGAAATTAGACGGAGCAGACTAGCAGTGCGGCCACTTAGCATGTCTCACAGTTTTGACTTGTCAGATGTCACTACGCCAGAATCTCCAAAGAATGTTGGAGAATCATCTATGGTGAATGGAGGCTTAACATCTCAAACAAAAGAAAATGGGCTGAGCGCTGCCCAGCAGGGGTCMAQPSSPGGEGPQLGAAGGPRDALSLEEILRLYNQPINEEQAWAVCFQCCGSLRAAAARRQPHRRVRSAAQIRVWRDGAVTLAPAAAAAAEGEPPPASGQLGYSHCTETEVIESLGIIIYKALDYGLKENEERELSPPLEQLIDQMANTVEADGSKDEGYEAADEGPEDEDGEKRSISAIRSYQDVMKICAAHLPTESEAPNHYQAVCRALFAETMELHTFLTKIKSAKENLKKIQEMEKGDESSTDLEDLKNADWARFWVQVMRDLRNGVKLKKVQQRQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMVNGDVPPRLKKSAHEVILDFIRSRPPLNPVSARKLKPTPPRPRSLHERILEEIKAERKLRPVSPEEIRRSRLAVRPLSMSHSFDLSSPKNVGESSMVNGGLTSQTKENGLSAAQQGSAQRKRLLKAPTLAELDSSDSEEEKSLHKSTSSSSASPSLYEDPVLEAMCSRKKPPKFLPISSTPQPERRQPPQRRHSIEKETPTNVRQFLPPSRQSSRSLDVTTPEVPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSKEEFCYPVECLALTVEEVMHIRQVLVKAELEKYQQYKDVYTALKKGKLCFCCRTRRFSFFTWSYTCQFCKRPVCSQCCSDEELQFPKELMEDWSTMEVCVDCKKFISEIISSSRRSLVLANKRARLKRKTQSFYMSSAGPSEYCPSERTINEIKKMRLPSKPYSTLPIFSLGPSALQRGESCSRSEKPSTSHHRPLRSIARFSTKSRSVDKKIND domainWH2 domains Alternate exon BAlternate ExonCSpire boxmFYVE domainSpire1 shRNA constructs were generated by cloning the sequences into Clontech's pSingle-tTS-shRNA vectors using the HindIII/XhoI restriction sites. The sequences cloned into the vector to knockdown Spire1C were 5\u2032-TCGAGGGATTAGACGTAGCAGATTATTCAAGAG ATAATCTGCTACGTCTAATCTTTTTTACGCGTA-3\u2032  and 5\u2032-TCGAGGCGAATAATCTCCTGACTAATTCAAGAGATTAGTCAGGAGATTATTCGTTTTTTACGCGTA-3\u2032 . Oligonucleotides for human INF2 siRNA were previously described in spire1C gene. A mouse tissue cDNA panel  was used as a template for amplification using Primers 7 and 8. Amplified DNA containing ExonC was \u223c400 bp, while DNA lacking this exon was \u223c200 bp. Multiple oligomer sets were utilized to eliminate non-specific amplification while capturing as many on-target amplifications as possible. PCR reactions were run on a 2% agaorse gel, and bands of the appropriate size were excised from the gel and purified using a QIAquick gel extraction kit . Purified DNA was cloned into the pCR II-TOPO vector using the TOPO-TA cloning kit (Invitrogen). Sequence analysis was used to confirm the sequence of the amplified and inserted DNA.Oligos flanking ExonC were designed to amplify the The Spire1C gene was amplified from mouse cDNA as described above. Vectors used for protein purification include a modified avidin-6x his-MBP-TEV-3x FLAG-Precision construct under the P1 promoter, as well as a modified pGEX vector for N-terminal GST fusion proteins. All vector backbones were gifts of Dr. Aaron Straight and are described in ExonC and the C-terminal 50 residues of mouse Spire1 were cloned into the avi-his-MBP-TEV-3xFLAG-precision vector described above or a modified pGEX vector  using standard techniques . Avi-hisg 4\u00b0C. Supernatant was applied to hydrated glutathione resin (2 ml bed volume per liter culture), protein bound for 1 hr at 4\u00b0C, and resin was washed extensively with lysis buffer. Protein was eluted with elution buffer  and loaded onto a HiTrapQ anion exchange column. A salt gradient of 150 mM to 1 M was used for protein elution.GST fusion proteins were used for affinity column construction for affinity purification of antibodies. These proteins were purified by using single colonies of transformed Rosetta (DE3) cells to inoculate 400 ml Terrific Broth  cultures containing 100 \u03bcg/ml carbenicillin, 34 \u03bcg/ml chloramphenicol, which was grown overnight at 37\u00b0C. This culture was diluted into 2 l of fresh TB with antibiotics and grown to an O.D. of 0.8\u20130.9, at which time it was moved to 23\u00b0C. After 1 hr at 23\u00b0C, cultures were induced with 0.5 mM isopropyl \u03b2-D-1-thiogalactopyranoside for 3\u20134 hr and harvested as described for purification of avi-his-MBP-TEV-3xFLAG-Precision proteins above. Cells were thawed in lysis buffer , sonicated, and lysates were centrifuged for 30 min at 125,000\u00d7Spire1 affinity columns were made using GST-fusion proteins following the method of Two rabbit polyclonal antibodies described above and three commercially available antibodies were used for examining expression patterns of Spire1 protein in various cell types. The antibodies discussed are: (1) Rabbit polyclonal anti-Spire1 C-term (affinity-purified), (2) Rabbit polyclonal anti-Spire1 ExonC (whole antisera), (3) Sigma mouse monoclonal anti-Spire1, (4) Abcam  mouse monoclonal anti Spire1, and (5) Abnova  rabbit antisera to Spire1. Notably, all of the commercially-available antibodies were targeted to residues 482\u2013584 of the Spire1 isoform lacking ExonC (NP_064533), and thus could only detect non-ExonC containing isoforms.Western blots were performed with 5\u201350 \u03bcg cell lysate and antibodies/antisera was tested at various concentrations, temperatures, and lengths of time for best conditions. Optimized conditions for all antibodies used in this work are described below. HRP-conjugated goat anti-rabbit secondary antibody was used at 1:20,000 in all cases.2, 1 mM EGTA, 10 mM Hepes-HCl pH 7.4, 1 mM DTT, 0.02% thesit , 10 \u03bcg/ml aprotinin, 2 \u03bcg/ml leupeptin, 0.5 mM benzamidine). INF2-CT  and INF2-NT  were purified as described (Spire-KIND (amino acids 1\u2013234) was expressed as a GST fusion protein in bacteria, and purified on glutathione-sepharose  followed by Superdex200 gel filtration (GE Biosciences) of the glutathione-eluted GST-fusion protein. GST-KIND or GST alone was re-bound to glutathione-sepharose in binding buffer  at 23\u00b0C for 1 hr before reading fluorescence anisotropy in an M-1000 fluorescence plate reader (Tecan Inc) at 530 nm excitation and 585 nm emission.INF2 C-term  was expressed in bacteria, purified and labeled on its N-terminal amine with tetramethylrhodamine succinimide as described . LabeledCells were washed in phosphate buffered saline  then fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 30 min before being blocked overnight with 4% BSA at 4\u00b0C. The next day, cells were incubated with primary antibody for 2 hr, rinsed three times with PBS for 10 min each, then incubated with secondary antibodies (Invitrogen) for 1 hr, rinsed three times with PBS for 10 min each, then counterstained with phalloidin (Invitrogen) for 30 min, then rinsed with PBS three times, then mounted using ProLong Gold antifade reagent.Confocal images were acquired with an Apochromat 63\u00d7 1.4 NA objective lens  on a Marinas spinning disk confocal imaging system  using an EM charge-coupled device camera , or a 100\u00d7 Apo TIRF 1.49 NA objective  on a Yokogawa CSU-X1 spinning disk system using an EM charge-coupled device camera . Cells were imaged in HEPES-buffered growth media. Confocal z-stacks were taken using 200 nm steps. Images were deconvoluted using Slidebook 6. Individual 16-bit tiff image files were exported, then processed using ImageJ.GFP-Spire1C photobleaching experiments presented in SIM imaging of fixed cells was performed using an ELYRA SIM (Carl Zeiss) with an Apochromat 63\u00d7 1.4 NA oil objective lens. Five angles of the excitation grid with five phases each were acquired for each channel and each z-plane, which were spaced at 110 nm each. SIM processing was performed using the SIM module of the Zeiss Zen software package. 16-bit grayscale tiffs were subsequently exported to ImageJ for quantification and processing into rendered colored images. Channels in maximum projection images were aligned in the xy-plane using maximum projection images of fluorescent beads.http://rsb.info.nih.gov/ij/plugins/mbf/index.html). When calculating Pearson's values, the mitochondria channel was used as a mask for colocalization. ER-mitochondria intersection sites were visually identified as regions where ER tubules could be clearly visualized crossing mitochondria\u2014these regions were always in the periphery of the cell, significantly restricting the total number of intersections that could be reliably identified. Mitochondria constriction sites were visually identified as regions defined by relative narrowing of mitochondria diameter or reduced fluorescence. Magnifications of boxed regions were generated using ImageJ. Color images of merged 16-bit tiffs exported from the microscope were generated using the ImageJ \u2018merge channels\u2019 function. Statistical analysis was performed using Excel . p-values were determined using the unpaired Student's t-test or ANOVA, as appropriate.All image analysis and processing was performed using ImageJ. Mitochondria lengths were measured manually by first setting the scale according to pixel size, drawing a line along the length of the mitochondria, then using ImageJ's \u2018measure\u2019 function. Colocalization analysis and rendering was performed using the colocalization plugin included in the MacBiophotonics ImageJ plugin bundle  value r = R, and (ii) the tubule profile remains parallel to the tubule axis. While the tubule length L = 680 nm was required to remain constant during the deformation, the tubule surface area was free to change. This means that the membrane lateral tension was taken to be zero, which guaranteed that the membrane bending energy was the sole contribution to the membrane elastic energy.The boundary conditions for the energy minimization consisted in the requirements that at the tubule left and right edges (i) the tubule cross-sectional radius, The energy minimization and the shape determination for each pressure value were performed using Brakke's \u2018Surface Evolver\u2019 program . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Pekka Lappalainen, is a member of our Board of Reviewing Editors. One of the three reviewers, Liza Pon, has also agreed to share her identity.Thank you for submitting your work entitled \u201cA mitochondria-anchored isoform of the actin-nucleating Spire protein regulates mitochondrial division\u201d for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Previous studies revealed that constriction of mitochondria, which precedes Drp1-mediated mitochondrial fission, occurs at sites of close contact between ER and mitochondria, and is driven in part by INF2-mediated actin polymerization. Here Manor et al. show that a specific splice-variant of an actin nucleating protein Spire (named Spire1C) localizes to the mitochondria outer membrane through a specific a-helical motif encoded by its alternate exon C. Importantly, they demonstrate that Spire1C promotes actin filament assembly at the mitochondrial surfaces and modulates mitochondrial fission through its actin-binding WH2 domains and its KIND domain, which specifically binds to INF2 formin. Collectively, these studies provide evidence that Spire1C and INF2 cooperate to form mitochondria-ER intersections, and to promote efficient actin filament assembly specifically at these sites.The majority of the data presented in the manuscript are convincing and the study provides fundamentally important new insights into the mechanisms of mitochondrial fission. However, there are few important issues that should be addressed to confirm the conclusions presented and to further strengthen this study.Essentials revisions:1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission  and \u2018mitochondrial constriction\u2019 assays  also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript  either show normal or even elongated mitochondria.2) The lipid specificity assay  is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis  ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a \u2018hairpin\u2019, which spans the outer membrane twice, or that this region would instead just \u2018horizontally\u2019 penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript). 1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission  and \u2018mitochondrial constriction\u2019 assays  also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript  either show normal or even elongated mitochondria.We have performed additional experiments using Spire knockdown cells (using two different shRNA constructs), and added the fission and constriction data to the manuscript , all of which is consistent with our other data.With regards to apparent inconsistencies in mitochondrial phenotype in Spire1C overexpressing cells: We found that in all conditions there was a range of mitochondrial lengths within each cell, as well as between cells. While the average mitochondrial lengths significantly changed depending on Spire or INF2 activities, nearly every cell has some mitochondria that are very short, and at least a couple that are longer. For the sake of clarity in some of our experiments , we used cells with longer mitochondria in order to be able to make measurements as necessary . In order to clarify our data in this regard, we have added as a supplemental figure a histogram showing the distribution of mitochondrial lengths for each condition .2) The lipid specificity assay  is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.We agree that co-flotation and/or co-sedimentation assays would better establish the lipid specificity of ExonC. This will require many more experiments that go beyond the scope of this work. Therefore, we have removed the lipid dot blot and will attempt to establish ExonC\u2019s lipid specificity in a future study.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis  ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a \u2018hairpin\u2019, which spans the outer membrane twice, or that this region would instead just \u2018horizontally\u2019 penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript).We think the reviewers may have overlooked our control protein in the protease protection assay in making his/her statement \u201cthis assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM.\u201d By definition, if our treatment conditions are not affecting the integrity of the OM, then any IMS protein would not be affected. This is exactly why we used the OMI-mCherry construct as a control in all of our experiments. OMI-mCherry localizes to the IMS \u2013 if the OM was being degraded by our digitonin treatment, then OMI-mCherry would escape into the cytoplasm. Similarly, if the OM was degraded enough that trypsin was able to digest proteins in the IMS, then OMI-mCherry would be depleted upon addition of trypsin. In none of our experiments did we observe depletion of OMI-mCherry mitochondrial fluorescence, indicating that the OM was indeed intact in all of our experimental conditions.We agree that it is important to be careful not to make any claims about the C-terminus of Spire1C being in the IMS, since our FPP assay only informs us that the C-terminal region of ExonC (which is in the middle of the full-length Spire protein) is protected from trypsinization. While our FPP assay leaves us confident that the C-terminus of ExonC is protected from the cytoplasm, we were unable to draw any conclusions from these assays as to the localization of the Spire1C C-terminus. While the secondary protein structure prediction software PHYRE identifies two putative alpha-helices within ExonC, only one of those helices was predicted to be a transmembrane domain by predictive software, so we chose not to postulate that there are two transmembrane domains in ExonC. However, we agree that the hairpin model the reviewers have proposed is just as likely to be correct, and is furthermore appealing since this would allow for the C-terminal domain of Spire1C to interact with other cytoplasmic proteins.We agree that an additional FPP assay using a C-terminus GFP-tagged Spire1C construct would ideally clarify the topology of the Spire1C C-terminus. In order to address the reviewers\u2019 comments, we performed an additional FPP assay using a new Spire1C-GFP construct with GFP on the C-terminus. Unfortunately, when we expressed this construct, the protein no longer localized properly to mitochondria, instead displaying a mostly cytoplasmic localization pattern (with barely detectable accumulation on mitochondria). Due to this disrupted localization pattern, we were unable to draw any conclusions from an FPP assay; upon addition of digitonin, all of the GFP fluorescence was almost immediately depleted, even without subsequent addition of trypsin. While this result may give some clue as to how Spire1C localizes to the OM, many more experiments that go beyond the scope of this work would have to be performed in order to determine what this result is telling us. Thus, we have mentioned these latest findings in the text, and modified our interpretation of our FPP data to include the likely possibility that Spire1C\u2019s ExonC localizes in a hairpin or horizontal fashion on the outer leaflet of the outer mitochondrial membrane."}
+{"text": "MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5\u2032-fragments were 3\u2032-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5\u2032-3\u2032 degradation or other molecular fates. Some 3\u2032-5\u2032 decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation. HOX B8 mRNA, directed by the perfectly base-matched miR-196, is one of only a few cases of miRNA-directed mRNA decay reported in mammalian cells1011MicroRNAs (miRNAs) are noncoding RNAs that have been shown to posttranscriptionally regulate gene expression and protein synthesis, particularly in mammalian cells, by partially base-pairing to complementary sequences in the 3\u2032-untranslated regions (3\u2032UTRs) of their target mRNAs124et al.It is very important to provide direct evidence for miRNA-mediated target mRNA cleavage and degradation to access the precise mechanism of action in the miRNA-mediated suppression of target gene expression in mammalian cells. The techniques currently used to study the individual or global effects of miRNA action on its target mRNA, such as 5\u2032RACE, ribosome profiling, and RNA-deep sequencing, are powerful and informative2810TUSC2, also known as FUS1), to demonstrate the mammalian miRNA-mediated target mRNA cleavage and regulatory activities in human cells using a novel SLA\u2013RT-PCR assay  could be posttranscriptionally added to the 3\u2032 ends of miRNA-cleaved 5\u2032-mRNA fragments in various organismsragments . These 3ragments  using toragments .TUSC2 target site  that is effective in all let-7 miRNA members. The expression of let-7 miRNAs was depleted in the cells transfected with miR-98 LNAi 48\u2009h after treatment, as shown by qRT-PCR analysis using let-7\u2013specific SL-RT primers  and the 3\u2032 supplementary pairing region, and they share a conserved short stretch of base-paired (7\u201310 nt) sequences at the seed region  were used as a negative control. The cel-miR-67 was confirmed to have minimal sequence identity with miRNAs in human, mouse, and rat and shown to have no significant biological impact when ectopically expressed in those mammalian cells. Overexpressed miR-622 and let-7d mRNAs were detected by qRT-PCR in H1299 cells transfected with a miR-622 or a let-7 expression vector, respectively  mRNA targeted by miR-142-3p activity in HeLa cells by a U-track\u2013specific SLA\u2013RT-PCR and Sanger sequencing , were detected by agarose gel electrophoresis  promoter, immediately followed by a 3\u2032UTR with two identical copies of let-7 target sequences (T1 and T2) directly derived from TUSC2 mRNA sequences arranged in tandem and a BGH poly(A) signalling sequence  reporter plasmid-based model system with a defined let-7 target and SLA\u2013RT-PCR primer binding sequences and a fully functional mammalian mRNA structure to monitor the precise action of miRNA on its target in a time- and space-dependent manner. The reporter plasmid (pLJ-T214) consisted of an sequence .TUSC2 let-7 target sites were produced: one copy in the 3\u2032UTR of the endogenous TUSC2 mRNA transcript and two copies in the 3\u2032UTR of the exogenously expressed pLJ-T214 EGFP reporter transcript. Endogenous let-7\u2013mediated mRNA cleavage of those target sites would produce 5\u2032-mRNA fragments with identical 3\u2032 termini, which could be detected competitively by the same SL-RT primer in RT. The origins of the cleaved mRNA fragments could be determined by subsequent PCR amplification with either the endogenous or the pLJ-T214 transcript\u2013specific PCR primers, which would generate SLA\u2013RT-PCR amplicons of different sizes. The predicted sizes of the SLA\u2013RT-PCR products derived from target sites T1 and T2 were 240-209\u2009bp and 532-500\u2009bp, respectively, and those derived from the endogenous target site were predicted to be 230-198\u2009bp.In pLJ-T214\u2013transfected H1299 cells, three identical copies of TUSC2 mRNA fragments and internal references for defining the spatial effect of target sites on endogenous let-7 activity by the SLA\u2013RT-PCR assay or SLA-qRT-PCR. Total RNAs from pLJ-T214\u2013transfected H1299 cells at different time points after transfection were examined with the same set of SLA-RT primers used for the endogenous TUSC2 mRNA fragment detection  by SLA-qRT-PCR in the T1 site were consistently stronger than those in its T2 counterpart at any given time; however, intensity patterns at both sites displayed similar trends over the course of transfection. The accumulation patterns of the 3\u2032-uridylated T2 fragments at 16 h after transfection  catalyse the transfer of UMP residues to the 3\u2032 hydroxyl group of RNA and are involved in bulk degradation of histone mRNA in human cells2228A\u2013RT-PCR . Total RA\u2013RT-PCR . The unmA\u2013RT-PCR . The siRe3 genes  showed me3 genes . The 3\u2032-e3 genes . In conte3 genes . No appae3 genes . The oliTUSC2 mRNAs at various sites in partially paired miRNA:mRNA sequences, predominantly within the miRNA seed region or in the 3\u2032 supplementary pairing region. Most of the cleaved mRNA 5\u2032-fragments were oligouridylated at their 3\u2032-termini by TUTase2 in the miRISC, while some 3\u2032-5\u2032 decayed mRNA fragments could also be intercepted by miRISCs stationed on their targets and oligouridylated by other TUTases in the proximity of the miRISC. These 3\u2032-oligouridylated mRNA 5\u2032-fragments accumulated in cytosol and appeared destined for 5\u2032-3\u2032 decay. These findings provide direct evidence for mammalian miRNA-mediated gene silencing by target mRNA recognition, cleavage and 3\u2032-uridylation, leading to degradation of the protein coding sequence\u2013containing 5\u2032-mRNA fragments via a possible bulk 5\u2032-3\u2032 decay pathway.Multiple models have been proposed for the molecular mechanics of miRNA-mediated posttranscriptional repression of target gene expression either by inhibition of mRNA translation, with no apparent impact on mRNA stability, or by initiation of mRNA decay1278307G cap-binding motif has been identified on Argonaute MID domains but was shown to be weak and could not compete with the cap-binding eIF4E to sustain translational suppression33Our findings suggest that the mRNA cleavage and 3\u2032-oligouridylation activities mediated by mammalian miRNAs with partial base-pairing to their target mRNA sequences in Ago2-miRISCs may be a common molecular mechanism for miRNA-targeted gene silencing. We have shown that target gene silencing mediated by let-7 family miRNAs and other human miRNAs could be initiated by Ago2-catalysed endonucleolytic cleavage on base-paired miRNA:mRNA target sites, which is consistent with the fact that the Ago2 endonucleolytic RNase H domain prefers paired bases as substrates15Most of the cleaved 5\u2032 mRNA fragments were oligouridylated at their 3\u2032-termini in the miRISC. Those 5\u2032 mRNA fragments derived from the initial cleavage sites on the same transcript that escaped oligouridine modification in the miRISC could be subjected to the exonucleolytic RNA 3\u2032-5\u2032 decay pathway and would then be intercepted and oligouridylated by other TUTases in proximity to the miRISC. These oligouridylated mRNA fragments could be recognised by the cytoplasmic heptameric Lsm1-7 complex and accumulated in P-bodies2836373839414243Our results provide new insights into the molecular mechanics of mammalian mRNA-mediated gene silencing by showing a cooperative action of miRNA, Ago2/TUTases and other essential molecular components in miRISCs mediating mRNA degradation initiated by mRNA cleavage and concurrent 3\u2032-uridylation. Further studies are needed to understand the precise mechanism of miRNA action in association with specific co-factors in miRISC on posttranscriptional regulation of target gene expression in a complex mammalian cellular system.RNase inhibitor was acquired from New England Bio-Labs (M0314L). DNA primers and RNA oligos were synthesized by Sigma-Aldrich. High-capacity cDNA first-strand synthesis kit, TaqMan Gene Expression Master Mix and TaqMan probes for miRNA quantification were purchased from Applied Biosystems (Life Technologies). The miRCURY LNA inhibitor against hsa-miR-98 was obtained from Exiqon. ExoSAP-IT for PCR Product Cleanup was obtained from Affymetrix. TRIzol LS Reagent, SYBR Green I and 1\u2009Kb-Plus DNA Ladder were obtained from Invitrogen (Life Technologies). Rabbit anti-Ago2 monoclonal antibody was purchased from Cell Signaling Technology. SiGENOME SMARTpool-EIF2C2 anti-Ago2 and ON-TARGET plus non-targeting siRNA as nonspecific controls (siR-NSCs) were purchased from Thermo Scientific.Human H1299 large cell lung cancer and HeLa cervical cancer cells were grown in Roswell Park Both Memorial Institute 1640 medium and Dulbecco modified Eagle medium supplemented with 10% foetal bovine serum (FBS). For transfection, plasmid DNA was adjusted to 0.5\u2009\u03bcg/\u03bcL and siRNAs (25\u2009\u03bcM) were dissolved in sterile water. Plasmid DNA or siRNA was mixed with an equal volume of 8\u2009mM DOTAP:Cholesterol liposome to make DOTAP:Chol:DNA complexes. DOTAP:Cholesterol liposomes were prepared in-house as described previouslySchizosaccharomyces pombe Cid1 enzyme, which showed that TUTase1 and TUTase3 are involved in histone mRNA uridylationmiRNA let-7 knockdown was carried out in H1299 cells transfected with 10\u2009\u03bcM of miRCURY LNA anti-miR-98 inhibitor packaged in a DOTAP:Chol:siRNA complex. After 24\u2009h of initial transfection, the medium was refreshed and cultures were returned to incubation for an additional 24\u2009h. Cells were then subjected to direct lysis in TRIzol LS Reagent, and total RNAs were isolated according to the vendor\u2019s recommendation. The let-7 miRNA levels were measured by qRT-PCR assays. Several putative TUTases have been identified in the human genome on the basis of a homology comparison with the U6-terminal transferase and the TUTase mRNA levels were measured by end-point RT-PCR assays as reported by Mullen et al.To determine the effect of TUTase knockdown on miRNA-mediated fragment distribution, we synthesized all TUTase siRNAs and PCR primers according to the ref. RNAs (50\u2009\u03bcg) were treated with 10\u2009\u03bcL of RNase-free DNase I  and 20\u2009\u03bcL of RNase inhibitor in 100\u2009\u03bcL of reaction solution for 5 min at 37\u2009\u00b0C, 10\u2009min at 80\u2009\u00b0C and 15\u2009sec at 95\u2009\u00b0C. RT master mixes were prepared using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and aliquots containing 0.25\u2009\u03bcg RNA were mixed with 2.5\u2009\u03bcL of the appropriate 100\u2009nM SL-RT primers. The reactions were incubated by the following program: 1\u2009min at 20\u2009\u00b0C and 10\u2009sec at 37\u2009\u00b0C for 60 cycles, 30\u2009min at 37\u2009\u00b0C, 20\u2009min at 42\u2009\u00b0C, 5\u2009min at 80\u2009\u00b0C and hold at 4\u2009\u00b0C. The RT products were then subjected to further PCR amplification for end-point PCR evaluation or to real-time PCR for quantitation. Samples not subjected to RT were used as negative controls to avoid any potential DNA contaminants in the RNA preparations.For end-point PCR analyses of cDNA products primed by SLA-RT primers, PCR was performed in TaqMan Gene Expression PCR Master Mix with universal primer and transcript-specific primers. The RT product (1\u2009\u03bcL) was mixed in 30\u2009\u03bcL of PCR mixtures containing 0.5\u2009\u03bcM PCR primers. The PCR program started at 95\u2009\u00b0C for 10\u2009min, followed by 30 cycles of 15\u2009sec each at 95\u2009\u00b0C, 1\u2009min at 68\u2009\u00b0C and 20\u2009sec at 72\u2009\u00b0C for an additional 5 cycles, chased with 15\u2009sec at 95\u2009\u00b0C, 2.5\u2009min at 68\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C. PCR products were analysed by electrophoresis on 3% agarose gels in 1\u00d7 Tris-Boric acid buffer\u2206Ct method, where normalised \u2206Ct\u2009=\u2009Ct of Sample\u2009\u2212\u2009Ct of NTC (non-template control). The qRT-PCR conditions for miRNA detection were modified to 10\u2009min at 95\u2009\u00b0C, 5 cycles of pre-amplification of 15\u2009sec each at 95\u2009\u00b0C, 2.5\u2009min at 61\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C, then 40 cycles of 15 sec at 95\u2009\u00b0C, 1\u2009min at 60\u2009\u00b0C and 1\u2009sec at 72\u2009\u00b0C. Let-7 family miRNA, hsa-miR-622, hsa-miR-30a and hsa-RNU44 in H1299 cells were determined by SLA\u2013RT-PCR methods. The data were first normalised against hsa-RNU44, and then the changes in miRNA expression level were calculated by the following formula: \u2206\u2206Ct\u2009=\u2009\u2206Ct of miRNA target \u2212\u2009\u2206Ct of miRNA negative control4546For profiling of relative quantitative mRNA fragment abundance, a real-time SLA-qRT-PCR with ether the TagMan probe-based or SYBR-Green-based method was performed with 1\u2009\u03bcL of RT product primed by each SLA-RT primer in triplicate in 40\u2009\u03bcL of TaqMan Gene Expression PCR Master Mix with or without containing SYBR Green I, 0.5\u2009\u03bcM of universal primer and 0.5\u2009\u03bcM of transcript-specific primer. PCR reactions started at 95\u2009\u00b0C for 10\u2009min, followed by 10 cycles of pre-amplification of 15\u2009sec at 95\u2009\u00b0C, 2.5\u2009min at 68\u2009\u00b0C and 2.5\u2009min at 72\u2009\u00b0C, then 40 cycles of 15\u2009sec at 95\u2009\u00b0C, 1\u2009min at 68\u2009\u00b0C and 20\u2009sec at 72\u2009\u00b0C on a CFX384 Real-Time PCR Detection System (Bio-Rad). Melting curve analysis was used to confirm a single PCR product in each reaction. RNU44 was measured as a sample control, and RNA fragment relative abundance was calculated by the 2TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an EGFP expression cassette as 3\u2032UTR under a CMV promoter. The transcript 3\u2032UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T214 transcript are illustrated in et al.47A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame \u2009\u2009\u2009\u2009\u2009\u2009\u2009stop codonTGTACAAGTACGGCATGGACGAGCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAATTAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCCTAGTCAGGCTAGCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCAGATCTCTAGTCAGGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an eGFP expression cassette as 3\u2032UTR under a CMV promoter. The transcript 3\u2032UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T722 transcript are illustrated in A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame \u2009\u2009\u2009\u2009\u2009\u2009\u2009stop codonTGTACAAGTACGGCATGGACGAGCAAGCGGCCGCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCATTTCAGCCGTTTGCTACCTCGGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCTACCTGCCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGTAGATCTGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.PCR was performed on genomic DNA extracted from normal human bronchial epithelial cells with the following primers flanking the miR-622 precursor:GGACTAGTGAATTCTTTAGAGAAGCTGGACAAGTACTACTTGTAACTCGAGAAAAGTGTCATCTCAGCAGCTCSpeI/EcoRI and cloned into a CMV promoter\u2013driven expression plasmid with BGH poly(A) signal. The plasmid expression cassette was verified by Sanger sequencing.PCR fragments were digested with CMV promoter\u2013driven expression plasmid with BGH poly(A) signal was digested by EcoRI/XhoI and purified as a cloning vector. The correct clones were screened and verified by Sanger sequencing as a let-7d expression vector.The following oligonucleotides were synthesized by Sigma-Aldrich and annealed as inserts. A AATTCCCTAGGAAGAGGTAGTAGGTTGCATAGTTTTAGGGCAGGGATTTTGCCCACAAGGAGGTAACTATACGACCTGCTGCCTTTCTTAGGCTCGAGCCTAAGAAAGGCAGCAGGTCGTATAGTTACCTCCTTGTGGGCAAAATCCCTGCCCTAAAACTATGCAACCTACTACCTCTTCCTAGGGet al.TUSC2 5\u2032-mRNA fragment detection and quantification and nU-track detection, and the TUTase-siRNAs for each experiment, are summarised in SLA-RT primer was composed of stem loop and probe as reported by Chen TUSC2 mRNA fragments with various uridine tails were detected by qRT-PCR assay. The A4 RNA fragments of pLJ-T214 transcripts were represented by 528-bp and 234-bp amplicons and detectable in both unmodified and oligouridylated forms in total RNA samples from H1299 cells transfected with plasmid pLJ-T214. The A4 RNA fragments were further tested with SL-RT primers with different probes to confirm the oligouridine detection methods. Total RNAs from pLJ-T214-transfected H1299 cells were reverse-transcribed with 5 SL-RT primers with different probe compositions to compare their priming capabilities, and the results are shown in 18RNA fragment abundance varies with oligouridine lengths see . Let-7\u2013mHow to cite this article: Xu, K. et al. MicroRNA-mediated target mRNA cleavage and 3\u2032-uridylation in human cells. Sci. Rep.6, 30242; doi: 10.1038/srep30242 (2016)."}
+{"text": "Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce \u2018TEMTAC\u2019, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2. Recent work provided evidence that quantitative rather than qualitative differences in the proteomic inventory between different cell types and tissues are the cause of functional differences123456Gene silencing based on short interfering RNAs (siRNA) has been demonstrated in several organisms, including in cultured mammalian cells11in vitro, optionally in high-throughput by robotics15Plasmid-based gene delivery systems have become essential for molecular and cell biology. However, the number of available selection markers and the physical space that needs to be available to allow entry of multiple plasmids into cells are technically limiting. These problems are exacerbated by imbalanced delivery if multiple plasmids are used, resulting in heterogeneous cell populations which interferes with read-out. These impediments can be circumvented by combining independent modules containing genes of interest, regulatory elements and other desired functionalities into a single multifunctional multigene delivery plasmid. This is a viable option if the combination of the modules is sufficiently straight-forward. Addressing this challenge, we previously developed the \u2018ACEMBL\u2019 system to enable fast and flexible generation of multigene delivery constructs by an automatable technique called tandem recombineering (TR) to combine \u2018Donor\u2019 and \u2018Acceptor\u2019 plasmid modules via reversible Cre-loxP fusion Tunable Endogenous Mammalian TArget Complementation by multiplexed plasmid-based recombineering. Retaining the original ACEMBL concept, we developed this novel system which affords the means to simultaneously knockdown endogenous targets via RNA interference89in vitro exploiting the Cre-Lox recombination reaction, which can be carried out optionally in high-throughput by robotics applying automated routines we have implementedHere, we introduce TEMTAC, a novel system for http://www.invivogen.com/sirna-wizard). Donor D1 containing the shRNAs was transiently transfected in HEK293 or HeLa cells. After 24 and 48\u2009hours, cells were lysed and endogenous protein levels detected by Western blotting, real time PCR, and deep sequencing. We observed the best silencing effect 48 h post transfection and no  down-regulation using random (\u2018scrambled\u2019 scrb) shRNAs as a control  contains the silencing cassette for knocking down the endogenous gene by RNA interference . In one 192021 control . IncomplE. coli) mutant and three VP16 (transcription activation domain of herpes simplex virus). In the presence of the inducer doxycycline (dox), the TETon3G-dox complex binds to the TRE minimal promoter (TRE-pMCVmin) and induces the expression of the gene of interest (GOI).Donor D2.1 (pMDS-GOI-TETon3G), constitutively expresses the TETon3G transactivator , a fusion protein of a Tet repressor (Donor D2.2 (pMDS-GOI-TEToff) is based on the TEToff advanced inducible expression system (Clontech). This vector expresses the TEToff advanced transactivator (TetR) and also contains a TRE-based module that expresses high levels of the GOI in the absence of dox.Donor D2.3 (pMDS-GOI-TETon-PGK), is a modification of Donor D2.1, where the expression of the rtTA is downstream of a minimal phosphoglycerate kinase (PGK) promoter .Donor D2.4 (pMDS-GOI-TETon-tTS) is a modification of TETon to reduce basal expression levels. This is achieved by adding an internal ribosome entry site (IRES) and a tTS , which is a fusion of the TetR and the KRAB-AB silencing domain of the Kid-1 protein, after the transactivator (rtTA). As tTS competes with rtTA by binding specifically to the TRE, it suppresses the transcription in the absence of dox.Achieving expression levels that are close to endogenous levels is a crucial prerequisite for analysing the physiological effect of mutations. This is particularly important when studying signalling proteins because of their frequent involvement in downstream activation and feedback loops, which often function at very precise protein levels that need to be maintained meticulously. A major problem in many TET regulated systems is basal expression (without the inducer being present), especially for proteins that are present at markedly low abundance in cells. Furthermore, depending on the protein of interest, different dynamic expression ranges of mutants would be highly desirable to properly recapitulate physiological events. To address this, we created as Donor D2 variants four different TET-related expression system versions with defined properties  in HEK293 cells grown in serum-containing medium. Exogenous expression of this oncogenic mutation tends to be higher as compared to wildtype (WT) . To rectify this, we took advantage of our TET-regulated system to tune the expression levels of WT and V600E to be close to identical  and Donor D2 (pMDS-GOI) were created based on the original plasmids of the ACEMBL system for protein complex expression in prokaryotic hostsde novo from scratch. All DNA elements . Sequences are provided in the shRNAs and genes of interest (GOI) were inserted into the TEMTAC system using standard restriction-ligation cloning methods or, alternatively, by Gibson cloningHEK293, HeLa and GH-HEK293 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with L-glutamine and 10% (v/v) heat-inactivated TET-free fetal calf serum (Clontech ref. 631106) .HEK293 or HeLa cells were seeded in 96-well-plates (Thermo Scientific 165306) and grown to 60% confluency in normal growth medium. Cells were transfected with 40\u2009ng of the four different Donor D2 plasmids containing luciferase as the GOI (Donor D2.1-D2.4) or with the fully Cre-assembled (A-D1-D2) pTEMTAC composite plasmid (containing siRNA scramble) using lipofectamine 2000 . 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich). 24\u2009hour later the luciferase assay was performed according to the manufactures instructions .HEK293 cells were seeded on 6 well plates and grown to 60% confluency in normal growth medium. Cells were transfected with 2.5\u2009\u03bcg of the fully Cre-assembled A-D1-D2 composite plasmid containing mCherry as the GOI (containing siRNA scramble) using lipofectamine 2000 (Invitrogen). 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline . 24\u2009h later YFP and mCherry fluorescence was imaged on an inverted DMI-6000 Leica wide-field fluorescent microscope equipped with a Leica DFC 350FX camera with a 20\u00d7 objective.GH-HEK293 cells  were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5\u2009\u03bcg of pTEMTAC (A-D1-D2) plasmid  or, alternatively, pMDC-RNAiDual (shRNA against SHP2 or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. 24\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in minimal medium (DMEM plus 2\u2009mM glutamine and 1% serum). 24\u2009hours after transfection, cells were stimulated with 125\u2009ng/ml growth hormone for 10\u2009minutes, washed with PBS and resuspended in 200\u2009\u03bcl of lysis buffer . Lysed cells were analysed by western blot.HEK293 were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5\u2009\u03bcg of pTEMTAC (containing BRAF/HRAS WT or V600E/G12V as GOI) or pMDC-RNAiDual (shRNA against BRAF/HRAS or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. 48\u2009hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in normal growth medium. 24\u2009hours after transfection, cells were washed with PBS and resuspended in 200\u2009\u03bcl of lysis buffer . Lysed cells were analysed by western blot.Cell lysates were loaded on SDS gels and separated by electrophoresis; gels were then transferred onto nitrocellulose membrane using the iBlot system (Invitrogen), and the blots were incubated 1\u2009h at room temperature in TBS, Tween 0.1%+5% milk. The primary antibody was incubated at 4\u2009\u00b0C overnight (1:1000 dilution), and the HRP-coupled secondary antibody (1:10 000 dilution) was incubated 1\u2009h at room temperature, both in TBS Tween 0.1%\u2009+\u20090.5% milk. Blots were developed using high sensitivity ECL reagent (Thermo) and visualized using the Fujifilm LAS-3000 developer. Bands were analyzed using ImageJ. The following antibodies were used for Western blotting: SHP2 , BRAF , total RAS , growth hormone receptor , phospho-ERK Thr202/Tyr204 , phospho-MEK Ser217 and Ser221  and \u03b2-actin .\u00ae Green RNA-to-CTTM 1-Step Kit, Applied Biosystems), 0.15\u2009\u03bcM of forward and reverse primers and 60\u2009ng of RNA following the manufactures instructions. For PCR the cycling parameters were (i) reverse transcription , (ii) activation of polymerase and initial denaturation , (iii) 40 cycles of denaturation , annealing, extension and read fluorescence , (iv) final hold (4\u2009\u00b0C). The following primers were used. For endogenous BRAF 5\u2032ATCCCAGAGTGCTGTGCTGT3\u2032 (forward) and 5\u2032TCTCCAACACTTCCACATGC3\u2032 (reverse), for exogenous BRAF 5\u2032AATGTTGCGCCGTTTACAG3\u2032 (forward) and 5\u2032TCTCCAACACTTCCACATGC3\u2032 (reverse), for endogenous HRAS 5\u2032TGCCATCAACAACACCAAGT3\u2032(forward) and 5\u2032ACGTCATCCGAGTCCTTCAC3\u2032 (reverse), for exogenous HRAS 5\u2032GAGGGCTTCCTGTGTGTCTT3\u2032 (forward) and 5\u2032TCTTTGACGCGCTTAATTTG3\u2032 (reverse), for endogenous SHP2 5\u2032CTTGTACTCCAACGCCACCC3\u2032 (forward) and 5\u2032CTGTGCTGAAGTTTTGGCAGG3\u2032 (reverse) and for total SHP2 5\u2032GGTGTGGAGGCAGAAAACCT3\u2032 (forward) and 5\u2032TTGATGTGGGTGACAGCTCC3\u2032(reverse).Cells were grown and transfected as before. RNA was isolated using miRNeasy Mini Kit (Qiagen) following the manufactures protocol including the optional DNase digestion step. PCR reactions were prepared using qPCR mix (Power SYBRTM II RT (Invitrogen) with random oligonucleotides. In the first PCR all target genes  were amplified for 5 cycles using Phusion DNA polymerase (New England Biolabs) with the following degenerated adaptor primers: for BRAF (region of V600E)RNA was extracted as described for real time PCR. cDNA synthesis was done using SuperScript5\u2032CCCTACACGACGCTCTTCCGATCTCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTcgtCTTCATGAAGACCTCACAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTagatCTTCATGAAGACCTCACAG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTcgtCTGTTCAAACTGATGGGACC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTagatCTGTTCAAACTGATGGGACC3\u2032(reverse), for HRAS (region around G12V)5\u2032CCCTACACGACGCTCTTCCGATCTGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTcaGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgctGACGGAATATAAGCTGGTGG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTatcaGACGGAATATAAGCTGGTGG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTcaGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTatcGTCGTATTCGTCCACAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTatcaGTCGTATTCGTCCACAAAGTG3\u2032(reverse), for SHP2 (region around D61G)5\u2032CCCTACACGACGCTCTTCCGATCTAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTtgaAGTAAAAGTAACCCTGGAGAC3\u20325\u2032CCCTACACGACGCTCTTCCGATCTctgaAGTAAAAGTAACCCTGGAGAC3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTtgaGACCAACTCAGCCAAAGTG3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTctgaGACCAACTCAGCCAAAGTG3\u2032(reverse), and for SHP2 (region around C459G)5\u2032CCCTACACGACGCTCTTCCGATCTGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTaGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTgaGAGGTGCACCATAAGCAGGAG3\u20325\u2032CCCTACACGACGCTCTTCCGATCTtgaGAGGTGCACCATAAGCAGGAG3\u20325\u2019CCCTACACGACGCTCTTCCGATCTctgaGAGGTGCACCATAAGCAGGAG3\u2032(forward) and5\u2032TTCAGACGTGTGCTCTTCCGATCTTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTgaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTtgaTCACAATGAACGTCCCTGTC3\u20325\u2032TTCAGACGTGTGCTCTTCCGATCTctgaTCACAATGAACGTCCCTGTC3\u2032(reverse).The resulting amplicons were used for determination of cycle numbers for the second PCR with the TruSeq dual barcoding primers. The primary PCR product was amplified using NEBNext 2x High Fidelity PCR Master Mix (New England Biolabs), with 200nM primer concentration, and 0.1x SYBR Green I  monitoring the amplification in a Roche LightCycler 480. The cycle number for the final library PCR was chosen to get a sufficient amount of product without reaching the plateau phase of the PCR. Then 2.5\u2009\u03bcl of the primary PCR product was used for amplification with the NEBNext Master Mix and barcode primers  but without SYBR Green, for the number of cycles determined from the qPCR. PCR products were purified using AMPure XP beads and concentration was determined on a Bioanalyzer DNA 1000 chip. Samples were sequenced using MiSeq v3 chemistry with 130 cycle single reads in a MiSeq sequencer (Illumina). Flanking reads were subtracted by mutant reads to calculate the reads corresponding to the endogenous expression level.How to cite this article: Beltran-Sastre, V. et al. Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering. Sci. Rep.5, 17432; doi: 10.1038/srep17432 (2015)."}
+{"text": "MAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1\u00b0 sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.HIV-2 and SIV MAC/SIVSM lineage, with restriction being greatest for SIVSM and the least for epidemic HIV-2. Here we show that this dominant-acting, antiviral activity is specific for the capsid and blocks the virus after it enters the nucleus. The evidence suggests that, in order to jump from sooty mangabey monkeys to humans, the capsid of these viruses changed in order to adapt to this antiviral activity. In keeping with the practice concerning anti-lentiviral activities we propose to call this new antiviral activity Lv4.HIV-1 and HIV-2, the two lentiviruses that cause AIDS in humans, are members of a family of such viruses that infect African primates. HIV-1 is a zoonosis that was transmitted to humans from chimpanzees. HIV-2 was transmitted to humans from sooty mangabey monkeys. In several documented cases of cross-species transmission of lentiviruses it has been shown that replication of the virus in the new host species necessitated that the virus adapt to species-specific antiviral factors in the host. Here we report that human blood cells possess an antiviral activity that exhibits specificity for viruses of the HIV-2/SIV CPZ), and perhaps from gorillas (SIVGOR) [CPZ itself is probably a recombinant virus that resulted from co-infection of a chimp with viruses transmitted from a red-capped mangabey (SIVRCM) and a greater spot-nosed monkey (SIVGSN) [CPZ did not cause disease in chimpanzees but extensive observation of feral animals has demonstrated that this is not the case [Human immunodeficiency virus type 1 (HIV-1) is the major cause of the acquired immune deficiency syndrome (AIDS) pandemic. Among the immunodeficiency viruses that infect at least 40 of the primate species in sub-Saharan Africa, the simian immunodeficiency viruses (SIVs) found in central African chimpanzees and gorillas are monophyletic with HIV-1 . Each of(SIVGOR) \u20136. SIVCP(SIVGSN) . Until rthe case .Cercocebus atys) on multiple occasions [SM, but cross-species transmission to another non-native host, rhesus macaques (SIVMAC), resulted in AIDS [HIV-2, a second AIDS-causing virus that has highest prevalence in West Africa, was transmitted to people from sooty mangabey monkeys  family can disrupt retroviral replication in a species-dependent manner [Though transmission of primate lentiviruses to humans has occurred on multiple occasions and may still be occurring , these et manner \u201318. TRIMt manner . Moreovet manner ,21 and it manner . TRIM5\u03b1 t manner  and has t manner . Inhibitt manner \u201327. Addit manner \u201330. Finat manner ,30\u201332.MAC and SIV from African green monkeys (SIVAGM) [HIV-1 is inhibited by TRIM5\u03b1 from a number of African and Asian monkey species, such as rhesus macaques, African green monkeys, and sooty mangabeys ,33. The (SIVAGM) ,36\u201339.MAC replication are not inhibited by TRIM5\u03b1 in human cells. These experiments, however, all used immortalized adherent cell lines such as TE671 (rhabdomyosarcoma) [MAC or HIV-1 in a range of mammalian cell lines [MAC vectors in several human cell lines, e.g. Raji (B lymphocyte) and in the T lymphocyte cell lines Jurkat, HuT78 and CEM. This raised the possibility that lentiviruses could be inhibited in a cell-type specific fashion in human cells. In the work presented here, we investigated restriction to SIVMAC replication in peripheral blood lymphocytes (PBLs) as well as in various cell lines. Our data reveal a TRIM5\u03b1-independent restriction activity targeting SIVMAC, and the related SIVSM and HIV-2, in human blood cells.Thus, available data suggest that the early post-entry stages of SIVsarcoma) ,40,41, Hsarcoma)  or HeLa sarcoma) ,31,43. Hll lines . They foNL4-3 and SIVMAC239, as previously described [nef was replaced with GFP coding sequence, such that the fluorescent reporter was expressed from the respective LTR. The two vectors were produced in parallel by collecting supernatant from transfected 293T cells. The vector-containing supernatants were checked for reverse transcriptase activity [Human cell lines were challenged with VSV G-pseudotyped, single-cycle vectors derived from HIV-1escribed . In eachactivity , normaliactivity , and theMAC transduction efficiency was 4 to 20-times less than that of HIV-1NL4-3 when the two vectors were used to challenge any of a panel of T cell lines, including Jurkat, SupT1, and CEM-SS cells, the Burkitt lymphoma-derived B cell line Raji, or the myelomonocytic cell lines U937 and THP-1 . Based on these parameters, the decrease in apparent infectivity of SIVMAC did not appear to be explained by poor expression of the GFP reporter from the SIV LTR. The latter point was demonstrated more conclusively by using 3-part lentiviral vectors in which the GFP reporter was expressed from the HIV-1 and SIVMAC vectors using an identical spleen focus-forming virus (SFFV) promoter  were prepared, stimulated with PHA for three days, and challenged with the single-cycle vectors. SIVMAC transduction was less efficient than for HIV-1NL4-3 , had transduction activity more similar to that of SIVMAC239  is the retroviral determinant that confers sensitivity to several restriction factors, including Fv1 [MAC239 or HIV-2ROD. Neither of the two chimeras had transduction activity on CRFK cells or on HeLa cells.The experiments described above suggest that SIVding Fv1 , TRIM5 [ding Fv1 ,45, and ding Fv1 \u201357, the ROD and SIVMAC239 CA back to amino acid 202, using HIV-1 CA sequences to encode amino acids 203 to 230 . As with the 2-part vectors, no infectivity was observed unless CA amino acids 203\u2013230 were provided by HIV-1. Among the chimeras generated, representatives from Groups AB, A, D, E, F, and H, and from a primary SIVSM, were sufficiently infectious to evaluate CRFK-normalized transduction efficiency on Jurkat T cells. As a general trend, chimeras generated with CA from the epidemic Groups (A and B) were the most infectious on Jurkat T cells, those from the non-epidemic Groups  were less infectious, and that from SIVSM was the least infectious  bearing restriction-sensitive cores [MAC-specific restriction activity in Jurkat T cells with SIV VLPs were also unsuccessful.Given the results described above, evidence was sought that the cell type-specific defect in SIVve cores . Flat, eve cores ,60. Atte2O3 rescues retroviruses from CA-specific restriction by TRIM5 but has no effect on retrovirus transduction efficiency in the absence of TRIM5-mediated restriction [2O3 blocks TRIM5-mediated restriction is not known, though the effect results in increased reverse transcription and correlates with disruption of mitochondrial membrane potential [Astriction ,47,61,62otential ,61.MAC transduction of human blood cells might be restricted by TRIM5, or by a cellular factor with similar properties, the effect of As2O3 on SIVMAC transduction was assessed. As2O3 had no effect on the transduction efficiency of VSV G-pseudotyped, 2-part vectors for either SIVMAC239 or HIV-1NL4-3 in TE671 , and the other which is not restricted (B-MLV) . The throbserved . Also, aved + T cellswo weeks .+ T cells from one of two representative blood donors are shown in + T cells, was not sufficient to assess the efficiency of TRIM5 knockdown. Instead, a lentiviral vector derived from the equine infectious anemia virus (EIAV-GFP) was utilized [NL4-3 infectivity , or the isogenic vector bearing the SIVMAC239 CA (sCA-GFP), that were shown schematically in Human blood cells such as Jurkat T cells might be less permissive for SIVA flow cytometry-based assay was established that discriminates infected heterokaryons from those cells that fail to form heterokaryons . PrimaryAs a control, HeLa-RFP cells were engineered to express TvA (HeLa-RFP-TvA); in these cells, transduction with SIV CA-GFP was 1.6-fold higher than with HIV-1 CA-GFP . ChallenMAC239 transduced human blood cells less efficiently than did HIV-1. Lower SIVMAC239 transduction efficiency relative to HIV-1 was observed with all human blood-derived cells tested here, including cell lines of lymphoid and myeloid lineage, human PBMCs and primary CD4+ T cells, and, as previously described [MAC239 as by HIV-1.The characteristics of a previously unreported retroviral restriction activity in human blood cells are described here. The first clue to the existence of this restriction activity was that SIVescribed ,50,74, mMAC239-specific, restriction factor in human blood cells\u2014as opposed to the lack of a cofactor for SIVMAC239 replication in these cells\u2014was supported by the finding of a block to SIVMAC239 replication in Jurkat/HeLa-heterokaryons of equal magnitude to the block in Jurkat T cells. Similar heterokaryon experiments demonstrated the presence of a dominant restriction activity prior to the cloning of the retroviral restriction factors APOBEC3G, TRIM5, and TETHERIN [The presence of a dominant-acting, SIVTETHERIN ,43,75,76TETHERIN , and theTETHERIN .MAC239-specific restriction activity described here will be called Lv4. Whether this activity is due to a single factor, or due to a multi-factor complex, remains to be determined. Knockdown experiments presented here showed that Lv4 is distinct from TRIM5 , 731744GCTCAGCAAGCAGCAGCTGACACAGGAAACAACAGCCAGGTCAGCCAAAATTACCCAGTGCAACAAGTAGCTGGCAATTATGTCCATGTGCCGTTAAGTCCCCGAACCTTAAATGCCTGGGTAAAATTAGTGGAGGAAAAGAAGTTCGGGGCAGAAATAGTACCAGGATTTCAGGCACTATCAGAGGGATGTACCCCTTATGATATCAATCAAATGCTAAATTGTGTGGGAGAACACCAGGCAGCCATGCAAGTCATTAGAGAAATAATCAATGAAGAGGCGGCAGACTGGGACCAGCAACACCCGATACCAGGTCCACTGCCAGCAGGACAACTTAGAGACCCCAGAGGATCAGATATAGCGGGAACCACCAGCACAGTAGAGGAACAAATACAGTGGATGTACAGGGGTCAAAATTCCGTCCCAGTGGGGAACATTTATAGAAGATGGATTCAATTAGGATTGCAGAAATGTGTCAGGATGTACAATCCTACTAATATACTAGATGTAAAACAAGGGCCAAAAGAACCCTTCCAAAGCTATGTAGATAGATTCTACAAAAGCCTACGGGCAGAACAAGCAGACACAGCCGTGAGAGCATGGATGACAGAAACACTACTGGTCCAGAATGCTAACCCAGATTGCAAGCTAGTACTC>HIV-2(A), GH123TGTACAACAGACAGGCGGTGGCAACTATATCCACGTGCCACTGAGCCCCCGAACTCTAAATGCTTGGGTAAAATTAGTAGAGGACAAGAAGTTCGGGGCAGAAGTAGTGCCAGGATTTCAAGCACTCTCAGAAGGCTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGATCACCAAGCAGCTATGCAAATAATCAGAGAGATTATCAATGACGAAGCAGCAGATTGGGATGCACAGCACCCAATACCAGGCCCCTTACCAGCAGGGCAGCTTAGAGACCCAAGGGGGTCTGACATAGCAGGAACAACTAGCACAGTAGAAGAACAGATCCAGTGGATGTATAGGCCACAAAATCCCGTGCCGGTAGGGAACATCTACAGAAGATGGATCCAGATAGGGCTACAGAAGTGTGTCAGGATGTACAACCCAACTAACATCTTAGACGTAAAGCAGGGACCAAAGGAACCGTTCCAGAGCTATGTGGACAGGTTCTATAAAAGCTTGAGGGCAGAACAAACAGATCCGGCAGTAAAGAACTGGATGACCCAAACGCTGCTAATACAGAATGCCAACCCAGACTGCAAGTTAGTACTA>HIV-2(D), L33083AGTGCAGCAAGTCGGCGGAAATTATGTCCACCTACCGCTGAGTCCCAGAACATTAAATGCATGGGTTAAGTTAGTGGAGGACAAAAAATTCGGGGCAGAGGTAGTGCCAGGGTTTCAGGCACTATCGGAAGGCTGCACTCCGTATGACATCAATCAGATGCTAAATTGTGTAGGAGAACATCAGGCAGCCATGCAGATCATAAGGGAAATAATCAATGATGAGGCAGCAGATTGGGATCAGCAGCATCCACAACCAGGCCCACTACCAGCAGGACAGCTCAGAGATCCACGAGGATCTGATATAGCAGGAACCACTAGCACAGTGGAGGAACAAATACAGTGGATGTACAGGCAGCAGAATCCCATACCAGTTGGAAATATCTATAGGAGATGGATCCAGCTAGGGTTACAGAAATGTGTCAGAATGTACAACCCAACTAACATTCTGGATATAAAACAAGGGCCAAAAGAGACGTTCCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(E), L33087AGTGCAACAGATAGGAAATAACTATGTGCACTCTCCACTGTCCCCAAGAACATTGAATGCATGGGTCAAATTAGTAGAAGAAAAGAAATTTGGAGCAGAGGTAGTGCCAGGCTTCCAGGCATTATCAGAAGGATGCACCCCGTATGACATCAACCAGATGCTTAATTGCGTGGGGGAACATCAGGCAGCCATGCAAATTATCAGAGAGATAATCAATGAAGAAGCAGCAGATTGGGACGTACAGCATCCAAGAGGGCAACCGCCAGCACAGGGCCTAAGAGACCCATCAGGATCAGACATAGCAGGGACAACCAGTACCCCCGCAGAACAAATAGAGTGGATGTACAGGAATCCAAATCCAATCCCTGTGGGAGACATCTATAGAAGATGGATCCAGCTAGGGCTCCAGAAATGTGTCAGAATGTATAATCCAACAAACATTCTGGACGTCAAACAGGGGCCCAAAGAATCTTTTCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(F), U75441AGTGCAGCAGGTAGGAGGAAATTACACCCATATTCCTCTGAGTCCGAGGACATTAAATGCTTGGGTTAAATTAGTAGAGGAAAAGAAATTTGGGGCAGAAATAGTGCCAGGCTTCCAAGCATTGTCAGAAGGCTGCACCCCTTATGATATTAATCAAATGTTAAATTGTGTAGGGGAACATCAGGCAGCCATGCAAATAATCAGGGAAATAATCAATGAAGAAGCAGCCGACTGGGATCAGAATCATCCAAGGCAGCTGCCAGCGCCACCAGGGCTGCGTGATCCGTCAGGATCTGACATTGCAGGAACAACTAGTACAGTACAAGAACAGATAGAATGGATGTACAGACAGGGTAACTCAATCCCAGTAGGGGACATTTACAGAAGATGGATCCAAATAGGCCTTCAAAAATGTGTAAGAATGTACAATCCTACTAATATCCTAGATGTAAAACAGGGACCAAAAGAACCATTTCAAAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(H), AY5308GGTGCAGCAGATAGGTGGCAATTATGCCCACCTACCTCTAAGTCCTAGAACACTCAATGCCTGGGTAAAACTGGTAGAGGAGAAAAAATTTGGAGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAGAGGGCTGCACGCCCTATGATATTAATCAAATGTTAAATTGCGTGGGAGAACATCAAGCTGCTATGCAAATTATCAGGGAAATAATTAATGATGAAGCAGCAGATTGGGACACACAGCACCCAAACCAAGGCCCACCACCAGCAGGGCAACTTAGAGAGCCAAGAGGTTCTGATATTGCAGGAACAACTAGCACAGTGGAAGAGCAGATACAGTGGATGTACAGGCCGCAAAATCCAATACCGGTGGGTAACATCTATCGGAGATGGATCCAATTGGGCCTACAAAAATGTGTTAGAATGTACAATCCAACTAATATCTTAGATATAAAGCAAGGGCCAAAGGAGCCATTTCAAAGTTATGTAGATAGATTCTACAAAAGTTTGAGAGCAGAACAAACAGATCCAGCAGTGAAAAATTGGATGACTCAGACGCTGCTGATTCAGAATGCTAACCCAGACTGCAAACTCGTGTTA>SIVSME041, HM059825AGTGCAGCAAGTAGGTGGCAATTATACCCACCTACCCTTAAGTCCAAGAACATTAAATGCTTGGGTAAAATTGATAGAAGAGAAAAAATTTGGGGCAGAAGTAGTGCCAGGATTCCAAGCACTATCAGAAGGCTGCACTCCCTATGACATCAATCAGATGCTAAATTGTGTAGGGGAGCATCAATCAGCCATGCAAATTATTAGAGAAATTATAAATGAAGAAGCTGCTGATTGGGATTTACAACACCCACAGCCAGGTCCAATACCAGCAGGACAACTTAGAGACCCGAGAGGATCAGACATTGCAGGAACTACTAGCACAGTAGAAGAACAAATTCAATGGATGTATAGGCAGCAAAACCCTATACCAGTAGGTAACATTTACAGAAGGTGGATCCAATTAGGGCTGCAAAAATGTGTAAGGATGTATAATCCAACAAACATTTTAGATGTGAAACAAGGACCAAAAGAGCCATTTCAAAGCTATGTAGATAGATTCTACAAGAGTCTAAGAGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACTCAAACACTGCTGATTCAAAATGCTAACCCAGATTGCAAATTGGTGCTCgag-pol plasmid and pONY8.0 is an EIAV GFP-packaging vector [pMD2.G encodes the vesicular stomatitis virus glycoprotein (VSV G) and psPAX2 encodes HIV-1 Gag and Gag-Pol . pCIG3N g vector .env glycoprotein for virion pseudotyping was expressed from pAB6 [pAPM is a lentiviral vector expressing puromycin-resistance and a miR30-based knockdown cassette from the spleen focus forming virus LTR ,63,64. Trom pAB6 . Far redrom pAB6  was clonCell lines were either grown in DMEM  or RPMI , supplemented with 10% fetal calf serum as described before ,104,105.PBMC were separated by Ficoll density centrifugation, stimulated with PHA for 3 days, and cultured in RPMI supplemented with antibiotics, 10% fetal bovine serum, and 20 IU/ml hIL-2 ,106.+ T lymphocytes were enriched from PBMC by positive selection using magnetic beads (Miltenyi Biotec). Typically the resulting population was >99% CD4+. Cells were stimulated for 24 hrs on NUNC maxisorp plates that had been coated with 2 \u03bcg/ml anti-CD3 antibody and 2 \u03bcg/ml anti-CD28 antibody (BD Biosciences) in RPMI with 10% FBS, glutamax (Invitrogen), and 20 IU/ml hIL-2. Two wks after primary stimulation, cells were re-stimulated using plate-bound anti-CD3 and anti-CD28 antibodies.CD4MAC239, and the CA chimera vectors described above, were prepared by co-transfection of the indicated plasmids with pMD2.G in 293T cells, as described [6 cells were plated per 10-cm plate. The next day, cells were transfected using Lipofectamine 2000 (Invitrogen) and 20 \u03bcg of pAPM, 15 \u03bcg of psPAX2 and 5 \u03bcg of pMD2.G. Supernatant was collected and passed through a 0.45 \u03bcM filter at 48 hrs and at 72 hrs post-transfection, and used immediately to transduce target cells.VSV G-pseudotyped viral stocks of HIV-1, SIVescribed . Virion escribed  and by tescribed . For pro4 of the indicated target cells, in 0.4 ml media per well, in 24-well plates. As2O3 (Sigma) was prepared as described [Reporter virus-containing supernatant was titrated onto 4 x 10escribed  and, wheescribed .+ T cells were spinfected with shRNA-encoding APM vectors twice, at 24 hr and 48 hr after stimulation with plate-bound anti-CD3 and anti-CD28 antibodies. Spinfection was done at 1,130 rcf for 90 mins, using 2 ml of freshly produced virus supernatant for each well of a 6-well plate containing 5 x 105 stimulated lymphocytes. Cells were put in 5 \u03bcg/ml of puromycin for 72 hrs, 2 days after the first spinfection.Jurkat cells or primary CD44)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 \u03bcM dNTPs. All reactions and quantitation of product were carried out with a Biorad CFX96 cycler. The RT step was 42\u00b0C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95\u00b0C for 5 s, annealing 55\u00b0C for 5 s, extension at 72\u00b0C for 20 s and acquisition at 80\u00b0C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).Virus-containing supernatant was harvested 48 hr post-transfection, clarified by low-speed centrifugation, and filtered through 0.45 \u03bcm pore filters (Sarstedt). Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay ,109. VirCell-free virions were normalized by RT-activity and incubated with CRFK, Hela or Jurkat cells in 6-well plates for 12 hrs, for full-length linear cDNA and 2-LTR circles, or 48 hrs, for Alu PCR. For each virus and cell type, infections were also performed in the presence of 40 \u03bcM AZT, to control for contamination of plasmid DNA in the PCR reaction. Cells were harvested and washed extensively with PBS. Total DNA was extracted , quantified, and subjected to real-time PCR with a Biorad CFX96 cycler.4)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 \u03bcM dNTPs. The PCR was programmed for 40 cycles of denaturation at 95\u00b0C for 5 s, annealing 55\u00b0C for 5 s, extension at 72\u00b0C for 20 s and acquisition at 80\u00b0C for 5 s. Provirus was quantified by Taqman-based ALU-PCR according to the protocol described by Butler et al. [Full-length linear retroviral cDNA and 2-LTR circles were detected with SYBR-Green I based reactions using 100 ng template DNA and 320 nM of each primer pair  in 20 mM Tris-Cl pH 8.3, 5 mM , at 37\u00b0C. Cells were incubated for another 2 mins and then 2 ml of serum-free DMEM was added slowly over a period of 4 minutes at 37\u00b0C with constant, gentle agitation. An additional 5 ml of serum-free DMEM was added and cells were incubated for 5 min at 37\u00b0C. Cells were then pelleted and resuspended in complete medium before seeding in 24-well plates. 6 hours later, cells were challenged with ALV-A Env-pseudotyped vectors. A negative fusion control sample was also produced with no PEG addition. Infected cell cultures were analyzed using a FACS-Canto (BD) 48 hrs after vector challenge. Fluorescence acquisition was performed using blue (488 nm) and red (633 nm) lasers. Dead cells were excluded from the analysis based on propidium iodide staining.2 x 10http://grants.nih.gov/grants/policy/hs/faqs_aps_definitions.htm.Human peripheral blood mononuclear cells (PBMC) were obtained from anonymous, untraceable blood donors. This research is therefore considered non-human subjects research by our Institutional Review Board, based on NIH guidelines (45 CFR 46.102(f)):"}
+{"text": "TRHR) gene, rs1800795 in the interleukin-6 (IL6) gene and rs6552828 in the coenzyme A synthetase long-chain 1 (ACSL1) gene. To gain insight into their functionality (which is yet unknown), here we determined for the first time luciferase gene reporter activity at the muscle tissue level in rs7832552 and rs6552828. We then compared allele/genotype frequencies of the 3 abovementioned variants among centenarians  and healthy controls  of the same ethnic and geographic origin (Spain). We also studied healthy centenarians  and controls  from Italy, and centenarians  and healthy controls  from Japan. The THRH rs7832552 T-allele and ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the C and G-allele, respectively (P = 0.001). Yet we found no significant association of EL with rs7832552, rs1800795 or rs6552828 in any of the 3 cohorts. Further research is needed with larger cohorts of centenarians of different origin as well as with younger old people.There are several gene variants that are candidates to influence functional capacity in long-lived individuals. As such, their potential association with exceptional longevity  deserves analysis. Among them are rs7832552 in the thyrotropin-releasing hormone receptor ( The oldest old population (\u226585 years) is rapidly expanding among westerners Waite, . HoweverMSTN) gene   gene), might also influence the likelihood of reaching EL  gene, rs16892496 and rs7832552  in the interleukin-6 (IL6) gene, where the G-allele is associated with higher transcription in vitro   analysis of 379,319 SNPs in US Caucasians of both genders (aged 50 years on average) revealed an association of lean body mass with 2 SNPs in tight linkage disequilibrium within the thyrotropin-releasing hormone receptor (2peak)], a recent GWAS study in sedentary Caucasians found that, among 324,611 SNPs, the strongest association with the VO2peak response to exercise was found to acyl coenzyme A synthetase long-chain 1 (ACSL1) gene polymorphism rs6552828 \u2014among Spanish centenarians (cases) and healthy controls matched by ethnic and geographic origin and also in 2 other geographically and ethnically-independent replication cohorts .In order to analyze their functionality at the muscle level, we measured for the first time luciferase gene reporter activity in MluI/NheI in the 5\u2032 and XhoI in the 3\u2032 (see below -in bold) of the sequences obtained from the genomes of:A-allele and one individual homozygous for the rs16892496 C-allele (see below -underlined)one individual homozygous for the rs16892496 AArs16892496 AGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCCCrs16892496 CGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCC-allele and one homozygous for the rs7832552 T-allele (see below -underlined)one individual homozygous for the rs7832552 CCrs7832552-CGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTATTrs7832552-TGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTAA-allele and one individual homozygous for the rs6552828 G-allele (see below -underlined)one individual homozygous for the rs6552828 rs6552828-AAAAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGArs6552828-GGGAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGAThe fragment, including the allele, was directly inserted into the pGL3-promoter at the restriction recognition sites We used mice skeletal muscle C2C12 cell lines to study muscle-specific expression. We performed cell cultures, transfections and dual-luciferase reporter assays following the procedures previously reported by our group , University of Pavia , and National Institute of Health and Nutrition, (Japan) Medical Research Institute and Keio University (Japan)] and the study followed the tenets of the Declaration of Helsinki for Human Research. Written consent was also obtained from each participant.Two groups of Spanish subjects were assessed: (i) 138 cases ; and (ii) 334 healthy controls . All the subjects were of the same Caucasian (Spanish) descent for \u22653 generations. The major diseases among the centenarians were osteoarthritis (66%), hypertension (57%), dementia (51%) and cardiovascular disease . The DNA of a convenience sample of 355 younger disease-free controls with no reported family history of high longevity (>90 years) was collected during 2008-2012 in the European University of Madrid.Two groups of subjects from Northern Italy (mainly from Lombardy and Piedmont) were studied: (i) 79 cases ; and (ii) 316 healthy controls . All patients and controls were Caucasian whites of Italian descent for \u22653 generations The Italian centenarians were free of major age-related diseases, i.e., severe cognitive impairment, clinically evident cancer, CVD, renal insufficiency or severe physical impairment  descent were assessed: (i) 742 cases ; and (ii) 499 healthy controls . The group of cases was gathered from 2 cohorts, which are described in detail elsewhere  because the genotype distributions of both SNPs are completely linked according to available HapMap for both European and Asian populations (sorted as a Supplementary file 1) and previous research has shown that both SNPs are in strong linkage disequilibrium (r2 = 0.98). All genotyping was performed only for research purposes with the researchers who performed the genotyping being blinded to the participants' identities. For quality control, a random ~20% of the samples of each cohort were genotyped again, with no differences in the results compared with the initial genotyping.As mentioned above, only one DNA was extracted from the participants' buccal cells  using a standard phenol chloroform protocol and the genotype analyses were performed in the Biomedicine laboratory at the European University, Madrid (Spain). The DNA samples were diluted with sterile water and stored at \u221220\u00b0C until analysis. Genotyping was performed by Real-Time PCR and using the TaqMan\u00ae rs7832552, rs6552828, and rs1800795 SNP genotyping assays with a Step One Real-Time PCR System .Genomic DNA was purified from blood leukocytes using the QiaAmp DNA Mini kit  according to the manufacturer's protocol. Genotyping was performed at the Cellular Pathophysiology and Clinical Immunology Laboratory (University of Pavia) using the TaqMan\u00ae rs7832552, rs6552828, and rs1800795 SNP genotyping assays .Total genomic DNA was extracted from blood leukocytes with a QIAamp DNA Blood Mini or Maxi Kit . Genotyping of rs7832552, rs6552828, and rs1800795 was performed at the Institute of Health and Sports Science and Medicine (Juntendo University) with Real Time Thermocycler  using TaqMan SNP genotyping assay method. PCR 384-well plates were read on LightCycler 480 using the end-point analysis mode. Allelic discrimination analysis was performed with a LightCycler 480 SW software version 1.5.1.62 .2-test. Genotype/allele frequencies of cases vs. controls within each cohort  were compared using the \u03c72-test with Yates' correction and the association between genotypes/alleles and EL within each of the 3 cohorts was analyzed with logistic regression analysis after adjusting for sex. All statistical analyses were performed using the PASW  and corrected for multiple comparisons using the Bonferroni's method -that is, the threshold P-value was obtained by dividing 0.05 by the number of studied polymorphisms (P = 0.05/3 = 0.017).One-way analysis of variance was used to compare the relative luciferase activity in the different plasmids of each SNP. Allele frequencies were calculated by gene-counting. We tested Hardy-Weinberg equilibrium (HWE) using \u03c7P \u2264 0.001); the THRH rs16892496 A-allele up-regulated luciferase activity compared to the C-allele (upper panel), the THRH rs7832552 T-allele up-regulated luciferase activity compared to the C-allele (middle panel), and the ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the G-allele (lower panel).The results of luciferase report analyses are presented in Figure THRH rs7832552, 97.2% in cases and 100% in controls; ACSL rs6552828, 97.2% in cases and 99.1% in controls; IL6 rs1800795, 100% in cases and 94.9% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for IL6 rs1800795 in the control group (P < 0.01).Rate of genotyping success was as follows: 2 = 1.21, P = 0.27) or genotype frequency distributions of THRH rs7832552 did not differ between cases and controls . Using logistic regression analysis, no significant associations were found between EL and rs7832552, including when analyzing both sexes separately (data not shown). No differences were found for IL6 rs1800795 in allele  or genotype distributions , with no significant association with EL after adjusting for sex or when analyzing both sexes separately -data not shown) or for ACSL1 rs6552828 .The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table P > 0.05), except for THRH rs7832552 in the control group (P = 0.04).Rate of genotyping success was 100% for all gene variants. The distribution of all genotypes was consistent with the HWE in both groups  and genotype frequency distributions of THRH rs7832552 did not differ between groups  and no significant association was found between this polymorphism and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown). Similar results were found for IL6 rs1800795  and ACSL1 rs6552828 , with no significant association between these two polymorphisms and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table THRH rs7832552, 97.0% in cases and 100% in controls; IL6 rs1800795, 98.7% in cases and 100% in controls; ACSL1 rs6552828, 95.9% in cases and 99.2% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for rs6552828 in centenarians (P = 0.02).Rate of genotyping success was as follows: THRH rs7832552 , rs1800795 , or rs6552828 , with no significant association between any of the SNPs and EL using logistic regression adjusted by sex or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table in vitro approach) the potential functional consequences of the rs16892496, rs7832552, and rs6552828 SNPs, with the A-allele, T-allele and A-allele up-regulating luciferase activity compared to the other alleles, respectively. The THRH rs7832552 and ACSL1 rs6552828 SNPs are intronic genomic variants and, as such, could potentially alter the stability and/or alternative splicing of mRNA, as well as transcription factor binding  might favor EL. Yet a GWAS study reported that the TRHR rs7832552 SNP was associated with lean body mass in US Caucasians  genotype had 2.55 kg higher lean body mass compared to the other subjects. There is some scientific rationale in postulating that higher TRHR expression might help preservation of muscle mass in long-lived individuals: TRHR stimulates the hypothalamic-pituitary-thyroid axis, thereby leading to the release of thyroxin, a hormone that plays an important role in the development of skeletal muscle as well as in attenuating age-related changes in tissue function , we used convenience samples, which increases the risk of bias induced by population stratification. The SNPs rs1800795 and rs7832552 did not meet HWE in Spanish and Italian controls, respectively. In this regard, deviation from HWE does not necessarily reflect genotyping errors  none of them was associated with EL. Similarly, no association was found for rs1800795. More research is needed in the field with other cohorts, using larger population samples, as well as younger elderly  to assess the potential link between these genetic variants and the human aging process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers. Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4\u00a0F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies.To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5\u2032UTR complexity determines Silvestrol-sensitivity and altering 5\u2032UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity.Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol\u2019s anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.The online version of this article (doi:10.1186/s13059-014-0476-1) contains supplementary material, which is available to authorized users. Energetically, protein synthesis is the most costly step on the path toward gene expression and is thus a rigidly controlled process. In eukaryotes, protein synthesis occurs in three phases: translation initiation, elongation and termination. Although translation is controlled at multiple stages, regulation is primarily exerted at initiation, the phase in which 80S ribosomes assemble onto mRNA transcripts. Regulation of initiation is mediated by multiple factors, many of which converge on the assembly of the eukaryotic initiation factor 4F (eIF4F). This heterotrimeric complex is composed of eIF4E, the rate-limiting protein which binds the 5\u2032-7-methylguanosine cap on cellular mRNA transcripts; eIF4A, a DEAD-box RNA helicase; and eIF4G, a scaffolding protein which bridges eIF4E and eIF4A, and recruits eIF3 and the 43S pre-initiation complex. Formation of eIF4F is tightly controlled by multiple mitogenic signaling pathways, namely mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and has been shown to stimulate translation of mRNAs involved in cell proliferation, growth, survival, cell cycle progression, and DNA damage repair \u20133. Moreoin vitro [Significant progress has been made toward understanding the machinery that drives protein synthesis. However, the underlying mechanisms by which individual eIF4F components contribute to translation regulation in the cell remain ambiguous. Emerging methods that allow for global dissection of translation have bolstered the long standing knowledge that translation is subject to considerable regulation and thus plays a key role in regulating gene expression \u201313. Studin vitro . These din vitro and in vivo, thereby impairing translation initiation [eIF4A is an integral part of the heterotrimeric eIF4F complex and the only component with known enzymatic activity. While several eIF4A-regulated genes have been identified, in-depth studies have yet to provide a genome-wide description of the eIF4A target gene landscape. We sought to comprehensively define the cellular mRNAs regulated by eIF4A and investigate the mRNA features that confer dependence on this helicase by directly blocking its activity. To achieve this, we employed the potent and specific eIF4A inhibitor Silvestrol. This compound has been shown to selectively target the RNA helicase activity of eIF4A both itiation ,22, and itiation ,24. Moreitiation . Here we35S-methionine incorporation into newly synthesized proteins. To mitigate off-target or secondary effects of drug treatment, we chose to treat cells with a low concentration of Silvestrol for a short time period. We selected a concentration of 25 nM Silvestrol, which had negligible effects on global translation after 2\u00a0hours of exposure  and a difference in distribution of \u2206G values from genes with increased TE was also notable compared with the insensitive pool (P =4.25\u2009\u00d7\u200910-7)  while those from genes with increased TE were comparatively shorter  and slightly higher in those from genes with increased TE (P =8.08\u2009\u00d7\u200910-6)  and ROCK1 , were significantly more sensitive to Silvestrol than unstructured controls, CMV alone and PFN2   . The introduction of ARF6mut into our luc2CP reporter decreased the sensitivity of our reporter construct to Silvestrol  analysis of Silvestrol-sensitive genes revealed that a large proportion of eIF4A-dependent genes were involved in cell cycle progression 4 motif is present in both the ARF6 and ARF6mut 5\u2032 UTR reporter constructs .Cells were seeded at a density of 1\u2009\u00d7\u2009105 cells/well in 6-well format and grown overnight. 24\u00a0hours after seeding, cells were washed in 1\u00d7 phosphate-buffered saline  for 10\u00a0minutes prior to harvest. Cells were lysed in RIPA buffer  containing complete EDTA-free protease inhibitors , and phosphatase inhibitors . Total protein was trichloroacetic acid  and used to normalize radioactive counts in TCA precipitated protein.MDA-MB-231 cells were seeded at a density of 3\u2009\u00d7\u2009106 cells/plate in 15\u00a0cm plates and grown overnight. Cells were then treated with DMSO or Silvestrol (25 nM) for 2\u00a0hours prior to harvest. At time of harvest, 100\u00a0\u03bcg/ml cycloheximide  was added to each plate and incubated for 1\u00a0minute. Cells were washed twice in ice-cold 1\u00d7 PBS +100\u00a0\u03bcg/ml cycloheximide and lysed in ice-cold polysome lysis buffer , 1% TritonX-100 ). Sucrose gradients (10 to 50%) were created by layering 10% and 50% sucrose solutions , 10 or 50% RNase-free sucrose) into Seton tubes  followed by mixing with the BioComp Gradient maker set at an 81.5\u00b0 angle at 16\u00a0rpm . Lysates were layered on top of each gradient and subjected to ultracentrifugation in an SW41-Ti rotor at 35,000\u00a0rpm at 4\u00b0C for 3\u00a0hours. Polysome profiles were analyzed using a BioComp Gradient Station at constant speed with optical monitoring at a 260\u00a0nm wavelength. For polysome fractionation experiments, 1\u00a0ml fractions were collected and stored at -80\u00b0C for subsequent RNA isolation followed by quantitative RT-PCR.Cells were seeded at a density of 4\u2009\u00d7\u2009102, 1\u00a0mM DTT, 8% glycerol, 100\u00a0\u03bcg/ml cycloheximide (Sigma), 24 U/ml Turbo DNAse (Ambion)). Lysates were clarified, treated with RNAse I (Ambion) and overlaid on a 34% sucrose cushion. Monosomes were isolated by centrifugation at 69,000\u00a0rpm for 4\u00a0hours in a TLA110 rotor. Ribosome protected RNA fragments (RFs) were isolated from monosome fractions by acidic-phenol extraction and used to generate libraries for sequencing. The genome-aligned RF profiles in Additional file Ribosome profiling was performed as previously described ,41 with Total RNA was extracted from MDA-MB-231 cells using Trizol  following the manufacturer\u2019s guidelines. Polyadenylated RNA was isolated from the total fraction using Oligotex mRNA kit . The resulting mRNA was partially fragmented by alkaline hydrolysis with sodium carbonate to generate approximately 150-nucleotide fragments on average. RNA fragments of 40 to 100 nucleotides were isolated by gel extraction and used to generate libraries for mRNA-Seq. The genome-aligned mRNA profiles in Additional file et al. [Strand-specific libraries were generated as described ,41 with et al. . SamplesSequence analysis was performed as described . Briefly5\u2032 UTR sequences were retrieved from Ensemble using RefSeq mRNA numbers as query. Folding energies of the resulting sequences were analyzed using CONTRAfold  and McCaCyclinD1 and ROCK1 were reverse transcribed from total RNA isolated from MDA-MB-231 cells  and the resulting cDNA was amplified by high-fidelity PCR using 5\u2032 UTR-specific oligonucleotides. The 5\u2032 UTRs for ARF6, ARF6mut and PFN2 were synthesized by Blue Heron Biotechnology . 5\u2032 UTRs were subcloned into the NcoI site of vector pMH2 to create vectors pCR300, pCR301, pCR302, pCR303, and pCR304 . Luciferase assays were performed by adding 100\u00a0\u03bcl/well Luciferase Assay Reagent (Promega E1501) and incubating for 10\u00a0minutes at room temperature before measuring luminescence. For luc2CP mRNA analysis, 4\u2009\u00d7\u2009105 cells/well were seeded in 6-well plates and grown overnight. Cells were then treated with the indicated amounts of Silvestrol for 40\u00a0minutes and lysed using Trizol reagent. RNA was isolated from Trizol lysates using standard methods. The abundance of luc2CP and \u03b2-actin mRNA was subsequently analyzed by quantitative RT-PCR.293\u00a0T cells were co-transfected with 5\u2032 UTR-bearing Prior to RNA extraction from polysome fractions, 5\u00a0ng of Luciferase Control RNA (Promega) was added to each fraction. Samples were treated with 200\u00a0\u03bcg/ml Proteinase K for 1\u00a0hour at 50\u00b0C and RNA was extracted using the standard hot acid phenol method. RNAs were precipitated using isopropanol and resuspended in 10\u00a0mM Tris pH7.luc2CP mRNA) levels, transcript abundance was normalized to \u03b2-actin mRNA measurements.Total RNA was converted to cDNA using SuperScript III reverse transcriptase (Invitrogen) with oligo dT primers according to the manufacturer\u2019s instructions. Transcript levels were measured by quantitative PCR using SYBR green FAST PCR mix  and oligo pairs specific to each transcript. In polysome fractionation experiments, transcript levels were normalized to luciferase control RNA abundance. To measure luciferase reporter mRNA  antibody  at room temperature for 30\u00a0minutes, washed, resuspended in PI/RNase solution (BD Biosciences #550825) and analyzed by fluorescence-activated cell sorting (FACS).MDA-MB-231 cells were seeded in 6-well dishes at 3.5\u2009\u00d7\u200910MDA-MB-231 cells were treated with 25 nM Silvestrol and lysed in RIPA buffer  containing complete EDTA-free protease inhibitors (Roche #11836170001), and phosphatase inhibitors . Lysates were quantified using BCA reagent (Thermo Scientific #23228). Lysates were subjected to SDS-PAGE (NuPAGE) using 15 to 25\u00a0\u03bcg of cell lysate per well and transferred to PVDF (Invitrogen).The following antibodies were purchased from commercial suppliers: CyclinD1 , CyclinB1 , CyclinE1 , Bcl2 (BD Pharmingen #551097), Parp(Asp214) , Arf6 , GAPDH , \u03b2-Tubulin .Cell lines used in this study were obtained from ATCC. Cells were tested for mycoplasma by the Novartis Institutes for Biomedical Research and were mycoplasma-free._CATCTCCATGGGCAGAACTGGGAGGAGGAGT_3\u2032; oARF6rev: 5\u2032_GACCTCCATGGCGCGTCGGAGGAGCCGGGGCCG_3\u2032; oCCND1fwd:_5\u2032_GACCTCCATGGGCTTAACAACAGTAACGTCACACGG_3\u2032; oCCND1rev:_5\u2032_CAGCTCCATGGCTGGGGCTCTTCCTGGGCAG_3\u2032; oROCK1fwd:_5\u2032_GACCTCCATGGGCGCUGGUUCCCCTTCCGAGCGT_3\u2032; oROCK1rev:_5\u2032_CATCTCCATGGGTGTTGCTGCTGCTGTGACAATGCCCT_3\u2032; oPFN2fwd:_5\u2032_GAATCTCCATGGGCCGCTGGTTTGTCAGCC_3\u2032; oPFN2rev:_5\u2032_CTCGAGCCATGGCTTCGAGCCCTTCGC_3\u2032; oARFmut_ampF:_5\u2032_CCATGGTTGGGACGTGCACTGGCAGCCGGC_3\u2032; oARFmut_ampR:_5\u2032_GTGGGAGTTGCCTCCTAAGCTAATATGTGC_3\u2032oARF6fwd: 5\u2032_ATGTTGCAGGTGAGATGTGGT_3\u2032,ARF6_qP_F8: 5\u2032_TACCTGCTCCAGTCACCAATG_3\u2032,ARF6_qP_R8: 5\u2032_AGCCTCGCCTTTGCCGA_3\u2032,ActB_qP_F1: 5\u2032_GCGCGGCGATATCATCATC_3\u2032,ActB_qP_R1: 5\u2032_TTCTGCCCCTGCCAAATCTT_3\u2032,BCL2_qP_F3: 5\u2032_CATCTGAGAACCTCCTCGGC_3\u2032,BCL2_qP_R3: 5\u2032_TGAGGGACGCTTTGTCTGTC_3\u2032,CCND1_qP_F2: 5\u2032_CTTCTGCTGGAAACATGCCG_3\u2032,CCND1_qP_R2: 5\u2032_ACCTGCCCCTTACTCTGACT_3\u2032,CDK6_qP_F2: 5\u2032_AGCACCCAGTAAGACATCCAG_3\u2032,CDK6_qP_R2: 5\u2032_TGAAAGCCGCACTGATGGAT_3\u2032,ROCK1_qP_F1: 5\u2032_GCCATGAGAAAACACATTGCAG_3\u2032,ROCK1_qP_R1: 5\u2032_ATCCGGAAGCGACCAACGCC_3\u2032,Luciferase_F: 5\u2032_GTCGGGAAGACCTGCCACGC_3\u2032,Luciferase_R: 5\u2032_ATCCACCTTAACAGCCACGG_3\u2032,Luc2CP_F: 5\u2032_CAGGGTGTCTATCCATGCCG_3\u2032.Luc2CP_R: 5\u2032ARF6mut sequence. The (CGG)4 motif is shown in bold.Mutated residues are indicated by lower case lettering in the ARF6wt:_5\u2032_AGAACUGGGAGGAGGAGUUGGAGGCCGGAGGGAGCCCGCGCUCGGGGCGGCGGCUGGAGGCAGCGCACCGAGUUCCCGCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCGGCGCUCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGGGGGGCCUGGGCCUCUGCCCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3\u2032.ARF6mut:_5\u2032_CCAUGGuUGGGAcGuGcAcUgGcAGcCgGcAGaGAGCCCGCcCaCcGcuacGCGGCUuacccgucCcgAgCcAcUaggCcCGAGGAUCCAUGACCUGACGGGGCCCCGGAGCCGCGCUGCCUCUCGGGUGUCCUGGGUCGGUGGGGAGCCCAGUGCUCGCAGGCCGGCGGGCGGGCCGGAGGGCUGCAGUCUCCCUCGCGGUGAGAGGAAGGCGGAGGAGCGGGAACCGCGGCucuagaCGCGCGGCGCCUGCGGGGGGAAGGGCAGUUCCGGGCCGGGCCGCGCCUCAGCAGGGCGGCGGCUCCCAGCGCAGUCUCAGGGCCCGGGUGGCGGCGGCGACUGGAGAAAUCAAGUUGUGCGGUCGGUGAUGCCCGAGUGAGCGuauacagUcGaCaUaUuagCUUAGGAGGCAACUCCCACGCAGGCCGCAAAGGCGCUCUCGCGGCCGAGAGGCUUCGUUUCGGUUUCGCGGCGGCGGCGGCGUUGUUGGCUGAGGGGACCCGGGACACCUGAAUGCCCCCGGCCCCGGCUCCUCCGACGCGCCAUG_3\u2032.CyclinD1:_5\u2032_CCAUGGGCUUAACAACAGUAACGUCACACGGACUACAGGGGAGUUUUGUUGAAGUUGCAAAGUCCUGGAGCCUCCAGAGGGCUGUCGGCGCAGUAGCAGCGAGCAGCAGAGUCCGCACGCUCCGGCGAGGGGCAGAAGAGCGCGAGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCCUCCCCAGCUGCCCAGGAAGAGCCCCAGCCAUG_3\u2032.ROCK1:_5\u2032_GCUGGUUCCCCUUCCGAGCGUCCGCGCCCCGCAUGCGCAGUCUGCCCCGGCGGUCUCCGUUUGUUUGAACAGGAAGGCGGACAUAUUAGUCCCUCUCAGCCCCCCUCGCCCCACCCCCCAGGCAUUCGCCGCCGCGACUCGCCCUUUCCCCGGCUGGGACCGCAGCCCCUCCCAGAAGCUCCCCCAUCAGCAGCCGCCGGGACCCAACUAUCGUCUUCCUCUUCGCCCGCUCUCCAGCCUUUCCUCUGCUAAGUCUCCAUCGGGCAUCGACCUCGCCCUGCCCCACCGGACACCGUAGCAGCAGCCCCAGCAGCGACGGGACAAAAUGGGAGAGUGAGGCUGUCCUGCGUGGACCAGCUCGUGGCCGAGACUGAUCGGUGCGUCGGGCCGGGCCGAGUAGAGCCGGGGACGCGGGGCUAGACCGUCUACAGCGCCUCUGAGCGGAGCGGGCCCGGCCCGUGGCCCGAGCGGCGGCCGCAGCUGGCACAGCUCCUCACCCGCCCUUUGCUUUCGCCUUUCCUCUUCUCCCUCCCUUGUUGCCCGGAGGGAGUCUCCACCCUGCUUCUCUUUCUCUACCCGCUCCUGCCCAUCUCGGGACGGGGACCCCUCCAUGGCGACGGCGGCCGGGGCCCGCUAGACUGAAGCACCUCGCCGGAGCGACGAGGCUGGUGGCGACGGCGCUGUCGGCUGUCGUGAGGGGCUGCCGGGUGGGAUGCGACUUUGGGCGUCCGAGCGGCUGUGGGUCGCUGUUGCCCCCGGCCCGGGGUCUGGAGAGCGGAGGUCCCCUCAGUGAGGGGAAGACGGGGGAACCGGGCGCACCUGGUGACCCUGAGGUUCCGGCUCCUCCGCCCCGCGGCUGCGAACCCACCGCGGAGGAAGUUGGUUGAAAUUGCUUUCCGCUGCUGGUGCUGGUAAGAGGGCAUUGUCACAGCAGCAGCAACACCCAUG_3\u2032PFN2:_5\u2032_CGCUGCGGUAAGGAGCAGCCGCCACAGGCACAGCCGCUUCGCAGCCUCCCGCCGCUGGUUUGUCAGCCCCGCGGCUGCGGGCGGCCGGGCGGCCGAGCGCGCUCUGAGGUUCGUCCCUCAUCGCUGAACCCGCGUCCUCCCGCCGCAGCUCCUCGGGGAGGGGGGCGGUCGGUGCCUGCGCAGAGCCGCCUCCUCCCCGCCCCCGCCCCGCCUCCCCCCGCGCCGCCGCCGCCCGCUACCGCCGCCGCCGCCGCUGCGCCUGCUGCUCCUCGCCGUCCGCGCUGCAGUGCGAAGGGCUCGAGCCAUG_3\u2032.Monosome preparations pelleted in ultracentrifuge tubes were washed with 1\u00a0ml ice-cold ethanol by addition of the ethanol followed by a 5\u00a0minute incubation on ice and aspiration of the ethanol via a vacuum trap. The pellet was allowed to air dry. Monosome pellet was dissolved in 100\u00a0\u03bcl of a solution of 20\u00a0mM ammonium bicarbonate, 8\u00a0M urea and 2\u00a0mM Tris(2-carboxyethyl)phosphine hydrochloride. MS-Grade Lys C (Thermo) was dissolved in water to a concentration of 0.5\u00a0\u03bcg/\u03bcl and 2\u00a0\u03bcl was added to the dissolved pellet. The Lys C digestion was incubated for 4\u00a0hours at room temperature. The digestion was further diluted with an additional 98\u00a0\u03bcl of water. MS-Grade Trypsin (Thermo) was also dissolved in water to a concentration of 0.5\u00a0\u03bcg/\u03bcl and 2\u00a0\u03bcl was added to the diluted Lys C digestion. This trypsin digestion was incubated overnight at room temperature.Digestion samples were prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) by acidification. The digestion sample (20\u00a0\u03bcl) was acidified by the addition of 2\u00a0\u03bcl of 10% formic acid; 20\u00a0\u03bcl of this sample was injected onto a 2.1\u2009\u00d7\u200950\u00a0mm Extend-C18 column  at 400\u00a0\u03bcl/minute flow rate. Elution of peptides was over a 51\u00a0minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier, and a flow rate of 200\u00a0\u03bcl/minute. Data acquisition was on an Agilent 6530 Q-ToF instrument equipped with a dual electrospray source, and a reference mass of 922.009798 enabled. MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher and individual ions were excluded for 9\u00a0seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 50,000 counts or 333 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 (Agilent) and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 10\u00a0ppm and MS/MS tolerance of 0.1\u00a0Da.Digestion samples were prepared for LC-MS/MS by acidification. The digestion sample (10\u00a0\u03bcl) was acidified by the addition of 10\u00a0\u03bcl of 1% formic acid; 4\u00a0\u03bcl of this sample was injected onto a 4\u00a0mm 40\u00a0nL trap column at a flow rate of 3\u00a0\u03bcl/minute and analyzed on a 75\u00a0\u03bcm\u2009\u00d7\u2009150\u00a0mm column containing Zorbax 300SB-C18 (5\u00a0\u03bcm beads) at a flow rate of 400\u00a0nl/minute. Elution of peptides was over a 100\u00a0minute gradient from 5 to 40% acetonitrile with 0.1% formic acid as the modifier. Data acquisition was on an Agilent 6550 Q-ToF instrument equipped with the ChipCube source, and a reference mass of 1221.990637 enabled. MS survey scans were acquired at a rate of 5\u00a0Hz, and MS/MS spectra were acquired in Auto MS/MS mode with triggering of precursor ions of +2 charge state and higher. Individual ions were excluded for 18\u00a0seconds after triggering an MS/MS scan. Up to five precursors were selected per MS scan, and MS/MS scans were for 25,000 counts or 200 mS, whichever came first. LC-MS/MS data were exported to a Mascot Generic Format file using MassHunter Qual 5.0 and searched against SwissProt 51.6 with taxonomy limited to human, without modifications other than variable pyroglutamate formation at Q and E, MS tolerance of 5\u00a0ppm and MS/MS tolerance of 0.1\u00a0Da.5\u2032 UTR sequences were retrieved from Ensemble using RefSeq mRNA names as query. Sequences were broken into 20-nucleotide fragments in a stepwise manner beginning from the first nucleotide (n) and proceeding in 1 nucleotide steps  through the length of the UTR. The structure of each fragment was analyzed using the CONTRAfold algorithm and the resulting free energy values were plotted based on their position across the length of the UTR, as were the percentage GC values.5 cells in serum-free medium. Medium containing 5% fetal bovine serum was aliquoted to lower wells and cells were incubated for 22\u00a0hours. When measuring migration in the presence of Silvestrol, compound was included in the medium in both upper and lower chambers at the concentrations indicated. To assess cell migration after 22\u00a0hours, upper chambers were transferred to wells containing 1\u00d7 PBS +8\u00a0\u03bcM Calcenein AM (Life Technologies) and incubated for 40\u00a0minutes. Fluorescence was evaluated by excitation at 485 nM wavelength followed by measuring emission at 520 nM.Migration assays were performed using trans-well migration chambers (BD Biosciences). MDA-MB-231 cells were seeded into upper chambers at a density of 0.5\u2009\u00d7\u2009105 cells/well in RPMI supplemented with 10% fetal bovine serum and grown for 24\u00a0hours to generate a monolayer. The following day cells were scratched to produce a wound in the monolayer and wound closure was monitored by microscopy using the Incucyte Live Cell Imaging system .Cells were seeded into 24 well plates at a density of 6\u2009\u00d7\u200910The next generation sequencing data presented in this paper have been deposited in NCBI\u2019s Gene Expression Omnibus  and are"}
+{"text": "Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA\u2013MRE interactions at any point during development.MicroRNA (miRNA) target recognition is largely dictated by short \u2018seed\u2019 sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA\u2013target interactions to the regulation of biological processes  Identifying miRNA response elements (MREs) within target mRNAs can be done computationally but the functional validation of putative MREs remains challenging. Here, Bassett et al. describe applications of genome engineering to target and assess the functional significance of MREs in different organisms and stages of development. To date, the standard strategy used to validate miRNA targets has relied on artificial sensor assays whereby the 3\u2032-untranslated region (UTR) of the gene of interest (or a region flanking the predicted MRE) is coupled to a reporter gene. Although valuable, a critical limitation of this technology is its inability to infer the physiological relevance of a putative MRE in vivo, as these assays do not recapitulate the endogenous context and stoichiometry of miRNAs and their targets. An alternative strategy is provided by target protector (TP) oligonucleotides, which overlap with the seed region and a unique flanking sequence in the target 3\u2032UTR, and can therefore block miRNA access to the MRE8910in vivo, few studies have been performed to address potential toxicity or off-target effects of TP oligonucleotides. Furthermore, TP assays are in most cases limited to transient effects in systems where oligonucleotides can be supplied by injection or transfectionMicroRNA (miRNA)-mediated post-transcriptional silencing represents an essential regulatory layer underlying cellular function, development and disease12ry high ~0%67, andin vivo. To generate targeted insertion and deletion (indel) mutations within MREs, we use both transcription activator-like effector nucleases (TALENs) in zebrafish and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas9) in Drosophila. In both systems, indels are created as a result of inefficient non-homologous end joining repair at the nuclease cleavage site. Using this approach, we have accurately defined bona fide MREs in these two species and have directly addressed their functional relevance in an endogenous context in vivo. Furthermore, we describe a novel approach for the identification of active MREs in cell culture systems, using CRISPR-mediated homology-directed repair (HDR) with short oligonucleotide donors. This now allows the effect of defined deletions or alterations within MREs to be rapidly assessed in heterogeneous cell populations. Finally, we present an online algorithm that computationally predicts CRISPR target sites neighbouring all predicted MREs in several species, and thereby show that these techniques may be generally applicable for investigation of the vast majority of MREs.To address these limitations, we have used genome engineering technologies to genetically ablate endogenous MREs within putative target genes lefty2 (lft2) 3\u2032UTR, causing a midline development phenotype in the embryo as a consequence of impaired Nodal signallinglft2 DNA  of single experimental embryos showed that lft2 expression level at shield stage was significantly increased as compared with controls . miR-430 MRE disruption, as assayed by the resistance to Bsp1286I digest, was observed in DNA samples obtained from all tested embryos that exhibited mutant phenotypes , indicating highly efficient editing events at the targeted locus. These results establish the feasibility of this approach for analysis of miRNA\u2013target regulatory interactions during zebrafish embryonic development, and validate the functional relevance of this MRE in the lft2 3\u2032UTR. Notably, such mutants could be used to generate stable lines, allowing phenotypic analysis at all stages of development and during adult life.In zebrafish, morpholino TP oligonucleotides have been used to block a predicted miR-430 site in the lft2 DNA . Customilft2 DNA . The spaion site . In agreion site , with a bantam (ban) in the enabled (ena) gene in Drosophila. Previous work showed that overexpression of ban in the wing imaginal disc can reduce Ena levels in the context of a 3\u2032UTR reporter assay, and that this effect was dependent on a conserved ban target siteTo realize the potential of our strategy in elucidating the functional significance of MREs throughout development, we next investigated the effect of removing a well-studied target site for the miRNA ban target site in the ena 3\u2032UTR . The presence of the mutation in animals used for all subsequent steps was validated by PCR (ban using the decapentaplegic driver (dpp-GAL4) that expresses in a stripe of cells across the anterior/posterior boundary of the wing imaginal disc (ena-3\u2032UTR-sensor construct (ena-3\u2032UTR) and endogenous Ena protein levels (n>30). No change in Ena levels was observed in control discs expressing CD8-EGFP under the same GAL4 driver . This result indicates that overexpressed ban can directly target ena via this predicted MRE in its 3\u2032UTR.To create a genomic deletion in the predicted MRE, we designed a synthetic guide RNA (sgRNA) that would target the Cas9 nuclease to the predicted na 3\u2032UTR . The expquencing . We decid by PCR  and sequd by PCR . We overnal disc . Confirmn levels , n>30. N4 driver . When th protein , n>30. Tban regulates Ena expression on either side of the dorsal/ventral (D/V) boundary of the wing imaginal discsban-MREmut animals revealed no apparent changes to endogenous Ena expression pattern in the wing imaginal discs (compare n=10), or any detectable phenotypes in the adult wings (n=10). This indicates that although ban overexpression is able to downregulate Ena through this MRE, the endogenous expression pattern of Ena in the wing disc is not dependent on this target site. Thus, although it remains possible that ban participates in establishing the identity of the D/V boundary in wing discs, it is unlikely this activity is mediated by tuning Ena expression through this predicted MRE. These results emphasize the importance of directly establishing the in vivo physiological relevance of a MRE, and suggest that caution should be exerted when inferring its function from indirect assays.It was previously suggested that endogenous  compare . We also compare , n=10, olt wings , n=10. Tin vivo are critical for understanding the function of MREs in the context of the whole organism, cell culture systems offer a valuable tool for studying miRNA function in a well-defined context, and a means to directly investigate their role in human cells. We therefore developed a novel strategy to validate functional MREs in cultured cells, using the CRISPR/Cas9 system to enhance homologous recombination, and generate defined sequence alterations of a MRE of interest. The approach involves co-transfection of the Cas9/sgRNA targeting a MRE with two ~140\u2009nt single-stranded DNA (ssDNA) oligonucleotide templates for HDR. One of these HDR templates inserts a T3 \u2018barcode\u2019 downstream of the target MRE locus, but maintains the intact MRE . Interestingly, C9orf7 is targeted by miR-92a via a non-canonical motif through complementary base pairing with its 3\u2032 end, demonstrating that such interactions can be biologically relevant. The other putative MREs in PCMTD1 and MAPRE1 failed to show this effect, suggesting that they may not play a role in regulating the abundance of these transcripts, at least in this particular cell type. One consideration when designing this strategy is to determine whether integration of the barcodes creates or inadvertently removes endogenous MREs. To test this, the sequences generated upon integration of T7-MREmut and T3-MREWT barcodes were screened for miRNA target sites using the PITA algorithmmut barcode, as intended , and revealed that both of these MREs are functional in this cell type intended . All butell type .Based on these results, we propose that this strategy can be applied broadly to the identification of physiologically active MREs when the target mRNA is expressed at sufficient levels to allow detection by qPCR of homologous integration within the cDNA. In addition, our results suggest that care must be taken when extrapolating the functional relevance of a MRE from studies of miRNA binding to the target transcript or changes in gene expression resulting from miRNA inhibition.www.microrna.org, August 2010 release1921To establish the versatility of these technologies, and whether they can be generally applied to analysis of miRNA\u2013MRE interactions, we performed computational analysis to identify CRISPR target sites in the vicinity of all predicted MREs in several organisms . CRISPR http://miR-CRISPR.molbiol.ox.ac.uk/fulga/miR-CRISPR.cgi). Information is provided for five species: human, mouse, rat, Drosophila and Caenorhabditis elegans. Upon entering a gene symbol and/or miRNA identifier, CRISPR sgRNA target sites are generated for each predicted MRE, along with the distance between the PAM and MRE, the forward and reverse oligonucleotides required for sgRNA production, and the number and details of putative off-target sites.CRISPR target sites for all predicted MREs are available via the miR-CRISPR online web interface lft2 3\u2032UTR at the miR-430-binding site, separated by a 16-nt spacer. RVD arrays containing HD, NG, NI and NN monomers were assembled using the Golden Gate TALEN and TAL Effector Kit 2.0  as previously describedin vitro transcription of corresponding TALEN mRNAs. The RVD sequences of the left and right TALENs are:The TALE nuclease was designed using the TALEN Targeter algorithm , and capped TALEN mRNAs were in vitro transcribed using the mMESSAGEmMACHINE SP6 kit . RNAs were then polyadenylated with the polyA tailing kit  and purified with the RNeasy mini kit .Plasmid pMTB2-Goldy A measure of 25\u2009pg of each TALEN mRNA was injected into single-cell stage embryos, using the Picospritzer II microinjector (Parker Instrumentation). Embryos were incubated in E3 mediumlft2 antisense probe was generated by PCR amplification of cDNA from 12\u2009hours post fertilization wild-type zebrafish, using the following primers: SP6-lft2-F 5\u2032-GATTTAGGTGACACTATAGgaccacagcgatctcactca-3\u2032 and T7-lft2-R 5\u2032-TAATACGACTCACTATAGGGgactggagggattttgtcc-3\u2032. The PCR product was gel extracted and purified by ethanol precipitation. 1.5\u2009\u03bcg of template DNA was transcribed using T7 RNA polymerase  in the presence of digoxygenin (DIG)-labelled dNTPs . Probes were purified using G-50 micro-columns . Embryos were permeabilized with 1% H2O2, equilibrated with hybridization buffer dimethylammonio]-1-propanesulfonate (CHAPS), 100\u2009\u03bcg\u2009ml\u22121 Heparin) and incubated overnight with lft2 DIG-labelled probes. Embryos were blocked with 20% sheep serum+2% Boehringer Blocking Reagent . Probes were detected with an anti-DIG-alkaline phosphatase antibody  and the signal was developed using 30\u2009\u03bcg\u2009ml\u22121 of each nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate .The template for Bsp1286I  for 4\u2009h at 37\u2009\u00b0C. Digested products were then analysed by gel electrophoresis (1% agarose). To identify the nature of indel mutations, PCR products were cloned into pGEM-T easy vector  and DNA was isolated from individual clones and sequenced using an SP6 primer.gDNA was extracted by homogenizing single zebrafish embryos in 50\u2009\u03bcl of 50\u2009mM NaOH, followed by incubation for 20\u2009min at 95\u2009\u00b0C, cooling to 4\u2009\u00b0C and addition of 5\u2009\u03bcl of 1\u2009mM Tris-HCl pH=8 to neutralize the solutionFor coupled transcriptional/genomic analysis of single injected zebrafish embryos, total RNA and DNA were extracted using the RNAqueous Micro Kit . cDNA was synthesized using the Superscript III Reverse transcriptase kit . For quantitative PCR, the following gene-specific primers were used: Lft2-F (5\u2032-CTGGCAGGAATACTCAGGGG-3\u2032), Lft2-R (5\u2032-TGGCCTCCATGTCGAACA-3\u2032), ActB-F (5\u2032-AATCCCAAAGCCAACAGAGA-3\u2032), ActB-R (5\u2032-ACATACATGGCAGGGGTGTT-3\u2032). The levels of Lft2 transcript in individual embryo analysis were calculated using the standard curve method.20NGG). Potential off target sites within the Drosophila genome were identified by BLAST and using the CRISPR design tool ( http://crispr.mit.edu). The closest off-target site contained four mismatches to the 20\u2009nt target sequence. Cas9 mRNA was in vitro transcribed from plasmid MLM3613 (ref. TAATACGACTCACTATAGGCGTATGAGATCGTGTGCTTGTTTTAGAGCTAGAAATAGC-3\u2032) and a reverse oligonucleotide encoding the remainder of the sgRNA sequence (sgRNA-R=5\u2032-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3\u2032). Reaction solutions (100\u2009\u03bcl) were cycled on a GStorm thermal cycler , 72\u2009\u00b0C 10\u2009min, 10\u2009\u00b0C \u221e) and purified with a PCR purification kit . In vitro transcription was performed with 300\u2009ng purified DNA template for 4\u2009h at 37\u2009\u00b0C using the Megascript T7 kit , and sgRNA purified by phenol chloroform extraction and isopropanol precipitation. sgRNAs were diluted to 1\u2009\u03bcg\u2009\u03bcl\u22121 in water and stored in aliquots at \u221280\u2009\u00b0C  was used with an Injectman NI2 micromanipulator and Femtotip II needles . Embryos were incubated at 25\u2009\u00b0C for the remainder of the development. Putative mosaic flies were crossed individually to yw; Sco/CyO flies. These flies were removed after 7 days, and analysed for mutant tissue by HRMA ; ena-3\u2032UTR-sensor and UAS-bantam were a generous gift from Marco MilanDrosophila CRISPR injections are provided at the OxfCRISPR website ( http://www.oxfcrispr.org)A measure of 0.5\u2009\u03bcg sgRNA and 10\u2009\u03bcg Cas9 mRNA were precipitated with ethanol to purify and concentrate, and resuspended in water at 0.5\u20131\u2009\u03bcg\u2009\u03bcl by HRMA . Only cr by HRMA . During \u22121 proteinase K , and heating to 37\u2009\u00b0C for 30\u2009min or 60\u2009min, followed by inactivation at 95\u2009\u00b0C for 2\u2009min  and blocked in 5% normal goat serum in PBS-T. Discs were then incubated overnight at room temperature with primary antibodies mouse anti-Enabled , rabbit anti-GFP  and mouse anti-wingless , washed three times 20\u2009min each in PBS-T and incubated for 2\u2009h at room temperature with anti-mouse A568 , anti-rabbit A488  and DAPI . Samples were then mounted in Slowfade  and imaged on a Zeiss 780 confocal microscope with a \u00d7 25 oil immersion objective and \u00d7 0.7 digital zoom. Images were acquired using similar laser power, pinhole size and gain parameters, and adjusted post-acquisition for background intensity and contrast correction. For assessing Ena expression in the dpp-GAL4>UAS-ban background, a minimum of 30 third instar larval wing imaginal discs were analysed. The results displayed in ban-MREmut lines. To confirm the nature of ban-MREmut indels in animals processed for immunofluorescence and imaging, half of each larvae was snap frozen immediately after dissection and subjected to gDNA extraction followed by PCR validation and sequencing.20NGG were chosen as close as possible to the predicted MREs in the 3\u2032UTRs of PCMTD1 (5\u2032-GGCAATATATGAGTGCAATA-3\u2032), MAPRE1 (5\u2032-CCTTGGGATCTGCCAGGCTG-3\u2032) and C9orf7 (5\u2032-GTGGCATCTGAGGCCGGGAG-3\u2032) in human cells, and Pck (5\u2032-TCCATAGTGCCTTTAACAAT-3\u2032) and CG13088 (5\u2032-CATTTCTACCTCAATCCGTC-3\u2032) in Drosophila cells. Target sequences were cloned into the pX330 vector Target sites of the form NPCMTD1 targetCCGGGCAATATATGA GTGCAATAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATG AAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTPCMTD1 ssOligoT7TAATACGACTCACTATAGGGAACAATACAAGATATTGAATAATTTAGCTTTAAA AAATCCCACAAATTTTATGAAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGAPCMTD1 ssOligoT3GTGCAATAATTAACCCTCACTAAAGGGAAACAATACAAGATATTGAATAATTTAGCTTTAAAAAATCCCACAAATTTTATGAAATTTT-3\u20325\u2032-TTACTTTTGTTGCAATCACTGTTGTTGGGTTGCTGTATATATATTGCGGGCAATATATGA MAPRE1 targetGGCAGGCTGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCA CTGTAATCCGAATACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT7TAATACGACTCACTATAGGGGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAA TACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCMAPRE1 ssOligoT3CAGGCTGGGAATTAACCCTCACTAAAGGGAGTGTTTTCGGTATCTGCTGTTCACAGCTCTCCACTGTAATCCGAATACTTTGCCAGTGCA-3\u20325\u2032-CTTTCTGGACCTCTGGCAAAGGGAGTGGTCAGTGAAGGCCATCGTTACCTTGGGATCTGCC9orf7 targetAGGCCGGGAGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTT TTCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT7TAATACGACTCACTATAGGGGTGGCATCTGAGGCCAGGAGTGGCAGGCTGGTGGGCTGGGCGTG GGGTTTTCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGC9orf7 ssOligoT3AGGCCGGGAAATTAACCCTCACTAAAGGGAGTGGCATCTGAGGCCAG GAGTGGCAGGCTGGTGGGCTGGGCGTGGGGTTT TCTGGGCCCT-3\u20325\u2032-AACTGTTTCCCAGGAACACCTCTCGGGCCCATCTGCGTCTGAGGCTGGGAGTGGCATCTGPCK targetCCGTCCATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAG ATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT7TAATACGACTCACTATAGGGATAGTGCCTTTAACAATCGACCATATGTATCTATATACA CGCCGACTCAGCCGAGATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAATCGACGPCK ssOligoT3CCGTCCAATTAACCCTCACTAAAGGGAATAGTGCCTTTAACAATCGACCATATGTATCTATATACACGCCGACTCAGCCGAGATCAG-3\u20325\u2032-ATTGGATCTTAGCTTAAGTTTTCAGACGCAATCTCGTGCCTCGAATCTGCGCAAT CGACGCG13088 targetC TCCGTCGGTATACGAATATATATGTAGATGGAGATCCAA ATGATATATCCTGAGTAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT7 TAATACGACTCACTATAGGGGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAG TAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAACG13088 ssOligoT3TCCGTCCAATTAACCCTCACTAAAGGGAGGTATACGAATATATATGTAGATGGAGATCCAAATGATATATCCTGAGTAAAATGTTGTA-3\u20325\u2032-GAATTCAGTCCAGACTCGTAGTAGTCATTTGAAAAGACTTAAATGACATTTCTACCTCAA 2 in DMEM with 25\u2009mM glucose , 10% fetal bovine serum  and 1% penicillin-streptomycin . S2R+ cells were cultured at 25\u2009\u00b0C in Schneider\u2019s Drosophila medium , 10% fetal bovine serum  and 1% penicillin-streptomycin . Cas9/sgRNA expression vectors (1\u2009\u03bcg) and homology oligonucleotides (0.5\u2009\u03bcg each T3/T7 oligo) were co-transfected into HEK293T cells in a six-well dish using polyethylenimine (Sigma-Aldrich) as previously describedHEK293T cells were cultured at 37\u2009\u00b0C and 5% CO\u22121 of proteinase K , and incubation at 55\u2009\u00b0C for a minimum of 2\u2009h. Proteins were removed by phenol/chloroform extraction, and DNA precipitated with 2.5 volumes ethanol. DNA was quantified with a Nanodrop spectrophotometer and diluted to a concentration of 100\u2009ng\u2009\u03bcl\u22121 before analysis. RNA was extracted using a miRNeasy mini kit  including the additional DNAse treatment step. cDNA was synthesized from 1\u2009\u03bcg RNA with the Quantitect Reverse Transcription kit  and diluted twofold before analysis.gDNA was extracted from zebrafish embryos by addition of 300\u2009\u03bcl lysis buffer  containing 200\u2009\u03bcg\u2009ml\u2212\u0394Ct.Quantitative PCR was performed with gene-specific forward primers , and either T3as (5\u2032-TCCCTTTAGTGAGGGTTAATT-3\u2032) or T7as (5\u2032-CCTATAGTGAGTCGTATTA-3\u2032) reverse primers. Amplification was performed with SybrGreen JumpStart Taq Ready Mix  and detected on an ABI 7500 Fast thermal cycler. Results are shown as a ratio of the signal with the T7 primer to that with the T3 primer for gDNA and cDNA for three independent biological replicates, each analysed in technical triplicate. The final signal ratio was obtained by calculating the difference in Ct between T7 and T3 primers (\u0394Ct) during the exponential amplification phase, and subsequently transforming the resulting values using 2www.microrna.org, August 2010 Release) for four species, Drosophila, human, mouse and rat. A custom perl script was used to identify potential CRISPR target sites within a 200-bp window around each of the MREs from the appropriate genomic sequences . Potential off-targets for each of the CRISPR target sites were identified using the BatMis algorithmMREs for conserved miRNAs with a \u2018good mirSVR score\u2019 were predicted using the miRanda algorithm .Supplementary Figure 1 and Supplementary Table 1"}
+{"text": "The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression. Published 19 March 2015TTCGATGATTCCCAGATACTAGTACGGTCAG-3\u2032 (WIZ \u2018WT DNA target\u2019).There was an error in the WIZ binding sequence used for the EMSA experiments. The incorrect sequence was 5\u2032-GAGTCTCACTCACGCGCCATTCCATTCCATTCAGATACTAGTACGGTCAG-3\u2032 (WIZ \u2018WT DNA target\u2019).The correct sequence is 5\u2032-GAGTCTCACTCACGCGCThe article has been corrected accordingly."}
+{"text": "TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR\u2019s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.The DOI:http://dx.doi.org/10.7554/eLife.13964.001 Prostate cancers are among the most common types of cancer in men, which, like other cancers, are driven by genetic mutations. Roughly half of all prostate cancers contain a genetic change that incorrectly fuses two genes together, causing the cells to produce abnormally high levels of a protein called ERG.ERG is a transcription factor, a protein that binds to specific sequences of DNA to influence the activity of nearby genes. ERG represses genes that help to prevent prostate cancers from growing, and so promotes prostate cancer development. Like most other transcription factors, ERG is difficult to target with drugs and no therapies that directly prevent the activity of ERG currently exist.Mounir et al. wanted to find out whether ERG cooperates with other proteins to cause prostate cancer cells to grow, with the hope that these proteins could be more easily targeted with a drug. By using various biochemical techniques in human prostate cancer cell lines, Mounir et al. found that ERG interacts with an enzyme called PRMT5. This interaction enables PRMT5 to chemically modify other proteins to change their activity. In the case of prostate cancer cells, PRMT5 inappropriately modifies the androgen receptor, a protein that regulates the growth of normal prostate cells. This abnormal modification contributes to the excessive growth of the cancer cells.Although PRMT5 will be easier to target with drugs than ERG, it also has many other roles besides those described by Mounir et al. Much more work is therefore needed to investigate whether PRMT5 could be safely targeted to treat patients with prostate cancer.DOI:http://dx.doi.org/10.7554/eLife.13964.002 TMPRSS2:ERG fusion  is highly prevalent and lethal . Drugs tG fusion . TMPRSS2 in mice . ERG is  in mice , suggest in mice . We explTMPRSS2:ERG positive PC cells, we performed a pooled short hairpin RNA (shRNA) screen in TMPRSS2:ERG and AR-positive VCaP prostate cancer cells, using ERG-negative 22Rv1 cells as a control . The shRNA pool targets 648 genes involved in transcriptional and epigenetic regulation . Identified proteins  includedTMPRSS2:ERG positive NCI-H660) and one PRMT5 hairpin likely to have minimal off-target effects. This shRNA shows a trend of sensitivity in ERG-positive lines, in agreement with our findings -inducible shRNA vectors targeting PRMT5 and a non-targeting control shRNA (NTC). PRMT5 knockdown was robust in all cell lines . Robust findings .PRMT5 is a protein arginine methyltransferase that regulates multiple signaling pathways through the mono- and symmetric di-methylation of arginines on its target proteins .To deterPSA, NKX3-1 and SLC45A3  to investigate ERG, AR, and PRMT5 recruitment to the AR targets cterized  binding cterized . We alsocterized . In VCaPcterized . As expecterized , ERG expcterized , and ERGcterized . Like ERcterized .To extend these findings to a genome wide scale, we performed AR and ERG ChIP-seq experiments in androgen (DHT or R1881) stimulated VCaP cells upon PRMT5 knockdown . ERG and AR recruitment were robust in these experiments, as judged by recovery of the canonical DNA binding sites of these proteins . In agrePSA or NKX3-1 loci  and symmetric di-methylation (SDMA) levels compared to wild-type controls  , suggest3-1 loci . We ther3-1 loci . To confenotypes . Like VCcontrols . On the sus VCaP , suggestsus VCaP . In thessus VCaP , suggestTo further understand if the observed AR methylation was directly dependent on PRMT5, we performed biochemical assays using purified PRMT5 (complexed with its requisite binding partner MEP50/WDR77) and AR LBD. PRMT5 activity, as judged by production of SAH (the by-product of SAM-dependent substrate methylation), was observed in the presence of AR LBD as substrate, but not in the presence of ERG ETS DNA binding domain or pointed (PNT) domain. PRMT5 activity in the presence of AR LBD further increased with the addition of ERG ETS domain protein to the reaction, but not with PNT domain . Unlike To identify the arginine methylation site(s) on AR, we cloned AR cDNA and mutated all arginines in the LBD to lysine . We expressed each construct in AR-negative, ERG-negative RWPE-1 prostate cells. R1881 stimulates exogenous AR to induce target gene expression in these cells. ERG expression represses this effect  and induPSA locus as a model AR-regulated gene. Relative to WT AR, the AR R761K mutant showed enhanced recruitment to PSA and increased PSA expression. Moreover, R761K mutation prevented ERG-dependent attenuation of AR recruitment to PSA and PSA expression  and tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza).Redundant siRNA Activity or RSA metric was used as described  were used for further analysis  targeting transcriptional and epigenetic regulators similar to a previously reported library . shRNA lescribed . Brieflyhttp://tools.proteomecenter.org/software.php]) using a false positive threshold of <1% for protein identifications (See Nuclear extracts of VCaP cells (~600 million cells) were pre-cleared using agarose beads  and used for pulldown with either an ERG antibody (Santa Cruz#353) or an anti-rabbit IgG antibody. All antibodies were pre-coupled to beads , washed then used for pulldown. Immunoprecipitations were eluted at pH2.5 followed by TCA protein precipitation, alkylated with iodoacetamide, and separated on a NuPage 4\u201312% Bis-Tris gradient gel (Invitrogen). Complete gel lanes were excised using a LEAP 2DiD robot and in-gel digested with trypsin (Tecan Freedom EVO 20). Peptide sequencing for the resulting resulting 16 digest samples was performed by liquid chromatography-tandem mass spectrometry using an Eksigent 1D+ high-pressure liquid chromatography system coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide mass and fragmentation data were searched against a combined forward-reverse IPI database (v3.55) using Mascot 2.2 (Matrix Science). Peptide and protein validation were done using Transproteomic pipeline v3.3sqall  was previously described, as were the sequences of the nontargeting control and ERG shRNA inserts and stable cell line generation. PRMT5 sPRMT5 shRNA#1:AGGGACTGGAATACGCTAATTCTCGAGAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#2:AGGGACTGGAATACGTTAATTGTTAATATTCATAGCAATTAGCGTATTCCAGTCCCTPRMT5 shRNA#3:GCGGATAAAGTTGTATGTTGTGTTAATATTCATAGCACAGCATACAGCTTTATCCGCAll shRNA were expressed from a puromycin resistant vector. Lentiviral production and cell transduction was as previously described .CGCGATTGGAACACGTTGATT .The Dox-inducible ERG constructs named ERG or ERG DNAx used for stable and inducible expression in 22Rv1 cells were generated as previously described . The cDNCAGAATCAAGCTCTACGCCGT .For shPRMT5-2: the GCGGATAAAGCTGTATGCTGT target sequence was converted to Full sequence of scPRMT5 below:scPRMT5 sequence: ATGTACCCCTATGACGTGCCAGATTACGCCATGGCGGCGATGGCGGTCGGGGGTGCTGGTGGGAGCCGCGTGTCCAGCGGGAGGGACCTGAATTGCGTCCCCGAAATAGCTGACACACTAGGGGCTGTGGCCAAGCAGGGGTTTGATTTCCTCTGCATGCCTGTCTTCCATCCCAGGTTCAAGCGCGAGTTTATTCAGGAACCTGCTAAGAATCGGCCCGGTCCCCAGACACGATCAGACCTACTGCTGTCAGGACGCGATTGGAACACGTTGATTGTGGGAAAGCTTTCTCCATGGATTCGTCCAGACTCAAAAGTGGAGAAGATTCGCAGGAACTCCGAGGCGGCCATGTTACAGGAGCTGAATTTTGGTGCATATTTGGGTCTTCCAGCTTTCCTGCTGCCCCTTAATCAGGAAGATAACACCAACCTGGCCAGAGTTTTGACCAACCACATCCACACTGGCCATCACTCTTCCATGTTCTGGATGCGGGTACCCTTGGTGGCACCAGAGGACCTGAGAGATGATATAATTGAGAATGCACCAACTACACACACAGAGGAGTACAGTGGGGAGGAGAAAACGTGGATGTGGTGGCACAACTTCCGGACTTTGTGTGACTATAGTAAGAGGATTGCAGTGGCTCTTGAAATTGGGGCTGATTTGCCCTCTAATCACGTCATTGATCGCTGGCTTGGGGAGCCCATCAAAGCAGCCATTCTCCCCACTAGCATTTTCCTGACCAATAAGAAGGGATTTCCTGTTCTTTCTAAGATGCACCAGAGGCTCATCTTCCGGCTCCTCAAGTTGGAGGTGCAGTTCATCATCACAGGCACCAACCACCACTCAGAGAAGGAGTTTTGTAGCTACCTGCAGTACCTGGAATACTTAAGCCAGAACCGTCCTCCACCTAATGCCTATGAACTCTTTGCCAAGGGCTATGAAGACTATCTGCAGTCCCCGCTTCAGCCACTGATGGACAATCTGGAATCTCAGACATATGAAGTGTTTGAAAAGGACCCCATCAAATACTCTCAGTACCAGCAGGCCATCTATAAATGTCTGCTAGACCGAGTACCAGAAGAGGAGAAGGATACCAATGTCCAGGTACTGATGGTGCTGGGAGCAGGACGGGGACCCCTGGTGAACGCTTCCCTGCGGGCAGCCAAGCAGGCCGACCGCAGAATCAAGCTCTACGCCGTGGAGAAAAACCCAAATGCCGTGGTGACGCTAGAGAACTGGCAGTTTGAAGAATGGGGATCCCAGGTCACGGTAGTCAGCTCAGACATGAGGGAATGGGTGGCTCCAGAGAAAGCAGACATCATTGTCAGTGAGCTTCTGGGCTCATTTGCTGACAATGAATTGTCGCCTGAGTGCCTGGATGGAGCCCAGCACTTCCTAAAAGATGATGGTGTGAGCATCCCCGGGGAGTACACTTCCTTTCTGGCTCCCATCTCTTCCTCCAAGCTGTACAATGAGGTCCGAGCCTGTAGGGAGAAGGACCGTGACCCTGAGGCCCAGTTTGAGATGCCTTATGTGGTACGGCTGCACAACTTCCACCAGCTCTCTGCACCCCAGCCCTGTTTCACCTTCAGTCACCCTAATCGCGACCCCATGATTGACAACAACCGCTATTGCACCTTGGAATTTCCTGTGGAGGTGAACACAGTACTACATGGCTTTGCCGGCTACTTTGAGACTGTGCTTTATCAGGACATCACTCTGAGTATCCGTCCAGAGACTCACTCTCCTGGGATGTTCTCATGGTTTCCTATTCTGTTTCCCATCAAGCAGCCCATAACGGTACGTGAAGGCCAAACCATCTGTGTGCGTTTCTGGCGATGCAGCAATTCCAAGAAGGTGTGGTATGAGTGGGCTGTGACAGCACCAGTCTGTTCTGCTATTCATAACCCCACAGGCCGCTCATATACCATTGGCCTCTGA.Generation of the PRMT5 catalytic dead mutant was performed by site-directed mutagenesis  through mutation of G365 to A and R368 to A .2 incubator at 37\u00b0C. Doxycycline  was used at 100 ng/ml. Cell proliferation was measured in 6-well plates (Corning) using automated confluence readings . R1881 (Sigma) and charcoal-stripped serum (Omega Scientific) were used where indicated.VCaP, 22Rv1, LNCaP and RWPE-1 cell lines (ATCC) were grown in vendor-recommended media and maintained in a humidified 5% CO2\u20135\u00a0ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer\u2019s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). After coupling, 1\u00a0mg of the antibody/bead mixture was incubated with 1\u20135\u00a0mg of protein lysate overnight under rotation at 4\u00b0C. IP samples were then washed with RIPA buffer containing protease/phosphatase inhibitor cocktail for 3\u20134 washes and resuspended in non-reducing loading buffer, boiled and loaded on a gel for western blot analysis. Immunoprecipitations were performed using the following antibodies: anti-ERG antibody (Epitomics# 2805\u20131), anti-HA antibody (Roche#11815016001), anti-AR antibody (Thermo Scientific# MA5-13426) or anti-IgG antibody .Procedures were previously described  and the Generation of labeled cDNA, hybridization to Affymetrix U133plus2 human arrays, and data normalization were performed as described . For theBlack line  represents expressed probe set position and is ranked by average fold-change. Blue, green, and red lines indicate where the probe sets mapping to genes in the androgen receptor activation signatures appear in our data set and show the cumulative sum of the probe sets in the androgen receptor activation signatures that overlap with our gene list (only the highest expressing probe set was used per gene). The dashed line represents the hypothetical cumulative sum for a random list of genes that are unenriched.PSA, NKX3-1 and SLC45A3 and VIC-labeled probe for Beta-2-macroglobulin (B2M) as a normalization control. Samples were run on a 7900HT Real-Time PCR machine (Applied Biosystems) and data was analyzed and normalized according to manufacturer\u2019s instructions (2-\u0394Ct method).RNA isolation was performed as previously described . Taqman VCaP, 22Rv1 and RWPE-1 cells were treated as specified followed by cross-linking with 1% formaldehyde for 10\u00a0min. Cells were next lysed in 1% SDS and sonicated until DNA ladder is below 1\u00a0kb (Diagenode). Sheared chromatin was then used for IP with specific primary antibodies  pre-complexed with Protein A/G Dynabeads and incubated overnight under rotation at 4\u00b0C. The next day, ChIP samples were washed with RIPA buffer and TE followed by reverse crosslinking using 1%SDS and 30\u00a0ug/ml proteinase K (Invitrogen) at 65\u00b0C for 6\u2009hr with beads. The eluates were then purified using the QIAquick PCR purification kit (Qiagen) and used for qPCR with the following primer sets:PSA -4100: acctgctcagcctttgtctc AND ttgtttactgtcaaggacaatcgPSA -3800: agaattgcctcccaacactg AND cagtcgatcgggacctagaaPSA -100: cttccacagctctgggtgt AND aaaccttcattccccaggacPSA +700: agccccagactcttcattca AND atgcagatttggggaatcagNKX3-1 -2800: gagagcagctgttcctccac AND acgagccttttccacctttcNKX3-1 -200: agggaggagagctggagaag AND tcctccctaggggattcctNKX3-1 +2150: accaggatgaggatgtcacc AND cagggacagagagagccttgNKX3-1 -+10800: tctctcgttggctcctgatt AND ccagcttttgttccttcctgNKX3-1 +62100: cggtttattgcccatgaaga AND aacagggctcacagtgctttVCaP cells harboring Dox inducible shRNAs targeting PRMT5 where grown to 80% confluency and re-seeded (day 0) into full media containing 100\u00a0ng/ml Doxycycline. On day 3 media was replaced with \u2018hormone reduced\u2019 media, containing 100\u00a0ng/ml Doxycycline. Cells were stimulated with adding indicated ligands (DHT (Sigma), R1881 [Sigma]) or vehicle on day 4 and harvesting of cells was performed on day 5. Cells were harvested by fixation using 1% methanol free formaldedyde  in PBS at room temperature. Fixation was stopped after 8\u2009min by replacing fixation buffer with ice cold PBS containing 125\u00a0mM glycine and 5\u00a0mg/ml BSA. Cells were further washed once using ice-cold PBS and re-suspended into 500\u00a0ul of PBS containing Complete Protease Inhibitors (Roche). Cells where then pelleted and supernatant removed, and the resulting pellet either snap frozen in liquid Nitrogen or immediately re-suspended in lysis buffer for further processing.3 and 1% (w/v) SDS. Crosslink reversal was done at 65C\u00b0C for 6\u2009hr and ChIP DNA were isolated using DNA purification beads (MagBio). ChIP-seq libraries were generated using the KAPA HTP library preparation kit (Kapa biosciences). All handling of samples after sonication was done on using a Sciclone NGS Workstation . Sequencing was performed on an Illumina NextSeq500 instrument.For ChIP the resulting cell lysate was sonicated using a Covaris E210 instrument according to manufacturers recommendations. Each ChIP reaction was performed using soluble fraction chromatin corresponding to 7.5\u00a0ug purifed DNA and 4\u00a0ug of antibodies. Antibodies were allowed to bind overnight before capture on protein A magnetic beads . Bound beads where washed 4 times in RIPA buffer containing 500\u00a0mM LiCl, and 2 times with TE buffer before being re-suspended in containing 100\u00a0mM NaHCOReads passing Illumina standard QC were mapped to genome version Hg19 using BWA, and binding sites (\u2018peaks\u2019) were identified using MACS2, evolutionary conservation scores at peak locations was calculated using Phastcons and enriched DNA motifs using MDscan and Seqpos. These were performed using the ChiLin QC pipeline (liulab.dfci.harvard.edu/WEBSITE/software). Peaks from AR ChIP-sequencing samples with a MACS2 enrichment score higher than 10 were extended to a uniform 400\u00a0bp across all samples and overlapping peaks where collapsed to generate a union of all peaks. This resulted in a Cistrome of 25,593 peaks that were used in all genome wide ChIP analyses. Using the features of this Cistrome as GTF, read counts from BWA mapped Bam files were processed using the Qlucore 3.1.19 software. Heatmaps and statistical test (two-sided t-test using correction for multiple hypothesis testing) of differential binding scores on the 25,593 features were performed in Qlucore v3.1.19. The AR and ERG ChIPseq datasets are available at the NCBI Gene Expression Omnibus (Accession number GSE79128).RWPE-1 cells were treated as specified, fixed with 4% paraformaldehyde for 45 min at room temperature (Electron Microscopy Sciences), blocked with 5% goat serum, 0.5% Triton X-100 in PBS for 2\u2009hr and incubated with 1:50 dilutions of AR (LSBIO #LS-C87494) and symmetric di-methyl arginine antibodies (CST#13222) in 5% goat serum and 0.05% Triton X-100. Fixed samples were incubated overnight at 4\u00b0C in primary antibody before incubations with proximity ligation assay (PLA) secondary antibodies .The secondary antibody incubation, ligation, amplification and final wash steps were performed according to the manufacturer\u2019s specifications. Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .Homo sapiens androgen receptor (AR) sequence  was synthesized to include a 5\u2019 NotI and 3\u2019 BamHI sites and used as a template for the mutagenesis of each arginine in the ligand binding domain of AR into lysine . Following sequence verification to ensure mutation incorporation, each AR mutant sequence was cloned into the pLVX vector via the 5\u2019 NotI and 3\u2019 BamHI as previously described  and incubated with 1:50 dilution of AR antibody (LSBIO #LS-C87494). Confocal microscopy was performed using an LSM 510 META  with a 40x C-Apochromat objective, NA 1.2. Images were collected and processed using Zen software .The heterodimeric structure of PPAR\u03b3-RXR\u03b1\u00a0(PDB Code: 3DZY) was used as a template to overlay individual domain structures of AR including the DBD in complex with DNA (PDB Code: 1R4I) and the LBD in complex with coactivator peptide TIF2(iii) and ligand R1881 (PDB Code: 2AO6). A superposition was achieved using secondary structure matching in COOT . The AR \u00b0C. Cells were resuspended in buffer A containing 50\u2009mM Tris-HCl (pH 7.3), 150\u2009mM NaCl, 10% glycerol, 0.25\u2009mM TCEP, and 10 \u00b5M DHT, to which 50 \u00b5g/ml DNase I and protease inhibitor cocktail (Roche) were added. Cells were lysed using an M-110L Microfluidizer at 18,000 psi, followed by the addition of 0.5% CHAPS to the lysate prior to high speed centrifugation. For one-step batch purification, the soluble extract was incubated with 2\u2009ml of glutathione sepharose 4 fast flow medium  for 1\u2009hr at 4\u00b0C with rotational mixing. The sepharose medium was washed in buffer A with the addition of 0.5% CHAPS. Elution was accomplished by resuspending and incubating the media for 10 min in the wash buffer plus 10\u201320\u2009mM reduced glutathione. The eluted fractions were then combined and concentrated to 0.3 mg/ml.The gene encoding human AR LBD (residues 663\u2013919), was inserted into a pGEX-6P-1 vector and expressed as a GST-tagged fusion protein in BL21 Star (DE3) cells. Cells were grown in TB2 medium containing 10 \u00b5M dihydrotestosterone (DHT) and induced with 1\u2009mM IPTG for 14 \u201316\u2009hr at 16S-adenosyl-L-homocysteine (SAH) product formation utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). 0.5\u20131\u00a0uM of PRMT5/MEP50 recombinant enzyme  was incubated with 50\u00a0uM SAM, 2\u00a0uM GST-AR LBD and/or 5\u00a0uM ETS or PNT ERG protein for 2\u2009hr at 37\u00b0C. Reactions were quenched to 0.1% HCOOH followed by addition of -SAH (SAH-D4) in 20% DMSO as an internal standard for MS quantification. Samples were sonicated with a Hendrix SM-100 sonicator (Microsonics Systems) and centrifuged. SAH was separated from the reaction mixture by reversed phase chromatography using polar endcapped C18 reversed phase columns  and detected using a 4000 QTRAP Hybrid Triple Quadrupole/Linear Ion Trap LC-MS/MS system (AB Sciex).PRMT5 enzymatic activity was assessed by monitoring  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your work entitled \"ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see below, the reviewers have some concerns that will require quite an additional effort to address. Ordinarily, we allow just two months to return a revised version but we are concerned that this required experiments may take longer to complete. To help us assess the likelihood of a successful completion of the required additional work, please send us a note indicating how you plan to proceed and an estimate of the time it will take to complete the work.Summary:In this manuscript, the authors present two major findings: 1) PRMT5 methylates AR at R761 and these methylation results in decreased AR recruitment at target genes and decreased AR function and 2) ERG targets AR to be methylated. Overall, the findings address long-standing questions of ERG function in prostate tumorigenesis and possibly posit new therapies to ERG translocated prostate cancers. This would be certainly relevant to the prostate field. In addition, it is appropriate for a general audience for implications of a general enzyme that affects specific programs. The experiments are well done and logical. The manuscript is well written. However, at this point, there are several critical experiments that should be performed to solidify the conclusion.Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.2) One weakness of the manuscript, intrinsic to prostate cancer research, is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site. Essential revisions:1) Definition of the ERG and AR binding genome wide with and without PRMT5 and ERG knockdown and correlation with the gene expression is very important for the conclusion. Is AR binding specifically depleted in ERG co-bound sites? The changes genome wide may not reflect the selected sites, as highlighted by the effect of FOXA1 on AR. This does require time and effort. But AR and ERG ChIP-seq is well optimized and ChIP-seq today is cost effective so this is eminently doable.KLK3 (aka PSA) and NKX3-1, the ChIPseq dataset shows a significant increase of AR recruitment upon PRMT5 knockdown at 6% of the total AR-bound cistrome. Genome-wide recruitment of ERG was not significantly affected upon PRMT5 knockdown, including to the sites where AR recruitment was increased . This data agrees with our model that ERG recruits PRMT5 to attenuate AR activity to a significant subset of its target genes.We thank the reviewers for raising this important point. To extend our findings beyond individual genes, we performed ChIPseq for both ERG and AR in the presence of the androgen molecules R1881 and DHT, and in the absence or presence of PRMT5 knockdown. This data is provided in 2) One weakness of the manuscript, intrinsic to prostate cancer research is the use of a single ERG positive cell line. So any hit of the screen could be ERG specific, or even more likely, VCAP specific. Since there are no other available ERG dependent cell lines, the authors should note in the manuscript this major caveat. It would be very helpful if the authors could examine xenografts or perform some validation of the mechanisms in primary human tissue samples. For example, authors can IP AR from several ERG positive and ERG negative prostatectomy specimens to assess for methylation.TMPRSS2:ERG positive models, we have explored the ERG/PRMT5 interaction using additional model systems. First, in TMPRSS2:ERG transgenic mice vs. control mice ; AR immunoprecipitates from the TMPRSS2:ERG mouse prostates show increased SDMA and MMA signals at the correct size for AR. Fifth, we show in We thank the reviewers for acknowledging the challenges of obtaining appropriate prostate models and patient specimens. To address the general lack of We further attempted to confirm our findings in human tissue samples. In the absence of an AR-R761me2s-specific antibody for immunoblotting or IHC, we planned to use a chromogenic in situ proximity ligation assay (PLA)  with primary antibodies recognizing AR and symmetric di-methyl arginine (SDMA) to detect AR-R761me2s in human prostate cancer samples. First, we focused on optimizing PLA for formalin-fixed paraffin-embedded (FFPE) tissues, using FFPE RWPE-1 prostate cells, FFPE human prostate cancer samples, and two primary antibodies against Ki67 . While numerous individual spots representing positive signal were detected in the nuclei of RWPE-1 prostate cells, few, if any, spots were seen in the human tissues see , suggest3) Mass spec evidence of methylation at R761 would be helpful. Since you know what you're looking for, IP of AR in VCAP and another ERG negative line and looking for this should not be hard and would provide definitive evidence. The mutagenesis is highly suggestive, but could still change conformation in a way such that R761 is not the direct methylation site.We agree that mass spectrometry data would be an important support of our findings. We made several attempts at mass spec identification of the arginine site using AR immunoprecipitated from VCaP cells. Unfortunately, we could not identify this site by mass spectrometry. In our hands, arginine methylation has been generally difficult to detect, including on histones, which are 'classic' PRMT5 targets. We suspect that R761me2s does not occur on the entire population of AR within the cell lysate, which 'masks' the detection of methylated AR by mass spec. This may be in line with the observation that PRMT5 knockdown does not affect AR recruitment to all possible sites in our ChIPseq experiments.While not directly addressing the reviewers\u2019 question, we provide additional supporting data that PRMT5 can methylate purified AR ligand binding domain in vitro. These biochemical assays demonstrate PRMT5 activity  on purified AR but not ERG, and that addition of purified ERG DNA binding domain can facilitate greater PRMT5 activity on AR. This data is provided in"}
+{"text": "AbstractPhyllodonta latrata (Guen\u00e9e) has been applied to what is a complex of three undescribed species in Costa Rica. They are very similar in maculation, but can be differentiated by genitalic characters and barcodes. P. alajuela Sullivan, sp. n. occurs at lower altitudes in the northwestern part of Costa Rica whereas P. intermediata Sullivan, sp. n. and P. esperanza Sullivan, sp. n. are found at partially overlapping altitudes in the central mountain ranges.Historically, the name  PageBreakPhyllodonta Warren currently consists of approximately 26 species of medium-sized geometrid moths that occur from the southern United States to Argentina (Phyllodonta latrata (Guen\u00e9e) was described from Brazil and Colombia; in the British Museum the specimen from Nova Friburgo  is labeled as the type. It is unlikely that specimens from Colombia are conspecific. In Costa Rica, specimens that are very similar to Phyllodonta latrata occur from about 500\u20133000 m, but mtDNA barcode sequences of distinct clusters suggest a complex of species. Four additional species from Peru and Ecuador have been subject to barcode analysis but no haplotype clusters overlap those from Costa Rica. There are no specimens of Phyllodonta latrata from Brazil that have been barcoded nor have the types of Phyllodonta latrata nor Phyllodonta succedens   been dissected. Currently, Phyllodonta succedens is placed as a synonym of Phyllodonta latrata in the British Museum collections, although Phyllodonta nolckeniata (Snellen)  as a synonym.The genus rgentina . Currentrgentina . UnpubliPhotographic methods used herein are described in  Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMPageBreakPhyllodonta latrata (Gn.), Phyllodonta succedens (Wlk.) and Phyllodonta nolckeniata (Snellen) are distinguished by their rich brown color and will be referred to as the Phyllodonta latrata complex. The antemedial and postmedial lines are usually distinct and are wavy or undulating. On the underside, these lines are distinct and the characteristic whitish blotch  and Phyllodonta sarukhani Beutelspacher are similar in general color but the wing markings and genitalia are very different , Cartago Province, 1275 m, 7\u20139 July 2008, J. Bolling Sullivan (DNA voucher #11-CRBS-381) (INBio). Paratypes: 5\u2642, 4\u2640: 1\u2642, same data as holotype (DNA voucher #11-CRBS-288); 1\u2642, same data as holotype but 12\u201317 February 2005 (07-CRBS-1206), (dissection #JBS-3305); 1\u2642, Costa Rica, Parque National Volcan Poas , Alajuela Province, 2500 m, 7\u20138 August 2007, J. Bolling Sullivan (07-CRBS-359), (dissection #JBS-2006); 2\u2640, same data (07-CRBS-357), (JBS-2007); 3\u2642, Costa Rica, Villa Mills , Cartago Province, 2845 m, J. Bolling Sullivan , 2\u2640, same data . BMNH, INBio, JBS, USNM.Holotype male: Costa Rica, Tapanti Parque ; the vesica is unarmed with pouches emanating from the left side . In the female, the collar on the ductus bursae is narrow and the signum on the corpus bursae is crescent shaped, often with a medial kink and with the anterior and posterior sides narrowly separated. In the other two species the collar is wider and the signa are more oval shaped, never kinked.Maculation does not seem to be diagnostic for distinguishing this species. It is best characterized by barcode data and the genitalia. It has the highest altitude distribution but does overlap that of Male. . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by prominent cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Forewings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.0 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent postmedial line, prominent discal spot, small yellow line of scales distal to postmedial line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Twenty nine specimens have been barcoded and exhibit twelve haplotypes that differ from each other by a maximum of 0.8%. They differ from those of PageBreakGATGAACTGTTTATCCTCCTTTATCTTCTAATATTGCTCACGGTGGTAGTTCTGTTGACCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACTACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCAGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAATTTTATACCAACATTTATTTAACATTATATTTTATTTTTGGGATTTGAGCTGGAATAGTAGGAACATCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATCGGAGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCTTTCCCTCGAATAAATAATATAAGATTTTGATTACTTCCACCTTCTATTACATTATTAATTTCTAGAAGAATTGTGGAAAATGGAGCTGGGACAGKnown from above 1200 m in the Talamancas as well as the Central Volcanic and Tilaran ranges in Costa Rica. In flight throughout the year.Nothing is known about the biology of this species. Its range probably extends into the other mountain ranges in Costa Rican and perhaps in northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/FEE6C9E5-A097-4088-983C-D0573F1F0E1C9.43\u00b0N, 83.46\u00b0W) Cartago Province, 1475 m, 7\u20138 July 2008, J. Bolling Sullivan (10-CRBS-283) (INBio), Paratypes. 5\u2642, 1\u2640: 2\u2642, 1\u2640, same data as holotype , 10-CRBS-282 (JBS-5409)); 2\u2642, Costa Rica, Tapanti Parque , Cartago Province, 1275 m, 7\u20139 July 2008, J. Bolling Sullivan, (11-CRBS-286 (JBS-5416); 1\u2642, Costa Rica, Vera Blanca, La Paz Waterfall Garden , Alajuela Province, J. Bolling Sullivan (07-CRBS-1202), (JBS-3306) .Costa Rica, tunnel road, Tapanti Parque , the vesica is unarmed and pouches emanate from the right side . In the female, the collar on the ductus bursae is narrow (broad in Phyllodonta esperanza)and the signum on the corpus bursae is peanut-shaped with moderate spacing between the anterior and posterior sides .Maculation is not diagnostic for identifying this species. It is best characterized by barcode data and the genitalia. In the male, the distal side of the socius is usually straight . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 23.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Seven specimens have been barcoded and exhibit two haplotypes that differ from each other by a maximum of 0.5%. They differ from those of AACATTATATTTTATTTTTGGAATTTGAGCTAGAATAGTGGGAACGTCTTTAAGTTTATTAATTCGAGCAGAATTAGGGAATCCTGGGTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACTGCACATGCTTTTATTATAATTTTCTTTATAGTAATACCTATTATAATTGGGGGATTTGGAAATTGATTAATTCCTTTAATACTAGGGGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGGTTACTTCCACCTTCCATTACATTATTAATTTTTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCACCTTTATCTTCTAATATTGCTCATGGGGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGAGCTATTAATTTCATCACCACAATTATTAATATACGATTAAATAATTTATCTTTTGATCAAATACCTTTATTTGTATGAGCGGTAGGAATTACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACCGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGTGGAGGAGATCCAATTTTATACCAACATTTATTTKnown from between 1275 to 2280 m in the Talamancas, the Central Volcanic range, and the Tilaran range in Costa Rica. The moths are probably in flight throughout the year.Nothing is known about the biology of this species. Its range may extend into the other mountain ranges in Costa Rican and perhaps into northern Panama.Taxon classificationAnimaliaLepidopteraGeometridaeSullivansp. n.http://zoobank.org/F193BB75-F3BF-42EC-9292-C10D65E1533110.22\u00b0N, 85.62\u00b0W), Alajuela Province, 850 m, 7\u201311 February PageBreak2005, J. Bolling Sullivan  (INBio). Paratypes: 4\u2642, 1\u2640: 2\u2642, same data as holotype , 1\u2642, 1\u2640, Costa Rica, Upata. Estacion San Gerardo , Alajuela Province, 550 m, 17\u201321 July 2006, J. Bolling Sullivan , JBS-3314). 1\u2642, Costa Rica, Upata Bijagua, Alberque Heliconias , 800 m, Alajuela Province, J. Bolling Sullivan (JBS-3310).Holotype male: Costa Rica, San Ramon Reserva Biol. Alberto M. Brenes Estacion Biol. . The female has a broadened collar on the ductus bursae (narrow in Phyllodonta esperanza)and the signum on the corpus bursae is almost round with equal-sized spines around it .Maculation does not seem to distinguish this species. It is best characterized by barcode data and the genitalia. To date it has been found below 1200 m and with no other member of the Male. . Wings\u2013forewings warm brown with prominent antemedial and postmedial lines that undulate, in both cases forming two outward bulges. Antemedial bulges separated by cleft. Both lines black edged with lighter grayish scales proximally. Black scaling distal to reniform at costa and forming a diffuse line paralleling antemedial line. Reniform spot small, black, and forming center of a gray circle. Wings long, outer margin truncated at costa with a distinct notch medially, forewing length 24.1 mm . Hindwing with prominent postmedial line, margin with submedial notch. Underside of wings warm brown, forewing with postmedial line prominent, medial line visible, antemedial line absent. White blotch subterminal at notch, larger black blotch proximal to it. Small discal spot present. Hindwing underside with prominent PM line, prominent discal spot, small yellow line of scales distal to PM line on both wings. Cream streak from wing base widening to anal angle. Male Genitalia  is:Twenty eight specimens have been barcoded and exhibit 9 haplotypes that differ from each other by a maximum of 1%. They differ from those of PageBreakGAGATGATCAAATTTATAATACTATTGTAACTGCTCATGCTTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGGAATTGATTAGTTCCTTTAATATTGGGGGCCCCAGATATAGCTTTCCCACGAATAAATAATATAAGATTTTGATTACTTCCGCCTTCTATTACACTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCCGGAACTGGATGAACTGTCTACCCTCCTTTATCTTCTAATATTGCCCACGGTGGTAGTTCTGTTGATCTTGCTATTTTTTCATTACATTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACTTATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATCACTGCATTTTTATTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACATCTTTTTTTGACCCTGCTGGAGGAGGAGACCCAATTTTATATCAACATTTATTCAACATTATATTTTATTTTTGGAATTTGAGCTGGAATAGTAGGTACATCTTTAAGTTTATTAATTCGAGCGGAATTAGGAAACCCTGGGTCTTTAATTGKnown from 500 to 1150 m in the provinces of Alajuela and Guanacaste. Moths are probably in flight throughout the year.Witheringia solanacea L. H\u00e9r. in the Solanaceae. One record may represent a second species on Brugmansia \u00d7 candida (Solanaceae). The present range of Phyllodonta alajuela seems to be limited to the Northwestern provinces of Costa Rica and may extend into Nicaragua. Southward and eastward its range is unknown. Specimens of the Phyllodonta latrata complex from Anchicaya and Calima Dam in Valle Province of Colombia (four dissections) have similar male genitalia, but the basal spines on the vesica are larger; no specimens of it have been barcoded. Species of non-migratory moths common to Colombia and Costa Rica are often distributed in the western lowlands of both countries.Phyllodonta latrata (Gn.) complex is represented in Costa Rica by three new species that can be differentiated by barcodes and genitalic features. Phyllodonta alajuela occurs at lower altitudes whereas Phyllodonta intermediata and Phyllodonta esperanza overlap at intermediate altitudes; but are often separate at the altitude extremes of their ranges. At the conclusion of this study, two male specimens in the Phyllodonta latrata complex from the states of Bahia and Rio De Janeiro in Brazil were borrowed from the USNM and dissected. Both dissections showed characters not seen in any other members of the complex  and are supported by other less obvious characters and geographic separation.The complex nature of species grouped under the name otropics . ConstanPhyllodonta latrata complex in South America. Specimens from lowland western Colombia may be conspecific with Phyllodonta alajuela but proof is lacking. Two male specimens taken above 2500 m in the central Andean range (Alto Rio Quindio) were dissected and differed from each other and all species from Costa Rica. Two specimens from coastal Brazil also were unique. Additional specimens barcoded from Ecuador and Peru are distinct. Thus, it is likely that the type locality for Phyllodonta latrata (Brazil) and high altitude locations throughout the Andes will provide a surprising number of additional species in the complex.Barcodes and genitalic analyses also support the differentiation of other populations of the PageBreak"}
+{"text": "AbstractDisphragis notabilis (Schaus) species-group are reviewed, including the types and their dissected genitalia. Disphragis hemicera (Schaus), stat. rev., is elevated to species rank, D. normula (Dognin) is retained as a synonym of D. notabilis, D. sobolis Miller is confirmed as distinct from D. hemicera, and D. bifurcatasp. n., is newly described. Both D. hemicera and D. bifurcata occur in Costa Rica. The known ranges of the other species are outlined. Defining characters of each species are presented and a key to species is provided. Unusual variation in the genitalia is noted.The four described taxa in the  Disphragis notabilis (Schaus), described from French Guiana, has been applied to prominent moths throughout Central and South America. Miller described Disphragis sobolis from Ecuador and indicated that genitalic characters reveal yet another member of the complex in Ecuador  from Costa Rica and Disphragis normula (Dognin) from Peru, so it is necessary to examine all named taxa in order to classify the species found in Costa Rica.The name  Ecuador . CollectPhotographic methods used herein are described in  Natural History Museum, London, UKBMNH Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio J. Bolling Sullivan collection, Beaufort, North Carolina, USAJBS National Museum of Natural History, Washington, District of Columbia, USAUSNMHeterocampa notabilis Schaus was named from French Guiana. Both Heterocampa hemicera Schaus  from Costa Rica and Heterocampa normula Dognin  from Peru are listed as synonyms of Disphragis notabilis by Disphragis sobolis from the mountains of eastern Ecuador and noted that it was easily separated from specimens of Disphragis notabilis by its darker color and more mottled appearance , Limon Province, 354\u2019, 1\u20134 July 2008, J. Bolling Sullivan. INBio. Paratypes: 11\u2642, 3\u2640: 4\u2642, same data as holotype ; 1\u2640, 22 March 2003, Monty Wood (JBS-3030); 1\u2642, Costa Rica, Est. Biol. La Selva , Heredia Province, 50\u2013150 m, 21\u201330 June 2003, Monty Volovsic (JBS-3040), 2\u2642, 29 Aug. \u20132 Sept. 2003, J. Bolling Sullivan (JBS-3038); 2\u2642, Costa Rica, Upata Estacion San Gerardo , Alajuela Province, 550 m, 17\u201321 July 2006, J. Bolling Sullivan, B. Espinosa (JBS-3035); 1\u2642, Costa Rica, Puriscal Chires, Mastatal , San Jose Province, 400 m, 16\u201318 Oct. 2011, J. Bolling Sullivan; 1\u2642, Costa Rica, Verugua Rainforest Campamento , Limon Province, 400\u2013500 m, 12\u201316 March 2010, J. Bolling Sullivan (11-CRBS-2066), (JBS-5427); 1\u2640, Costa Rica, Tuis, 2500\u2019, June, W. Schaus 1910-110. (BM-); 1\u2640, Costa Rica, Cashi, 8\u201310 1912 (Lankester), Rothschild Bequest, B. M. 1939-1. (BM-). Holotype male: Costa Rica, Reserva Hitoy Cerere . Longest rami 0.44 mm. Thorax a blend of fine brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing (17.5 mm N = 10) elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak paralleling costa. White streak below basal dash; warm brown patch distal to white streak bordered by white; wavy antemedial (AM) and postmedial (PM) lines. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Hind wing fuscous with darker margin and veins, weak darker brown anal markings almost a spot at anal angle. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and well differentiated. Legs a mixture of brown and white scales, appearing almost yellowish, with white scales forming rings at distal end of tarsal segments. Tibial spines 0-2-4. Male genitalia  and with fasciculate antennae. Female genitalia  is:Five barcoded specimens exhibit two haplotypes that differ from each other by a maximum of 0.15%. They differ from AACCTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCGAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCTCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCTGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTGTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTCCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakKnown from Guatemala to Colombia , and probably extending south into northern Ecuador.Disphragis hemicera.This species occurs at lower altitudes and moderate elevations (1000 m) where it occurs with Taxon classificationAnimaliaLepidopteraNotodontidaestat. rev.Heterocampa hemicera Schaus, 1910, Annals and Magazine of Natural History 6: 582.Costa Rica.Disphragis hemicera and Disphragis sobolis from Disphragis notabilis and Disphragis bifurcata. Their appearance is mottled, grayish brown with a distinct dark band next to the PM line. Males may be distinguished by the shape of the phallus, which in Disphragis sobolis has a distinct dorsal projection. Females can be separated by the shape of the genital plate, which in Disphragis sobolis is bifurcate at the distal tip and in Disphragis hemicera has a middle phalanx with lateral \u201cwings\u201d from the base. Geographic distribution also separates Disphragis hemicera from Disphragis sobolis, with Disphragis hemicera in Central America and western Colombia, and Disphragis sobolis along the western slopes of the Andes.Maculation will usually separate Male. . Rami noticeably longer than in Disphragis bifurcata, longest rami 0.53 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax with a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin . Gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings forming something of a spot at anal margin. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales appearing somewhat yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:Fifty eight barcoded specimens exhibit seven haplotypes that differ from each other by a maximum of 0.30%. They differ from those of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTCTTTTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATCGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTCCCTTTAATACTAGGAGCACCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGACTATTACCCCCTTCTTTAATACTTCTAATTTCAAGAAGTATTGTAGAAAATGGAGCTGGTACAGGATGAACAGTTTATCCCCCACTGTCATCAAATATTGCTCACGGAGGAAGCTCTGTTGATTTAGCTATTTTTTCCCTTCATTTAGCGGGTATTTCCTCAATTTTAGGGGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTCTACTTTTACTTTCACTCCCAGTATTAGCTGGAGCTATTACTATACTTTTAACCGATCGTAATTTAAATACATCTTTTTTCGACCCTGCTGGGGGAGGAGATCCTATTTTATACCAACATTTATTTPageBreakDisphragis hemicera occurs throughout Costa Rica at moderate altitudes. It is found south along the western coast of Colombia and may extend to the west coast of Ecuador. The northern limits are unknown but it probably occurs at least into Nicaragua.Disphragis hemicera is by far the most common member of the group in Costa Rica and appears to be absent below 500 m. At moderate altitudes both Disphragis hemicera and Disphragis bifurcata occur together.Taxon classificationAnimaliaLepidopteraNotodontidaeHeterocampa notabilis Schaus, 1906, Proceedings of the United States National Museum 29: 253.Heterocampa normula Dognin, 1909, Annales de la Soci\u00e9t\u00e9 entomologique de Belgique 53: 81. Disphragis notabilis: French Guiana; Disphragis normula: PeruDisphragis notabilis and Disphragis bifurcata from Disphragis hemicera and Disphragis sobolis. Disphragis notabilis and Disphragis bifurcata are warm brown, not mottled or brownish gray like Disphragis hemicera and Disphragis sobolis. The male antennal pectinations are shorter in Disphragis notabilis than in Disphragis hemicera and Disphragis sobolis. Males of Disphragis notabilis are easily distinguished by their moderately wide socii, which taper to a single point with many ventral spines. In males of Disphragis bifurcata the socii are much broader and are bifurcate at the upturned apex. Females must be sorted by maculation and geography. Disphragis notabilis is Amazonian in distribution whereas Disphragis bifurcata occurs from central and western Colombia north into Central America.Maculation characters can usually be used to separate Male. . Longest rami 0.34 mm, shortest of all species. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad tan subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Basal dash below streak perpendicular to thorax, abbreviated relative to that of Disphragis bifurcata. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Chocolate shading from middle of wing below costal streak and forming a wedge to margin . Weak gray crescent on lower half of margin. Warm brown from patch expanded almost to margin and reducing size of chocolate wedge seen in Disphragis bifurcata. Hind wing fuscous with darker margin, weak darker brown anal markings almost forming a spot. Underside of forewing fuscous, anal margin and cell yellowish. Basal 3/4 of hind wing yellowish, margin brown and moderately differentiated. Legs a mixture of brown and white scales appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:Two barcoded specimens exhibit 2 haplotypes that differ from each other by 0.30%. They differ from those of AACTTTATATTTCATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTTCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGACCAAATTTATAATACTATCGTAACAGCTCATGCTTTCATTATAATTTTTTTTATAGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGACATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCTCCTTCTTTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCACCACTGTCATCTAATATTGCCCATAGAGGAAGCTCTGTTGATTTAGCCATTTTTTCCCTTCACTTAGCCGGTATTTCATCAATTTTAGGGGCTATTAATTTTATCACAACAATTATTAATATACGATTAAATAATATATCTTTTGATCAAATACCTTTATTTGTATGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTTTCTCTTCCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGTAATTTAAATACATCTTTTTTTGACCCTGCAGGGGGAGGAGATCCTATTTTATACCAACATTTATTTThis species occurs throughout the Amazon basin from western Venezuela eastward and southward to at least Bolivia.Disphragis notabilis is by far the most common member of the group in South America, however, earlier references to this species should be confirmed in light of the additional species described here.Taxon classificationAnimaliaLepidopteraNotodontidaeMiller, 2011Disphragis sobolis Miller, 2011. In Ecuador.Male. . Rami noticeably longer than in Disphragis hemicera, longest 0.59 mm. Thorax a blend of brown and cream scales giving a tan appearance. Metathorax bearing a central white spot with row of darker brown scales anteriorly. Abdomen with appressed brown scaling. Forewing  elongate, rounded apically and with broad light brown subcostal streak from base of wing to apex. Streak encloses chocolate reniform spot and has several slightly darker brown lines crossing obliquely from costa. Brown scaling throughout as well as several black streaks. Basal dash below streak perpendicular to thorax and greatly reduced in length. White streak below dash; warm brown patch distal to white streak bordered by white; AM and PM lines wavy. Distinct brown line bisecting warm brown patch. Chocolate shading from middle of forewing below costal streak and forming a wedge to margin  more extensive than in Disphragis hemicera. Prominent gray crescent on lower half of margin with distinct brown band inward to PM line. Hind wing uniformly fuscous with brown anal markings almost a spot. Light streak along anal edge. Underside of forewing fuscous with yellowish subapical crescent along costa. Basal half of hind wing yellowish, no well-differentiated margin. Legs a mixture of brown and white scales, appearing almost yellowish with white scales forming rings at distal end of tarsal joints. Tibial spines 0-2-4. Male genitalia  is:One specimen has been barcoded and differs from that of AACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTCTCCTAATTCGTGCTGAATTAGGAACCCCCGGGACTTTAATTGGAGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATACCCATTATAATTGGAGGATTTGGTAATTGATTAGTTCCTCTAATATTAGGAGCTCCAGATATAGCTTTCCCACGAATAAATAATATAAGTTTTTGATTATTACCCCCCTCTCTAATACTTTTAATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTGTCATCAAACATTGCTCATAGAGGAAGATCTGTTGATTTAGCTATTTTTTCCCTTCACTTAGCAGGTATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGATTAAATAACATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTAGGAATTACTGCTTTTTTACTTTTACTCTCTCTTCCAGTATTAGCAGGAGCTATTACTATATTATTAACCGATCGTAATTTAAATACATCTTTTTTTGACCCCGCTGGGGGAGGAGATCCTATTTTATATCAACATTTATTTPageBreakThis species appears to be limited to the eastern slopes of the Andes from Bolivia to Villavicencio, Colombia.Disphragis sobolis was recently described from Ecuador; the species appears to have a much greater geographical range and occurs to almost 3000 m. The lower altitude limits of its range are undefined, as is the southern boundary.PageBreakDisphragis notabilis complex is typical of many neotropical species. When studied in detail, they frequently are found to consist of a number of very similar species that do have structural differences, can be separated by barcoding, and occupy different altitudes or geographic ranges. The correct generic placement of this complex is not in Disphragis  and makes the delineation of species and their defining characters with female specimens extremely difficult. Fortunately, such extreme variation has not been observed in males.A second difficulty encountered in this study was the hyper-variation of the female genital plate in hemicera . This inDisphragis notabilis complex found in Colombia (and probably Ecuador) remains to be elucidated but should highlight the individual habitat requirements of each species. Neither larvae nor foodplants are known. The geographical area where Disphragis bifurcata and Disphragis notabilis come into contact should be particularly interesting because the two species differ by 1.4% in their barcodes, a magnitude lower than between most congeneric species. However, there are a number of characters that separate the two species and characters do not seem to intergrade in individuals from central Colombia and western Venezuela.The exact distribution of the four species of the"}
+{"text": "Nicotiana tabaccum L.) and rustica tobacco (Nicotiana rustica L.) were analysed between the third intron and the fourth intron of the propylene alcohol dehydrogenase gene. The results showed that the alcohol dehydrogenase gene is a low-copy nuclear gene. The intron sequences have a combination of single nucleotide polymorphisms and length polymorphisms between common tobacco and rustica tobacco, which are suitable to identify the different germplasms. Furthermore, there are some single nucleotide polymorphism sites in the target sequence within common tobacco that can be used to distinguish intraspecific varieties.A pair of primers was designed to amplify the propylene alcohol dehydrogenase gene sequence based on the cDNA sequence of the tobacco allyl-alcohol dehydrogenase gene. All introns were sequenced using traditional polymerase chain reaction (PCR) methods and T-A cloning. The sequences from common tobacco ( Nicotiana sp.) is one of many plants currently used as models for molecular studies and is an economically important crop. The interspecific phylogenetic relationships within tobacco are complicated due to complex hybridization events within the tobacco lineage. DNA molecular markers have been used to identify germplasms and to accelerate the breeding cycle. The second generation of molecular markers that include random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) has been employed broadly to differentiate tobacco varieties,[Tobacco .The specific primers were designed to target the boundary between the third intron and the fourth intron of the tobacco propylene alcohol dehydrogenase gene, and the IP sequences were identified as follows, IP-S: GTGCTGGAAGCAAAGAAAAG, IP-A: CAATTCCATCAGGGAAGTAC. The PCR materials included 5\u00a0\u03bcL of 10 \u00d7 PCR buffer, 4\u00a0\u03bcL of dNTP (2.5\u00a0mmol/L each), 1\u00a0\u03bcL of each primer (10\u00a0\u03bcmol/L), 1\u00a0\u03bcL of the DNA template and 1.5\u00a0U of PCR was performed at 94\u00a0\u00b0C for 5\u00a0min, then 94\u00a0\u00b0C for 30\u00a0s, 58\u00a0\u00b0C for 30\u00a0s, 72\u00a0\u00b0C for 1\u00a0min for 30 cycles, with a hold at 72\u00a0\u00b0C for 20\u00a0min. The PCR products were ligated to a pGEM-T vector  and sequenced on an ABI 3730 sequencer after electrophoretic detection in a 1.0% agarose/EB gel.The propylene alcohol dehydrogenase gene of sample C1 contained four introns and five exons that were 225, 178, 167, 83 and 379\u00a0bp long . The boundary sequence between the intron and exon segments were the sites of interest for which a primer was designed to target.Nicotiana rustica) exhibited two bands when both primers, IP-S and IP-A, were used, while those of common tobacco (sun-shined tobacco and flue-cured tobacco) exhibited only one band on the 1.0% agarose/EB gel .In the common tobacco, the shorter fragment was 559-bp long in the target region, and the two introns found were 223 and 253\u00a0bp. There were no length differences in this fragment among the 12 samples sequenced, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATCTTCATAGTGTTGAATCTATCTTCATTCTTCGTTATGTTAATCATTGTGCTCCGTCCACCATCAAACCCAAAGGTTGGGGTTGGGGTTGGGGTTAGGGTTGGGTGGGTTATGCCTTTGGGGGGAAAGAGAGGTTTTAGTTTAATAGACCATGATGTTTAAAACTCTTGTTATTTAATGTCTCTTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAGGCTTTTAACTATAAAGAGGAGCAGGATTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGCTACAAAACAATCTACCTATTGTACAAAAGCAGTTAAAGATGTTCTGTGAAAAGTATGATGATATTATGTAGATAGTGTGAAACTTATAGAAAGACTTTAAGATATGCCTACGAAAACCGAGGAACATGGATCATTTCAAATCTGGCTTTTCATGAATATGGAACCCCATTCACTAGATTTTAGTGCGAAATTCCTAGACTTGAAGTGACTGGAAATACTGGTTTGTTATTAGwww.ebi.ac.uk/Tools/clustalw2/). The results indicated that there were two transitions and one transversion in addition to three gaps within the third intron. The two transitions and one transversion were also present within the fourth exon, but no amino acid replacement had occurred. There were four transitions and one transversion within the fourth intron as well . These polymorphic sites within the third and fourth intron may function as references to distinguish the varieties of common tobacco studied here.In the common tobacco, the longer sequence obtained was between 588 and 591-bp long in the target region, and the two introns were either between 194 and 197\u00a0bp or 311\u00a0bp in length. The sequences from 13 samples were analysed with the software ClusterW2 (In the rustica tobacco, the shorter sequences were 442-bp long in the target region, and the two introns found were 195 and 164\u00a0bp in length. No length polymorphisms were found in this fragment among the 12 samples sequenced, and the consensus sequence obtained was as follows:GTAAGTTCCTTCATAGTGTTGAAGCTCTCTTCATTCTTCGTTATGTTAATTATTGTGCTCCGTCCTCCATCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGGGTATACCTTTGGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTCTCTTTTTATATTCATTTCCAAAGGTTGATCTGTTGAGGAACAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTACTTGGCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCATCAATATTTCCATAGAATGATATTTATAATGGAAGTCCCAAAGTAAAGATGTTCAGTGAAAAGTATGCCTGTGAAAACTGAGGGAAATAATACTGGTTTCTTATTAGIn the rustica tobacco, the longer sequence was 583-bp long in the target region, and the two introns found were 220 and 280\u00a0bp in length. No polymorphisms were found in this fragment among the 12 samples sequenced as well, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATGTTCATAGTGTTGAAGCTATCTTCATTCTTCGTTATGTTAATCATTGTGTTCTGTCCACCGTCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGTATACCTTTGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTATCTTTTTATTTAATGTCTCTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATTCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGTTAGAAAACAATCTACCTATTGTAACAAACTTGCCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCCTTTTCATGTAAACGGAATCATCGATATTTCCATAGAATGATATTTATTCCTTGAACTGAAAAATAGAAGTCCCAAAGTAAAGATGTACAGTGAAAAGTATGAAGATTGTGTGAAAATTATAGAAAAGACTTTAGGATATGCGTATGAAAACTGAGGGAAATTCCTCCCTGGAATACTGGTTTCTTATTAGIn this study, the tobacco propylene alcohol dehydrogenase gene proved to be a low-copy nuclear gene. It could have formed as a result of divergent evolution after gene duplication, or by a parallel evolutionary process between the two sets of genomes, as the tobacco plants tested were all allotetraploid. Although the PCR product for the allyl-alcohol dehydrogenase gene of sample C1 was sequenced successfully, this does not mean that only one copy of the target gene is present in common tobacco. There were obvious intron length polymorphisms between common tobacco and rustica tobacco, which were mostly due to different modes of evolution of the two kinds of tobacco.Nicotiana sylvestris.[N. tomentosiformis,[N. otophora or from introgressive hybridization of N. otophora and N. tomentosiformis within the subgenus of common tobacco.[N. rustica originated from a natural chromosome duplication event after ancestral hybridization of N. paniculata and N. undulate.[N. rustica L. group and Nicotiana tabaccum L. group, after an analysis that used AFLP methods coupled with unweighted pair group method with arithmetic mean analysis, which was consistent with our results in this work. Furthermore, the two categories of tobacco can be distinguished using only one pair of primers according to the results of this work.It is thought that the maternal ancestor of common tobacco was lvestris. It is alsiformis, N. otoph tobacco. It has bundulate. Yang et\u00a0undulate. divided The two copies of the propylene alcohol dehydrogenase gene found in tobacco each contained four introns and five exons. The length polymorphisms of the intron existed intraspecifically as well as interspecifically. The primers produced here, IP-S and IP-A, were able to easily differentiate common tobacco from rustica tobacco in a 1% agarose gel. Some SNPs of the intron within common tobacco were found, and may be useful in the identification of varieties of this species.http://dx.doi.org/10.1080/13102818.2014.907651.Supplemental data and research materials for this article can be accessed at"}
+{"text": "Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from  Interferon-induced transmembrane proteins (IFITMs) encoded by a variety of vertebrates, including pigs, chickens, bats, fish, mice, and humans, have been shown to restrict cellular infection by specific viruses ,3,4,5,6.The essentiality of mouse IFITM3 in restricting influenza virus infection was demonstrated by showing that IFITM3 knockout mice succumb to sublethal doses of virus and exhibit higher lung titers and more severe lung pathology than wild type mice ,15. SimiAn extensive evolutionary study of IFITMs posited that vertebrate IFITMs originated from a horizontal gene transfer from bacteria to a common ancestor of choanoflagellates and metazoans . WhetherExpression constructs in the pCMV-HA vector or pCMV-myc vectors  encoding mouse and human HA-IFITM3, human myc-IFITM3, and mouse HA-IRGM1 have been previously described ,20,21. UMAV IFITMGAATTCCCATGACCCAACCACCTCCTCCCCCGCCACCACCAGGATATCCCCCACAACAGCCAGCCGCTCAGGCTCCTAATAACTATCTGGTGTGGTCTATCCTTGTCACTCTCTTCTGCTGCCTGCCGTTTGGCATTGTCGCTATCGTAAAGAGCTCTCAAGTGAACGGACTTTGGGCACAGGGTAGATATGCTGAGGCACAGGCCTCCGCAGACAGTGCCAAGAAATGGGTGATATGGAGCGCAGTTATAGGCGTCGTGGTGGGAATAATCTATGGAATCCTTATGGCCGTAGGCGCCCTCAACACAAATACAAACGCGGCCCTCGCCGCGATGTTTTAGTAAGTCGAC        MAB IFITMGAATTCCCATGAGTGATGAAACCAAAAGCGACGAGCCTACAGGCGCTATCACCACACCGACCCCTCCTCCCCCACCGGCTCCTGCCTCTGTGACTGGCCCACCCAAACCCCCACCCACTAACGTGGGTTGGGCCGTCGCTAGCGTGATTTTTTTCTGGCCTCTGGCATTTAGCGCATTCACCAATGCACTGAATGTGACTCAGTTTTGGCTGACGGGGCAGTATGATCGGGCCCAGGAGTCTAGCGATCGGGCCAAGCTCCTGGGAAAGATTGCCCTCCTGACCGGGTTGGTACTGCTGTTCCTGTTCATCACCCTCCGCATTGCCTGCGCCATCTGGTGGCACTCACATGGTGGGGGATGGGGTCATCATGGCGGATGGCATAGGAGTTGGGACGACGGCGGGTGGGATGGCCCTGGCCCCATCGGGCCTATGGGTAGGCCGGGTCGCGACAACTAGTAAGTCGAC2 at 37 \u00b0C. For imaging experiments, cells were grown on sterilized glass coverslips in 12-well plates. For all other experiments, cells were grown in 12-well or 6-well plates. Cells were transfected overnight using LipoJet  according to the manufacturer\u2019s instructions. For biochemical experiments, 2 ug/well of each plasmid was transfected into cells grown in 6-well plates. For infection experiments, 1 ug/well of each plasmid was transfected into cells grown in 12-well plates, with the exception of plasmid encoding HA-MAB IFITM for which 0.5 ug/well was transfected. For examination of palmitoylation, transfected cells were labeled with 50 uM alk-16 for 1 h as previously described in detail [HEK293T cells were obtained from the ATCC and were grown in DMEM supplemented with 10% FBS  in a humidified incubator with 5% COn detail ,22,23. In detail ,22,23. In detail . Sendai n detail . Cells wFor palmitoylation experiments, cells were lysed with 1% Brij Buffer , pH 7.4) containing EDTA-free protease inhibitor cocktail. For co-immunoprecipitation experiments, cells were lysed with 1% digitonin in PBS containing EDTA-free protease inhibitor cocktail. Immunoprecipitations were performed with either anti-HA or anti-myc EzView Affinity Gel (Sigma). For palmitoylation experiments, immunoprecipitations were washed 3 times with standard RIPA buffer , and for co-immunoprecipitation experiments, the immunoprecipitations were washed 5 times with 0.1% digitonin in PBS. All detergents were purchased from Sigma. Western blotting was performed with anti-HA antibody  or anti-myc antibody . For immunofluorescence and flow cytometry, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X100 in PBS for 10 min, and blocked with 2% FBS in PBS for 10 min. For immunofluorescence, cells were stained with the anti-HA or anti-myc antibodies described above that were directly labeled with Alexafluor 488 or 555 using 100 ug antibody kits from Life Technologies. Slides were mounted with Prolong Gold Antifade Reagent containing DAPI from Life Technologies and images were taken using an Olympus Fluoview FV10i confocal microscope. Quantification of Manders overlap coefficients was performed using the Just Another Colocalization Plugin (JACoP) for ImageJ software. For flow cytometry, cells were stained with anti-IFITM3 antibody  and anti-rabbit-Alexafluor-488 secondary antibody (Life Technologies), or with anti-HA antibody directly conjugated to Alexafluor-488 and anti-influenza NP antibody  directly conjugated to Alexafluor-647 as previously described [Mycobacterium abscessus (MAB) was significantly divergent from the majority of the remaining mycobacterial IFITMs (Mycobacterium avium (MAV) IFITM as a representative of the more typical mycobacterial IFITMs (Evolutionary studies of the IFITM gene family have identified numerous IFITM genes in different vertebrates ,26,27. Ol IFITMs A. For ful IFITMs A. Clustal IFITMs B, 65.6%,l IFITMs B. Howevel IFITMs D. One iml IFITMs B. We, anl IFITMs B.S-palmitoylated cysteines, including an S-palmitoylated di-cysteine motif, within the first hydrophobic domain [S-palmitoylation site on murine IFITM1 [Despite the major differences between the amino acid sequences of IFITM3 and mycobacterial IFITMs, a similar fundamental domain structure could be identified in each of these proteins. Two hydrophobic domains separated by approximately 30 amino acids are defining characteristics of IFITMs and were present in both MAV and MAB IFITMs B. Most vc domain ,31,32 (Fe IFITM1  (Figure e IFITM1 . InteresGiven the important differences and striking similarities between the mycobacterial IFITMs and IFITM3 B, we souWe synthesized codon optimized MAB and MAV IFITM genes for insertion into the pCMV-HA mammalian expression vector in-frame with the HA tag of the vector, and transfected these constructs into HEK293T cells. After transfection, we observed high expression of MAB IFITM, far greater than IFITM3 expression from the same vector, and a more similar expression level of MAV IFITM A. Thus, S-palmitoylation of IFITM3 is important for its proper membrane targeting and maximal antiviral activity [S-palmitoylation sites on IFITM3 (S-palmitoylation site present on murine IFITM1 [S-palmitoylation of these IFITMs (S-palmitoylation (S-palmitoylation of IFITMs is a driver of maximal antiviral activity [activity ,12,36. Uactivity ,23,45,46n IFITM3 B. MAB IFn IFITM3 B that ise IFITM1 . Using ce IFITMs B. We nexe IFITMs C. Similae IFITMs C. Consisoylation A,B, mutaoylation C. Overalactivity .IFITM3 was reported to homodimerize and also to heterodimerize with other IFITMs ,47. ThisN-terminus that interacts with endocytic adaptor complexes, resulting in the effective trafficking of IFITM3 from the plasma membrane to endosomes [S-palmitoylation that we observed for mycobacterial IFITMs compared to IFITM3 (IFITM3 has been reported to co-localize with early and late endosomal markers and with markers of lysosomes and multi-vesicular bodies ,29,36,48ndosomes ,29. Thisndosomes  (Figure o IFITM3 A,B, thiso IFITM3 C.Monosiga brevicollis [Monosiga brevicollis. IFITMs were also identified in multiple ancient bacteria species, and given the identifiable sequence similarities between bacterial IFITMs and metazoan IFITMs (S-palmitoylation (Though the exact evolutionary origin of vertebrate IFITMs is unknown, an IFITM with a splicing pattern that is conserved in all metazoans was identified in vicollis , a unicen IFITMs B, it wasn IFITMs C,D. Confn IFITMs D, and thoylation , which iS-palmitoylation level and that positioning of this domain evolved toward optimal modification in vertebrates. Alternatively, other determinants may affect interaction with the as yet unidentified palmitoyltransferase enzyme that modifies IFITMs. Further interrogation of MAV IFITM may shed light on these important questions.Many aspects of IFITM3 cell biology that are not fully understood may possibly be clarified by study of the mycobacterial IFITMs. For example, IFITM3 is known to be optimally active when expressed at high levels in cells, and its turnover is promoted by post-translational ubiquitination . HoweverThe precise mechanism of antiviral action of IFITMs is also currently unknown. Two potentially complementary models have been proposed for the anti-influenza virus activity of IFITM3. One, IFITM3 may directly alter fluidity of endolysosomal membranes ,10,49 th"}
+{"text": "AbstractHarpacticella Sars, 1908 is described from a tidal pool on Jeju Island, Korea. Harpacticellajejuensissp. n. is closely related to Harpacticellaitoi Chang & Kim, 1991, with regard to the structure of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the setal number of the P5 exopod in males. However, the new species is clearly distinguishable from Harpacticellaitoi by the combined following characters: six setae on the P5 exopod in females, one naked seta on the inner margin of P1 exp-2, the short endopod of P1 compared to the exopod, and a naked long seta on the proximal inner margin of the P5 exopod of males. The mtCOI partial sequence is provided as a DNA barcode for the new species.A new species of the genus  Harpacticella Sars, 1908 is a genus of harpacticoid copepods, family Harpacticidae Dana, 1846. The genus has been reported from various habitats , mostly in Asian waters , and Harpacticellaamurensis Borutzky, 1952 were described from freshwater, Harpacticellalacustris Sewell, 1924 and Harpacticellaitoi Chang & Kim, 1991 from brackish water, and Harpacticellaoceanica It\u00f4, 1977 from the marine environment.The first reported . Among tHarpacticella from a tidal pool. Here, we describe the new species and provide a key to species in the genus Harpacticella. Partial mtCOI sequence was also obtained as a DNA barcode for the new species.During a study of the harpacticoid community along the coast of Jeju Island of Korea, we collected a new species of Samples were collected by hand net (63 \u00b5m mesh-size) from a tidal pool on the coast of Jeju Island, Korea. Specimens were preserved in 99% ethanol. Specimens were dissected in lactic acid, and the dissected parts were mounted on slides with lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings were prepared using a drawing tube attached to an Olympus BX51 differential interference contrast microscope., antennuleA1; , antennaA2; , aesthetascae; , exopodexp; , endopodenp; , first to sixth thoracopodP1-P6; , proximal  segment of a three-segment ramusexp (enp)-1; , caudal ramiCR. Specimens were deposited in the National Institute of Biological Resources, Incheon, Korea (NIBR). Scale bars in figures are indicated in \u00b5m.Descriptive terminology is adopted from Molecular analysis. For DNA extraction, fixative materials (99% Et-OH) were removed from specimens by washing with distilled water, and DNA was extracted using a tissue DNA purification kit . Amplifications were performed in 20 \u00b5l reactions volumes containing extracted tissue DNA, primers LCO-1490 (5\u2019-GGT CAA CAA ATC ATA AAG ATA TTG G-3\u2019) and HCO-2198 (5\u2019-TAA ACT TCA GGG TGA CCA AAA AAT CA-3\u2019) , and PCRPageBreakTaxon classificationAnimaliaHarpacticoidaHarpacticidaehttp://zoobank.org/77BE96F4-597C-44D0-A07E-03A4D993A5F233\u00b013.949'N; 126\u00b030.653'E) on Beophwan beach, Seoguipo-shi, Jeju Island, Korea.A tidal pool (Holotype: 1\u2640 (NIBRIV0000304111) in 70% ethanol from the type locality. Paratypes: 5\u2640\u2640 (NIBRIV0000304112) in 70% ethanol, 2\u2640\u2640 (NIBRIV0000304113 \u2013 NIBR0000304114) dissected on 11 and 10 slides, respectively, and 2\u2642\u2642 (NIBRIV0000304115 \u2013 NIBR0000304116) dissected on 11 and 2 slides, respectively. All from the type locality and collected by R. Jeong on 3 March 2013.KM272559, 619 bp).DNA-barcode (mtCOI) sequence and trace were submitted to GenBank . Maximum width at posterior margin of cephalosome . Urosome gradually tapering posteriorly. Body surface armed with some sensilla Figs .Prosome Fig.  4-segmenRostrum Fig.  well devUrosome Figs , 2C 5-seGenital double somite Figs , 2C, 7A Anal somite Fig.  without PageBreakPageBreakPageBreakPageBreakouter corner; seta III as long as lateral seta II; apical seta IV unipinnate, slightly longer than urosome; apical seta V bipinnate, as long as whole body; apical seta VI similar in length to seta III; dorsal seta VII bare and bi-articulate at its base.Caudal ramus Figs , 2D wideAntennule 7-segmented Fig. . AesthetAntenna Fig.  3-segmenMandible Fig.  with larMaxillule Fig.  with praMaxilla Fig. : syncoxaMaxilliped Fig. : syncoxaSwimming legs 1\u20134 Figs , 5A\u2013B biP1 Fig. : coxa wiPageBreakP2 Fig. : coxa wiP3 Fig. : coxa wiP4 Fig. : coxa wiArmature formulae as follows:PageBreakP5 Fig. : exopod Male. Total body length of examined samples 631\u2013650 \u00b5m . Greatest width at posterior margin of cephalosome. Cephalosome with sensilla along lateral margin. Other prosomites also with sensilla along lateral margin Fig. .Prosome Fig. , 4-segmeUrosome Figs , 7E 6-seAntennule Fig.  6-segmenP5 Figs , 7D, basPageBreakPageBreakPageBreakP6 Fig. : small fmitochondrial oxidase subunit I; partial cds; 619 bpPageBreakGATTTTTGATGCCCTCTCTTATATTAATAATTATTAGAAGAGTTGTTGAAGGCGGGGCAGGGACAGGGTGAACTGTTTACCCCCCTTTAAGAAGAAATTTAGCACATGCAGGAGGCTCGGTGGATTTAGTAATTTTTTCTTTACATTTAGCAGGAGTTTCTTCCTTATTAGGGGCTGTAAATTTTATTAGGACTTTAAGAAATCTTCGAGTATTCGGGATGTATTTTGACCAAGTGCCGTTATTTTGTTGATCTGTCTTGGTTACAGCTGTTCTATTACTTTTATCACTGCCTGTATTAGCGGGGGCAATTACTATATTGTTGACCGATCGAAACATTAATTCAAGCTTCTATGATGTTAACTTTATATCTTTTAAGGGGGATATGAGCGGGAGTTATGGGGGCGGCAATAAGAGTTATTATTCGGCTTGAATTAGGACAGCCTGGGACTTTAATTAAGGATGAGCAAATTTATAATGTTTTAGTGACTTCGCATGCTTTTATTATAATTTTCTTTATGGTTATACCAATTTTAATTGGGGGGTTTGGAAACTGGTTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCCTTTCCTCGATTAAATAATTTGAGATTCTThe specific name refers to the type locality of Jeju Island, Korea.Harpacticella based on the combination of following character sets: a) 7-segmented antennule in the female, b) 2-segmented antennary exopod, c) 3-segmented P1 endopod and exopod, d) only one seta on the inner edge of P2 enp-2 and e) spiniform outer spine of exp-3 P3 and P4  six setae on the P5 exopod of females  P1 see Fig. ; (4) naksee Fig. , but plusee Fig. ; (5) naksee Fig. , but it see Fig. .PageBreakPageBreakHarpacticella species have a wide distribution ranging from freshwater to true marine environments, and have been found in Asian waters, American waters, and the Aldabra Atoll in the Indian Ocean  is the most ubiquitous species; it has been recorded in China, Japan, and the northwest coast of the USA  for use in future studies; no sequences have been obtained from congeners to date, even though it would be interesting to determine the phylogenetic relationships among congeners based on analysis of mtCOI sequences.DNA barcoding is an efficient tool to identify species, especially morphologically similar species . This ba"}
+{"text": "Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Nav\u03b22 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Nav\u03b22 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Nav\u03b22 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Nav\u03b22 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Nav\u03b22 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav\u03b22 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH. Since voltage-gated sodium channel (VGSC) is necessary in the initiation and propagation of action potentials in neurons, it is a valuable therapeutic target for neurological disorders, such as epilepsy and chronic neuropathic pain \u20133. Recen+ channels (VGSCs) are macromolecular protein complexes, which are composed of \u03b1-subunits (Nav1.1\u2013Nav1.9) and \u03b2-subunits , in which \u03b1-subunits are necessary for forming a functional ion-selective channel and \u03b2-subunits affect ion channel gating and trafficking to regulate the voltage-dependency and density of VGSC on the cell membrane. It has been reported that mutation of Nav1.1, a remarkable feature of Dravet syndrome, could induce higher seizure activity and cognitive dysfunction [BACE1-transgenic mice, accompanied with disturbed Nav\u03b22 [hAPP) transgenic mice [BACE1-null mice [BACE1) in BACE1-deficient or over-expressing mice [APP) mice [Voltage-gated Nafunction . Interesed Nav\u03b22 . And resnic mice . Furtherull mice . These sull mice \u201317, and ull mice . Interesing mice , and thePP) mice . HoweverChronic brain hypoperfusion (CBH)-mediated chronic cerebral ischemia and consequent cognitive impairment , 22 is mMicroRNAs are small non-coding RNA, which regulate protein synthesis. MicroRNA-9 (miR-9), enriched in central nerve system (CNS) , contribIn this study, our data provide strong evidence that miR-9 regulates Nav1.1/Nav1.2 trafficking by post-transcriptional regulating SCN2B gene under CBH status.P\u2009<\u20090.01 vs sham). In order to explore whether the decreased surface protein expression of Nav1.1/Nav1.2 was due to the reduction of total Nav1.1/Nav1.2 or the impairment of trafficking, the total protein levels of Nav1.1/Nav1.2\u00a0were evaluated. The data showed that the total protein levels of Nav1.1 and Nav1.2 were significantly increased in the hippocampi and cortices of 2VO rats  rather than reduced. These results suggested that the decreased expression of Nav1.1/Nav1.2 on the cell membrane were due presumably to the impaired trafficking of Nav1.1 and Nav1.2 protein from cytoplasm to cell membrane in 2VO rats rather than the changes of their total protein levels.Previous studies have reported that Nav1.1/Nav1.2 trafficking was changed after dementia , 20, 40.P\u2009<\u20090.01). However, their mRNA expression of Nav\u03b22 (SCN2B) was not changed consistently with the protein expression of Nav\u03b22 .MicroRNAs have long been known to control post-transcriptional gene regulation and are essential for neuronal function . MiR-9 ihttp://www.targetscan.org/), we found that SCN2B has a poorly conservative \u2018seed\u2019 sequence of miR-9 in its 3\u2019UTR and the length of 3\u2019UTR of SCB2B in rat was very short based on the database of the University of California Santa Cruz (UCSC) and the National Center for Biotechnology Information (NCBI) Genome Browsers. Since recent studies have reported that microRNAs regulate protein expression by binding to the coding regions of target protein [http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and found that the CDS domain of SCN2B is likely to serve as potential targets for miR-9. We next identified whether there are binding sites for miR-9 on the CDS domain of SCN2B gene at the position of 336\u2013358 and 575\u2013597 with highly conservative regions , however, scrambled negative control and inhibitor of miR-9 had no effect on luciferase activity compared with control group , and the difference of luciferase activity between inhibitor of miR-9 and scramble negative inhibitor control was also not detected . For each of the two binding sites, we generated and tested several variants by introducing silent mutations designed to disrupt the putative base pairings between the microRNAs and the corresponding predicted targets. Silent mutations introduced into the predicted targets at the position of 336\u2013358 of SCN2B CDS disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS . However, simultaneous introduction of silent mutations at the position of 575\u2013597 of SCN2B CDS did not abolish the downregulation of SCN2B by miR-9 . Silent mutations introduced into both two predicted targets at the positions of 336\u2013358 and 575\u2013597 of SCN2B CDS, MutSCN2B-1 & 2 disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS, strongly suggesting that miR-9 uniquely targets at the position of 336\u2013358, rather than 575\u2013597, of SCN2B CDS.Since the protein level of Nav\u03b22 was reduced in both hippocampi and cortices in 2VO rats, which is discrepancy with the unchanged SCN2B expression, the alternation of miR-9 is highly expected in this process. By searching the database of microRNA targets , implying that the SCN2B shows a great potential as the target for miR-9.To observe the influence of miR-9 on the protein translation of SCN2B, we analyzed the protein levels of Nav\u03b22 in primary cultured neonatal rat neurons (NRNs) co-transfected with miR-9 mimics. The successful transfection of miR-9 was identified Fig.\u00a0 by qRT-PP\u2009<\u20090.05) and Nav1.2  total protein. And they were prevented in the presence of AMO-9  was injected directly into CA1 region of bilateral hippocampus of each rat and significantly higher expression of miR-9 in both hippocampi and cortices was observed at 8\u00a0weeks after injection compared with the negative control oligonucleotide . The surface expressions of both Nav1.1 and Nav1.2 proteins were markedly reduced in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which was reversed by lenti-pre-AMO-miR-9 , even though the total protein levels of Nav1.1 and Nav1.2 were increased in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which were reversed by lenti-pre-AMO-miR-9 . These data suggested that miR-9 plays an important role in trafficking and cellular distribution of Nav1.1/ Nav1.2.To verify the functional role of miR-9 on Nav1.1/Nav1.2 trafficking NC, Fig.\u00a0. ImportaP\u2009<\u20090.01). And furthermore, the surface protein expression of Nav1.1 was increased and the total protein of Nav1.1 level was decreased by lenti-pre-AMO-miR-9 compared with that in 2VO control rats . Similarly, lenti-pre-AMO-miR-9 effectively improved the impaired trafficking of Nav1.2 from cytoplasm to cell membrane under the same experimental condition .Our data displayed that 2VO results in miR-9 increase, and over-expression of miR-9 then induces the trafficking defects of Nav1.1/Nav1.2 in vitro. In order to evaluate the protective effect of AMO-miR-9 on the onset of the trafficking defects of Nav1.1/Nav1.2 in 2VO rats, lenti-pre-AMO-miR-9 was injected into the hippocampus area. At 8\u00a0weeks after injection, miR-9 level was significantly decreased in both hippocampi and cortices compared with 2VO rats Fig.\u00a0. To our In this study, our observations have demonstrated, for the first time that CBH induces the trafficking defects of Nav1.1/Nav1.2 in hippocampi and cortices areas in 2VO rats, leading to the decrease in the expression of Nav\u03b22 protein. Further study has shown that the increased miR-9 negatively regulated the expression of Nav\u03b22 protein by binding to the target in CDS region of SCN2B gene. This observation provides a novel mechanism to modify the reduction in Nav1.1/Nav1.2 membrane trafficking, and careful monitoring the changes in miR-9 level and the expression for Nav1.1/Nav1.2 and targeted gene are considerably necessary during CBH.BACE1-trangenic mice although total Nav1.1 and Nav1.2 levels are elevated [BACE1-null mice [BACE1-trangenic mice [It has been known that Nav1.1, Nav1.2, and Nav1.6 are abundant in the central nervous system, whereas Nav1.3 is mostly present during embryonic stage . Nav1.6 elevated , 14, andull mice . In the nic mice .BACE1 affects Nav1.1 and Nav1.2 surface trafficking differentially [BACE1 activity leading to decrease in surface levels of Nav1.1 in neuronal cells [APP and BACE1 expression [APP and BACE1 is unknown and need to be elucidated further.Previous studies have demonstrated that Nav\u03b22 subunit, an auxiliary subunit of Nav channel, participates in channel trafficking, re-localization, and interaction of both Nav1.1 , 47 and entially . Furtheral cells . Interespression , 41. WheAs far as we know, microRNAs are newly discovered and commonly considered as modulators of protein expression at post-transcriptional level, which are associated with the pathogenesis in multiple kinds of diseases \u201353. In tOur study provides strong evidences that miR-9 increases in both hippocampi and cortices, and inhibited the expression of Nav\u03b22, which in turn blocked the trafficking of Nav1.1 and Nav1.2 from cytoplasm to plasma membrane Figs.\u00a0, 4 and 5In addition, our important finding here is that miR-9 regulates endogenous Nav\u03b22 expression by targeting its coding sequence (CDS) region rather than not 3\u2019UTR of SCN2B Fig. . Additioin vivo study supports the data collected from our in vitro observations that the upregulation of miR-9 induced by both CBH and lenti-pre-miR-9 could also disturb the trafficking of both Nav1.1/Nav1.2 by downregulation of Nav\u03b22 expression. On the contrary, lenti-pre-AMO-miR-9 injection into hippocampus markedly prevents the abnormal trafficking of both Nav1.1 and Nav1.2 following either CBH or lenti-pre-miR-9 treated normal rats accompanied by increased Nav\u03b22 expression. These results combination with our in vitro data suggested that the inhibition of miR-9 in hippocampi and cortices in CBH model rats would be a way to prevent sodium channel dysfunction after CBH. An understanding of miR-9-Nav\u03b22-Nav1.1/Nav1.2 trafficking pathway could yield to the potential therapeutic targets for the prevention of abnormal electrical activation induced by CBH.More importantly, our in vitro and in vivo, we did not provide evidence whether these changes could induce abnormal sodium channel currents and its dynamics characteristics in hippocampi and cortices of 2VO rats. These need to be studied further.In the present study, though we have demonstrated the regulation effect of miR-9 on the trafficking of Nav1.1/Nav1.2 by inhibiting the expression of Nav\u03b22 both MiR-9 plays a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav\u03b22 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially-valuable approach to prevent the Nav1.1/Nav1.2 trafficking disturbance induced by CBH.ad libium. Rats used for operation of permanent, bilateral common carotid artery occlusion (2VO) and stereotaxic injection of the lentiviral vectors were anesthetized with chloral hydrate  and maintained by administrating 0.5-1.0\u00a0% isoflurane. The depth of anesthesia was monitored by detecting reflexes, heart rate and respiratory rate. Samples for qRT-PCR, and Western blot assay were obtained from the hippocampi and cortices of rats after anesthetized with chloral hydrate  following by confirmation of death by exsanguination. Tissues for primary neuron culturing were from neonatal SD rats after administration of 20\u00a0% isoflurane and confirmation of death by cervical dislocation. All animal procedures were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No.HMUIRB-2008-06) and the Institute of Laboratory Animal Science of China (A5655-01). All procedures were conformed to the Directive 2010/63/EU of the European Parliament.Male Sprague\u2013Dawley rats  were housed at 23\u2009\u00b1\u20091\u00a0\u00b0C with 55\u2009\u00b1\u20095\u00a0% of humidity and maintained on 12\u00a0h dark\u2013light artificial cycle (lights on at 07:00 A.M.) with food and water available The method for preparation of 2VO rat was according to the previous study , 62. BriThe hippocampi and cortices regions were removed from the postnatal day 0 (P0) rat pups. After tissues were dissected and triturated, they were plated onto cell plates precoated with 10\u00a0\u03bcg/mL poly-D-lysine  and cultured in the culture media containing neurobasal medium  with 2\u00a0% B27 supplement  and 10\u00a0% fetal bovine serum . After 3\u00a0days, the neurons were treated with 5\u00a0\u03bcM cytosine arabinoside  to inhibit astrocyte proliferation. For all experiments, the neurons were used at 14\u00a0days after plating .MiR-9 mimics  and AMO-miR-9 (5\u2019-UCAUACAGCUAGAUAACCAAAGA-3\u2019) were synthesized by Shanghai GenePharma Co., Ltd . AMO-9 contains 2\u2019-O-methyl modifications at every base and a 3\u2019 C3-containing amino linker. Additionally, a scrambled RNA was used as a negative control . The Nav\u03b22-masking antisense oligodeoxynucleotides (ODNs) were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. Nav\u03b22 masking antisense-ODN-1 was 5\u2019-ATGCCTTCGTCTTCTAGCTGC-3\u2019, which masks the binding sites of miR-9, located in the position 336\u2013358 of SCN2B CDS (coding sequence) region; Nav\u03b22 masking antisense-ODN-2 was 5\u2019TCCTCTTCGGTCTTCAGGTCA-3\u2019\u2009, which masks the binding sites of miR-9, located in the position of 575\u2013597 of SCN2B CDS region. Five nucleotides or deoxynucleotides at both ends of the antisense molecules were locked by a methylene bridge connecting between the 2\u2019-O- and the 4\u2019-C atoms.Thirty pmol/mL miR-9 and/or AMO-9, ODNs or NC siRNAs were transfected into neonatal hippocampal and cortical neurons with X-treme GENE siRNA transfection reagent  according to the manufacturer\u2019s instructions. Forty-eight hours after transfection, cells were collected for total RNA isolation or protein purification.miR-9 (\u201ctop strand\u201d oligo: TGCTGTcTTTggTTaTcTagcTgTaTgaGTTTTGGCCACTGACTGACTcaTacagagaTaaccaaaga) and its complementary chain (\u201cbottom strand\u201d oligo: CCTGTcTTTggTTaTcTcTgTaTgaGTCAGTCAGTGGCCAAAACTcaTacagcTagaTaaccaaagaC); (2) pre-AMO-miR-9 (\u201ctop strand\u201d oligo: TGCTGTcaTacagcTagaTaaccaaagaGTTTTGGCCACTGACTGACTcTTTggTTcTagcTgTaTga) and its complement (\u201cbottom strand\u201d oligo: CCTGTcaTacagcTagaaccaaagaGTCAGTCAGTGGCCAAAACTcTTTggTTaTcTagcTgTaTgaC); (3) negative control (\u201ctop strand\u201d oligo: tgctgAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT) and its complement (\u201cbottom strand\u201d oligo: cctgAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTc). We then cloned the double-stranded oligonucleotides (ds oligo) generated by annealling the top and bottom strand oligos into the pcDNA\u21226.2-GW/\u00b1 EmGFP-miR vector and transformed the ligated mixture into competent E. coli. After colony was purified and identified as the correct expression clone, the pre-microRNA expression cassette was transferred to other Gateway\u00ae adapted destination vectors utilizing PolII promoters and formed a new miRNA expression clone containing attR substrates. The vector was identified after analyzing the plasmid sequence . The titers of the vectors used for experiments were 9.25\u2009\u00d7\u2009108 transducing U/ml. Virus suspensions were stored at \u221280\u00a0\u00b0C until use and were briefly centrifuged and kept on ice immediately before injection.Using the BLOCK-iT polII miR-RNAi expression vector with the EmGFP kit from invitrogen, three single-stranded DNA oligonucleotides were designed as follows: (1) pre-After anaesthetized, rats were placed onto a stereotaxic frame  described as previous study . InjectiBefore the luciferase activity assay, plasmid design and construction was shown as in the below figure: a 707\u00a0bp fragment from the coding region of SCN2B containing the putative binding sequences for miR-9 (position 336\u2013358 and position 575\u2013597 of SCN2B CDS) was amplified by PCR, cloned into the pSICHECK-2-control vector.GACGAAGGC)ATTTACAACTGCTAATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAGACCGAAGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGT; mutSCN2B CDS (bold nucleotide): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAAGTACGACGTGTCAGTGACTCTAAAGAA(CGTGCAGCTAGAAATCGATCGC)ATTTACAACTGCTACATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGTGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAATCGATCGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGTMutagenesis nucleotides were carried out using direct oligomer synthesis for the CDS region of Nav\u03b22-binding site 1 and Nav\u03b22-binding site 2. Point mutations were introduced into a possible miR-9 binding site located in the coding region of SCN2B (position 336\u2013358 and position 575\u2013597 of SCN2B CDS). MutSCN2B-1 represents that \u201cGACGAAGG\u201d was mutated \u201cATCGATCG\u201d in the position 336\u2013358 of SCN2B CDS, MutSCN2B-2 represents that \u201cGACCGAAG\u201d was mutated \u201cATCGATCG\u201d in the position 575\u2013597 of SCN2B CDS. MutSCN2B-1&2 represents that both sites were mutated. All constructs were sequence verified. Rat SCN2B CDS and mutSCN2B CDS sequences were shown as following: SCN2B CDS sequences (bold nucleotide showed the putative binding sequences for miR-9): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAGTACGACGTGTCAGTGACTCTAAAGAA as well as 0.5\u00a0\u03bcg of psi-CHECKTM-2-target DNA (firefly luciferase vector) and 1\u00a0\u03bcl blank plasmid using lipofectamine 2000  transfection reagent according to the manufacturer\u2019s instructions. After 48\u00a0h of transfection, Firefly and renilla luciferase activities, as indicated by relative luminescence units (RLU) were determined using luciferase assay kits  and luminometer  according to the manufacturer's instructions.The sequence of miR-9 mimic is 5\u2019-UCUUUGGUUAUCUAGCUGUAUGA-3\u2019 (synthesized based on the sequence of rno miR-9 (miRBase Accession No. MIMAT0000781)); that of miR-NC is 5\u2019-UUCUCCGAACGUGUCACGUAA-3\u2019; the sequence of the antisense 2\u2019-O-methyl (2\u2019-O-Me) oligonucleotide for miR-9 is 5\u2019-UCAUACAGCUAGAUAACCAAAGA-3\u2019, that of inhibitor-NC is 5\u2019UUCUCCGAACGUGUCACGUTT-3\u2019; HEK293T cells (plated at 40\u00a0%\u2009~\u200950\u00a0% confluence) were transfected with 20\u00a0\u03bcmol/l miRMiR-9 level was quantified by the TaqMan\u00ae MicroRNA Reverse Transcription Kit  and the TaqMan\u00ae Gene Expression Master Mix . The TaqMan qRT\u2013PCR probes and primers for miR-9, were designed by Applied Biosystems [SCN2B forward: CTCTCTGAACTGGACTTACC and SCN2B reverse: GGTTGGTGATGTAGCAGTTG; \u03b2-actin forward: GGAAATCGTGCGTGACATTA and \u03b2-actin reverse: AGGAAGGAAGGCTGGAAGAG. All reactions were performed in triplicate, and the expressions of microRNAs data were shown as Delta-Delta Ct method.Total RNA was purified with the Trizol Reagent , according to the manufacturer\u2019s instructions as described previously . MiR-9 losystems . U6 was Both the total protein and surface protein samples were extracted from hippocampi and cortices of rats or primary cultured neurons for immunoblotting analysis. For the total protein analysis, frozen tissue was homogenized with 1000\u00a0\u03bcl solution contained 40\u00a0% SDS, 60\u00a0% RIPA and 1\u00a0% protease inhibitor in each 200\u00a0mg brain tissue. The homogenate was then centrifuged at 13,500\u00a0rpm for 30\u00a0min and the supernatants (containing cytosolic and membrane fractions) were collected. The method of surface protein extraction was using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit  according to the manufacturer's instructions. Protein concentrations were measured spectrophotometrically using a BCA kit . Protein samples were fractionated by SDS-PAGE (10\u00a0% polyacrylamide gels for sodium channels) then transferred to PVDF membrane. The primary anti-Nav1.1, Nav1.2, Nav\u03b22 antibodies  were used and \u03b2-actin  \u03b2-actin  was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR)  was selected as an internal control of surface proteins. Western blot bands were captured on the Odyssey Infrared Imaging System  and quantified with Odyssey v1.2 software by measuring the band intensity (area\u2009\u00d7\u2009OD) in each group and normalizing to the internal control.The cultured neonatal rat neurons were transcardially perfused by 4\u00a0% buffered paraformaldehyde, pH7.4. After blocking, cultured neonatal rat neurons were incubated with the anti-\u03b2-Tubulin III  antibody  or anti- Nav1.1, Nav1.2, Nav\u03b22 antibodies  overnight at 4\u00a0\u00b0C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594  for 1\u00a0h at room temperature.t-test was applied for comparisons between the two groups. Multi-group\u2019s comparisions were performed by One-way ANOVA. SPSS19.0 software was used for all statistical analyses. P\u2009<\u20090.05 was considered significant.Data were described as mean\u2009\u00b1\u2009s.e.m for experimental data. The two-tailed Student\u2019s"}
+{"text": "There is an error in the third sentence of the \u201c16S library preparation\u201d subsection of the Materials and Methods. The first primer listed is incorrect. The correct sentence is: The V6 region was amplified with custom barcoded primers that consist of a region that anneals to the V6 region of interest, followed by a unique five basepair barcode, and the Illumina sequencing adapter [44]:V6-L [5\u2019- ACACTCTTTCCCTACACGACGCTCTTCCGATCTnnnnnCWACGCGARGAACCTTACC-3\u2019]V6-R [5\u2019-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTnnnnnACRACACGAGCTGACGAC-3\u2019]"}
+{"text": "Traditional fermented cheese whey (TFCW), containing probiotics, has been used both as a dairy food with ethnic flavor and a medicine for cardiovascular disease, especially regulating blood lipid among Kazakh. We therefore investigated anti-atherosclerotic effects of TFCW in atherosclerotic rabbits and identified lactic acid bacteria (LAB) and yeasts in TFCW.Atherosclerotic rabbits were induced by administration of atherosclerotic diet for 12\u00a0weeks and divided randomly into three groups and treated for 4\u00a0weeks with Simvastatin (20\u00a0mg/kg) or TFCW (25\u00a0mg/kg) and (50\u00a0mg/kg). In addition, a normal control group and an atherosclerotic group were used for comparison. All drugs were intragastrical administered once daily 10\u00a0mL/kg for 4\u00a0weeks. Body weight (BW), lipid profiles, C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were tested and theromatous plaques and the number of foam cells and infiltrating fibroblast cells in the thoracic aorta endothelium was evaluated by hematoxylin and eosin stainin. LAB and yeasts were isolated and purified by conventional techniques and identified using morphological and biochemical properties as well as gene sequences analysis.P\u2009<\u20090.05) compared to atherosclerotic group, and increased HDL-C (P\u2009<\u20090.05) compared to normal controls. Histological analysis showed TFCW reduced VCAM-1 expression and formation of atheromatous plaques on the aortic endothelium of atherosclerotic rabbits.After 4\u00a0weeks of treatment, high and low dose TFCW decreased serum TC, TG, LDLC, CRP, VCAM-1 and ICAM-1 (Seven classes of LBA from two different genera including Lactobacillus brevis, Lactobacillus kefianofaciens, Lactobacillus helveticus, Lactobacillus Casei, Lactobacillus plantarum, Lactobacillus kefiri and Lactococcus lactic as well as 2 classes of yeasts from two different genera including Saccharomyces unisporus and Issatchenkia orientalis were isolated and identified from TFCW. In summary, TFCW, containing 7 classes of LBA and 2 classes of yeasts, has significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium. The public health burden of cardiovascular disease (CVD) is substantial, as CVD remains the leading cause of mortality and morbidity worldwide and atherosclerosis is the major cause of CVD , 2.Traditional fermented cheese whey (TFCW), by-product of cheese-making, has been widely used as a traditional dairy medication for regulating blood lipid among Kazakh people . Indeed,Lactobacillus and Bifidobacterium and a few yeast species including Saccharomyces boulardi [S. boulardii, one of the probiotic yeasts, provides anti-inflammatory and host immunity stimulatory effects [Probiotics mainly include boulardi . Lactic boulardi  and atheboulardi  in anima effects  and lowe effects .However, anti-atherosclerotic effects of TFCW have not been experimentally demonstrated and no LAB or yeast has been found in TFCW. The aims of this study were to investigate anti-atherosclerotic effects of TFCW in a rabbit model of atherosclerosis and to identify LBA and yeast in TFCW.Traditional fermented cow\u2019s milk is the source of the cheese whey. Experimental TFCW samples were manufactured by standard procedures in 10\u00a0L vats in Altay Kanas Dairy Co. Ltd., . Fresh cow\u2019s milk samples were obtained from Jimunai Saur farm  and skimmed in centrifuging at 3000\u2009\u00d7\u2009g for 30\u00a0min, homogenized under the pressure of 1.5\u2009~\u20091.7 Mpa and pasteurized by high temperature short time (HTST) then cooled to about 30\u00a0\u00b0C and fermented by inoculation with traditional home made Kazak yogurt purchased from Jimunai Saur farm  at 37\u00a0\u00b0C for 12\u00a0h. After ferment, the whey was filtered in sterile gauze and dialyzed in cellulose membrane  under constant magnetic stirring at 8\u00a0\u00b0C, also performed lactose removal by periodic water exchange. The experimental TFCW was stored at \u221220\u00a0\u00b0C until further use.Sodium pentobarbital was purchased from Merck & Co., (Germany). Simvastatin was purchased from Merck Sharp & Dohme  Pty Ltd., . VCAM-1, ICAM-1 and CRP ELISA kits were purchased from Shanghai Senxiong Technology Co. Ltd., . Man Rogosa Sharpe (MRS) was purchased from Merck Sharp & Dohme  Pty Ltd., . All media for cultivation of zymocytes were purchased from Hangzhou Microbial Reagent Co. Ltd., .ad libitum access to food and water. After a week of adaptive feeding, all the rabbits were randomly divided into 5 groups with 12 in normal group and 12 in atherogenic group, the normal control groups were given regular die and the atherogenic models were developed using an atherogenic diet for 12\u00a0weeks. The atherogenic diet consisted of 3\u00a0% cholesterol, 0.5\u00a0% sodium taurocholate, 0.2\u00a0% propylthiouracil, 5\u00a0% sugar, 10\u00a0% lard, and 81.3\u00a0% standard laboratory rabbit chow, which were provided by Experimental Animal Center of Xinjiang Medical University, China. After developing atherogenic models, Group 1  was treated with saline in a matched volume; Group 2 (atherogenic group) had atherogenic rabbits treated with saline in a matched volume; Group 3 (positive control) had atherogenic rabbits administered with simvastatin 20\u00a0mg/kg; Group 4 and Group 5 were treated with TFCW 25\u00a0mg/kg and 50\u00a0mg/kg, respectively (low and high doses). Simvastatin and TFCW were intragastrical administered once daily 10\u00a0mL/kg for 4\u00a0weeks. All animals received care in compliance with the Chinese Convention on Animal Care, and the study was approved by the Institutional Ethics Committee of Xinjiang Medical University.Sixty male white New Zealand rabbits, weighing 1.95-2.05\u00a0kg, specific pathogen free (SPF), were provided by Experimental Animal Center of Xinjiang Medical University, China and placed in separate cages and maintained on a 12-h day/night cycle at an ambient temperature, with At the end of experiments, all rabbits were fasted for 12\u00a0h, weighed, anesthetized with sodium pentobarbital  and continually monitored until total loss of consciousness as indicated by a total lack of response after a foot pinch. Blood samples were collected from abdominal aorta, allowed to clot on ice and subsequently subjected to centrifugation (3500\u00a0rpm at 4\u00a0\u00b0C for 10\u00a0min), where after serum aliquots were stored at \u221280\u00a0\u00b0C for further analysis. Serum TC, TG, LDL-C and HDL-C were examined via an automatic biochemical analyzer . CRP was determined by rate nephelometry . Serum ICAM-1 and VCAM-1 were determined using commercially-available ELISA kits according to manufacturer instruction.Aorta was harvested from rabbits, placed immediately in formaldehyde 10\u00a0%, embedded in paraffin 24\u00a0h later, cut at 5\u00a0\u03bcm, stained with hematoxylin and eosin (H&E), and then scanned to assess pathological changes. For immunohistochemical staining, sections were incubated with anti-VCAM-1  and anti-F4/80  at 37\u00a0\u00b0C for 1\u00a0h, color developed with 3,3\u2032-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Samples in the absence of the primary antibodies were used as negative controls. Slides were observed under a light microscope, and images were subjected to statistical evaluation of positively stained cells in 10 random fields of view at a magnification of\u2009\u00d7\u2009400. The average numbers of positively stained cells were counted per high power field (HPF).2) production were analyzed. All isolates were presumptively identified as LAB strains based on their ability to grow on MRS agar plates, Gram-positive staining, and a catalase activity-negative phenotype [Agar plates with Man Rogosa Sharpe (MRS) broth suitable for lactobacillus growth were used for initial isolation of LAB single colonies. Single bacterial colonies were initially separated based on their morphological differences on agar plates. Cell morphology was observed under light microscopy after Gram staining. Catalase activity, carbohydrate fermentation, acidogenicity, aciduricity , and gas . Significance was defined as p\u2009<\u20090.05), whereas Simvastatin group and low and high dose TFCW group showed significantly lower serum TC, TG and LDL-C than did atherogenic group (p\u2009<\u20090.05). TFCW significantly increased HDL-C levels compared to normal control group (p\u2009<\u20090.05), but no significant difference was observed in treated groups with TFCW, compared with atherogenic group. Data indicated that TFCW affects lipid metabolic parameters and TFCW treatment could effectively improve lipid metabolism in atherogenic rabbits.Table\u00a0P\u2009<\u20090.05). Outstandingly, the simvastatin and low and high dose TFCW groups had significantly lower CRP (P\u2009<\u20090.05) at values 3.43\u2009\u00b1\u20090.80\u00a0mg/L, 3.33\u2009\u00b1\u20090.50\u00a0mg/L and 1.34\u2009\u00b1\u20090.90\u00a0mg/L, respectively, compared with the atherogenic group. CRP in low and high dose TFCW was significantly decreased, compared to atherogenic group (P\u2009<\u20090.05). Data indicate that TFCW affects CRP and TFCW treatment could effectively improve inflammatory status in atherogenic rabbits. No significant differences were recorded in treated groups with TFCW on body weight compared with atherogenic group.Serum CRP was measured to determine inflammatory status of experimental groups as shown in Table\u00a0p\u2009<\u20090.05), while Simvastatin and low and high dose TFCW group showed significantly decreased ICAM-1 and VCAM-1, compared to atherogenic group (p\u2009<\u20090.05). Data indicated that TFCW might affect ICAM-1 and VCAM-1 and TFCW treatment might effectively inhibit adhesion of circulating inflammatory cells to endothelial cell walls in atherogenic rabbits.Adhesion molecules ICAM-1 and VCAM-1 play important roles in perpetuation of inflammation. Table\u00a0Histopathological examination of aorta sections from experimental groups were shown in Fig.\u00a0Immunohistochemical evaluation of VCAM-1 expression in aorta section and histomorphometric analysis from experimental groups were shown in Fig.\u00a0Lactobacillus species was rod-shaped, slender short or long, and the majority of Lactobacillus exhibited chain-like arrangement. We successfully isolated and purified seven isolates of LBA and identified them as L. brevis, L. kefianofaciens, L. helveticus, L. casei, L. plantarum, L. kefiri and Lactococcus lactic based on morphological characteristics, physiological tests, biochemical tests, and 16S rDNA and 16S rRNA sequence homology with NCBI Reference Sequences NC 008497.1, NC 015602.1, NC 006814.3, NC 008526.1, NC 004567.2, NC 015428.1 and NC 017486. All the test results and sequence homology were shown in Table\u00a0L. brevis:GCTGACTCCCGAAGGTTATCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTAGCCTCACGACTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTGAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGTCTTATCTCTAAGATTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACTAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAACCCTTGAACAGTTACTCTCAAAGGTGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCATTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATCGTCTTGGTGGGCCTTTACCTCACCAACTAACTAATACGCCGCGGGATCATCCAGAAGTGATAGCCGAANCCACCTTTCAAACAAAATCCATGCGGATTTTGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTGCTTCTGGGCAGATTTCCCACGTGTTACTCACCAGTTCGCCACTCGCTTCATTGTTGAAATCAANGCAAGCACGTCATTCAACGGAAGCTCGTTCGACTL. kefianofaciens:CTGCTTAGACGGCTCCTTCCTTGCGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTAATAGCTATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGAATTGCTTGCCTTTGCAGGCTCGCTGCTCGTTGTGCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAGCCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTATTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCTACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAATTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGGTCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCACTGCCTTGGTAGGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGTCCATCCTTTAGCGACAGCTTGCGCCGCCTTTTAAAAGCTGTTCATGCGAACTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTTAAGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCCAGCGTCCTCACCGAAGTGATTCTGCTGGTTCCGCTCGCTCGACTTGCATGTATTAGL. helveticus:TTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCATCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTGCATGTATL. casei:TAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANNAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTCGTTCGACTGCATGTATAGCL. plantarum:GGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTGGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCACCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAGGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGL. kefiri:CTTAGACGGCTGGTCCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTTAAATGCAAGCATCTAAAATCAATAACGGAAACGCGTTCGACTTGCATGTATLactococcus lactic:GGTCTTACCTTAGGAAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTATGTTTAAAGTTGTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCTTCAAATTGGTGCAAGCACCAATCTTCATCGCTCAACTTGCATGATTAGSeven gram-positive, catalase-negative bacilli were isolated from TFCW. Most of the bacilli had yellow or white colonies with uneven edges and a rough and dull surface. Some of the colonies in the center were translucent, with neat edges. The type colony size was approximately 0.5-3.0\u00a0mm. Morphology of the separated S. unisporus and I. orientalis based on morphological characteristics, physiological and biochemical tests and 26S rDNAD1/D2 sequence homology with sequences AY 707865 and EU 019220 in Genbank. All the test results and sequence homology were shown in Table\u00a0S. unisporus:TGCATATTCAATAAGCGGAGGAAAAGAAACCAACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTAGTACCTTCGGTGCTCGAGTTGTAATTTGTAGAGGGATACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTATGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGCGCCCTCTGCTCCTTGTGGGTGGGGGAATCTCGCAGCTCACTGGGCCAACATCAGTTTTGGTGGTCGGATAAATCCGTAGGAATGTGGCTTGCCTCGGCAAGTGTTATAGCCTGCGGGAATACGGCCAGCTGGGACTGAGGACTGCCACTTTTGTCAAGGATGTTGGCATAATGGTTATATGCCGCCCGTCTTGAAACACGGACCAAI. orientalis:GCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTGAAGCGGCAAGAGCTCANATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTTGGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGAGCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAATGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAGGGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTCGTGGGCGGCGCTCTGGGCTTTCCCTGGGCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGTTCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGACCGAGGACTGCGGCCGTGTAGGTCACGGATGCTGGCAGAACGGCGCAACACCGCCCGTCTTGAAACACGGACCAWe identified two yeast isolates to be Major finding of the current study is that treatment with TFCW significantly modified lipid profile and reduced CRP, ICAM-1 and VCAM-1 in atherosclerotic rabbit model. Preventive effects of TFCW in atherogenic rabbits were also demonstrated by reduction in VCAM-1 expression and formation of atheromatous plaques on aortic endothelium. In fact, accumulation of cholesterol and lipids leads to foam cell formation, which is regarded as a critical process in development of atherosclerosis . OverwheL. casei [L. helveticus [L. plantarum [L. lactis [In this study, 7 potential probiotic lactobacillus species, including L. casei , L. helvlveticus , L. planlantarum  and L. l. lactis , which a. lactis  and redu. lactis , 23 both. lactis , 25 and . lactis . LAB or . lactis .Lactocillus fermenterum ME-3 improves antioxidant activities in human blood, thus providing antiatherogenic activity [In addition, goat milk fermented with activity . Consumpactivity , 29.S. unisporus is ubiquitously present in fermented milk, cheese and kefir-based milk products and may produce vitamins and interact with LAB, which may enhance LAB growth [S. unisporus contains middle chain fatty acids up to C 14:0 to 18:1 and produces a high percentage of palmitoleate. Palmitoleic acid, an omega-7 monounsaturated fatty acid, is a major constituent of human adipose tissues and is considered antioxidant [I.orientalis exhibits a higher tolerance for pH, bile, and heat stress for survival in gastrointestinal environment as a probiotic [I. orientalis commonly exists in cheeses and other fermentation milk products and exhibits ability to scavenge 1,1 diphenyl-2-picrylhydrazyl and to inhibit lipid peroxidation, thus presenting antioxidant activity as a potential probiotic in fermented milk products [We also identified two probiotic yeasts in TFCW. B growth . S. unisioxidant . I.orienrobiotic . I. orieproducts .Notably, oxidized LDL in vascular wall seems to be a key factor in atherosclerosis, because oxidized LDLs might recruit monocytes and favor their transformation into foam cells through a receptor-mediated intake (scavenger pathway). Moreover, cytotoxic oxidized form of LDLs are likely responsible for endothelial cell damage and macrophage degeneration in atherosclerotic human plaque . PolyunsIn conclusion, current study indicates that 7 LAB and 2 yeasts identified from TFCW and TFCW have significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium."}
+{"text": "Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial\u2019s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time . Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy  were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. To date, mitochondria have been thought to play an important role in myocardial ischemia-reperfusion (I/R) injury . It is wIn contrast, heme oxygenase-1 (HO-1), which belongs to the low-molecular weight heat shock protein HSP family, induction and expression are important protective mechanisms during cell stress \u20136. In raTherefore, in this study, we chose to use genetic engineering techniques to construct an Lv-HO1-H9c2 cell line that enables a steady increase in the expression of exogenous HO-1. After myocardial hypoxia-reoxygenation injury, the RFP-GFP-LC3 double-labeled adenovirus was used to detect autophagy, and flow cytometry was used to measure mitochondrial membrane potential and mitochondrial ROS levels to investigate the mechanism by which HO-1 protects mitochondrial function following myocardial hypoxia-reoxygenation injury.The effect of different durations of hypoxia/reoxygenation on myocardial cytotoxicity was measured. As shown in Cell viability was compared between the four groups . SignifiCell viability was compared between the four groups . An Lv-HCell viability in the four groups was compared . After ZHO is the initial and rate-limiting enzyme that catalyzes the oxidative metabolism of heme. Three HO isozymes exist, and HO-1 is the only isoenzyme that exhibits inductive expression. During cardiac ischemia-reperfusion injury, ROS generation can increase the expression of HO-1 , consistThe autophagic dysfunction of myocardial cells has attracted attention as one of the mechanisms underlying ischemia-reperfusion injury ,24,25. IIn addition, in this study, flow cytometry detection showed that during autophagy, the mitochondrial membrane potential was significantly higher  and the Therefore, clearing damaged mitochondria is extremely important. Autophagy, the major degradation pathway involved in mitochondrial quality control, has been reported as a cellular adaptive response to oxidative stresses . PreviouInterestingly, in our studies, rapamycin could up-regulate the expression of the HO-1 protein . It is aTo define the role of HO-1, most studies have used methods such as employing adeno-associated viruses to achieve instantaneous HO-1 over-expression ,14 or knWith HO-1 overexpression, LC3II levels were significantly increased after reoxygenation, and p62 protein expression was significantly decreased . MoreoveRecent studies have shown that an HO-1 activator can activate transcription factor EB (TFEB), a regulator that induces lysosome and autophagosome formation, to protect against LPS-induced oxidative stress in heart tissue . MeanwhiIn summary, we based our conclusions on evidence that showed that during myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect against myocardial hypoxia-reoxygenation injury. By studying the molecular and regulatory mechanisms of mitochondrial function protection, we will obtain further knowledge regarding the mechanism underlying the development of ischemic heart disease, information that will aid in for the development of new disease prevention and treatment strategies.Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) and other cell culture supplies, such as trypsinogen, were purchased from Invitrogen. Fetal calf serum was purchased from Gibco BRL . A Cell Counting Kit-8 was purchased from Dojindo . HO-1 and pAbR rabbit antibody were purchased from Enzo Life Sciences Inc. . p62 and LC-3 antibody were purchased from Abcam (USA), and GAPDH antibody was obtained from CST (USA). AntiRabbit was obtained from Sigma (USA). BSA was obtained from Invitrogen (USA). TritonX-100 was obtained from Sigma (USA). A JC-1  Mitochondrial Membrane Potential Detection Kit was purchased from Beyotime (China). Confocal microscopy and imaging systems (ZEISS-Axio) were used at the Wuhan University School of Basic Medical Sciences.34H32N4O4Zn and a molecular weight of 626.03 g/mol.Zinc protoporphyrin (ZnPP), a HO-1 inhibitor, was supplied by Sigma. The purity of ZnPP (\u226595%) was determined by HPLC; the inhibitor was soluble in water and has the molecular formula C2. The medium was replaced every 2\u20133 days, and the cells were subcultured or subjected to experimental procedures at 80\u201390% confluence. In all experiments, the cells were rendered quiescent by serum starvation for 24 h before treatment. For the H/R experiments, hypoxic conditions were created by incubating the cells in an anaerobic chamber equilibrated with 2.5% O2, 5% CO2 and 92.5% N2 at 37\u00b0C for 24 h. The cells were then reoxygenated under normoxic conditions in a humidified atmosphere (95% air/5% CO2) at 37\u00b0C for the indicated time . Normoxic control cells were incubated at 37\u00b0C under 95% air/5% CO2. In the Rapamycin experiments, the cells were pretreated for 1 hour with the autophagy inducer Rapamycin (100 nM) and then subjected to hypoxia and reoxygenation. In the ZnPP experiments, the cells were pretreated for 6 hours with the HO-1 inhibitor ZnPP (10 \u03bcM) and then subjected to hypoxia and reoxygenation.The H9c2 cardiomyocyte cell line (rat embryonic cardiomyoblasts) was maintained in our laboratory and obtained from the American Type Culture Collection . The H9c2 cells were maintained in high-glucose DMEM supplemented with 10% v/v fetal calf serum at 37\u00b0C in a humidified atmosphere containing 5% COA lentiviral vector was constructed, and a lentiviral shuttle plasmid and its secondary packaging of the original vector plasmid were prepared; the three plasmid vectors were subjected to endotoxin-free extraction to obtain materials of high purity. Next, the plasmid vectors were co-transfected into 293T cells. The medium was replaced with complete medium at 6 hours after transfection. The supernatant, which was rich in lentivirus particles, was collected after 48 and 72 hours of culture and filtered using a 0.45 \u03bcm filter (Millipore), and the virus was then concentrated by ultracentrifugation. Next, a high-titer lentivirus concentrate was obtained after concentration. A vector map of pHBLV-CMVIE-IRES-puro is shown in The pHBLV-CMVIE-IRES-puro vector contains a CMVIE promoter to initiate the expression of target genes and contains a puromycin resistance gene as a marker. The HO-1primer designs as ATGGAGCGTCCGCAACCCGACAGCATGCCCCAGGATTTGTCAGAGGCCCTGAAGGAGGCCACCAAGGAGGTGCACACCCAGGCAGAGAATGCTGAGTTCATGAGGAACTTTCAGAAGGGCCAGGTGACCCGAGACGGCTTCAAGCTGGTGATGGCCTCCCTGTACCACATCTATGTGGCCCTGGAGGAGGAGATTGAGCGCAACAAGGAGAGCCCAGTCTTCGCCCCTGTCTACTTCCCAGAAGAGCTGCACCGCAAGGCTGCCCTGGAGCAGGACCTGGCCTTCTGGTACGGGCCCCGCTGGCAGGAGGTCATCCCCTACACACCAGCCATGCAGCGCTATGTGAAGCGGCTCCACGAGGTGGGGCGCACAGAGCCCGAGCTGCTGGTGGCCCACGCCTACACCCGCTACCTGGGTGACCTGTCTGGGGGCCAGGTGCTCAAAAAGATTGCCCAGAAAGCCCTGGACCTGCCCAGCTCTGGCGAGGGCCTGGCCTTCTTCACCTTCCCCAACATTGCCAGTGCCACCAAGTTCAAGCAGCTCTACCGCTCCCGCATGAACTCCCTGGAGATGACTCCCGCAGTCAGGCAGAGGGTGATAGAAGAGGCCAAGACTGCGTTCCTGCTCAACATCCAGCTCTTTGAGGAGTTGCAGGAGCTGCTGACCCATGACACCAAGGACCAGAGCCCCTCACGGGCACCAGGGCTTCGCCAGCGGGCCAGCAACAAAGTGCAAGATTCTGCCCCCGTGGAGACTCCCAGAGGGAAGCCCCCACTCAACACCCGCTCCCAGGCTCCGCTTCTCCGATGGGTCCTTACACTCAGCTTTCTGGTGGCGACAGTTGCTGTAGGGCTTTATGCCATGTGAAfter H9c2 and Lv-HO1-H9c2 cells were cultured in 96-well plates and received the described treatments, 10 \u03bcl of CCK-8 solution was added to each well (1/10 dilution); the plates were then incubated for a further 3 h. The absorbance was measured at 450 nm using a microplate reader . The mean optical density (OD) of five wells in the indicated groups was used to calculate the percent cell viability according to the following formula: Percent cell viability = OD treatment group/OD control group\u00d7100%. The experiments were repeated 3 times.\u00aePremix Ex Taq\u2122 Perfect Real-Time Kit. To measure mouse gene expression, the following SYBR Green real-time PCR primers were used:Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) and purified using the RNeasy Total RNA Isolation Kit (Invitrogen). Real-time quantitative PCR was performed using the ABI 7900 Real-Time PCR System (Applied Biosystems) and the SYBR5\u2032- TGCACATCCGTGCAGAGAAT -3\u2032HO-1: Forward 5\u2032-CTGGGTTCTGCTTGTTTCGC -3\u2032; product length = 147\u2003\u2003\u2003\u2003Reverse 5\u2032\u2014CACCATCTTCCAGGAGCGAG -3\u2032GAPDH: Forward 5\u2032\u2014AAATGAGCCCCAGCCTTCTC -3\u2032; product length = 114\u2003\u2003\u2003\u2003Reverse Proteins were isolated from the cells that had been subjected to hypoxia and reoxygenation using standard protocols, and the protein concentrations in the lysates were determined using the Bradford Protein Assay Kit . Equal quantities of proteins (30\u201350 lg/lane) were subjected to 8%\u201312% SDS-PAGE, depending on the target proteins, electrotransferred onto nitrocellulose membranes, and incubated with primary antibodies against HO-1 , p62 , LC-3-II , GAPDH , and antiRabbit . After incubating the membranes with the corresponding secondary antibodies, protein bands were detected using a chemiluminescence imaging analysis system (Fujifilm), and quantitation was performed using Quantity One 4.4.0 software (Bio-Rad).4 cells/mL) was added. Cells were transfected with Ad-mCherry-LC3-GFP adenovirus (MOI = 50); 36 hours later, the plates were pre-incubated for 24 hours in an incubator . Next, 4% PFA  was used to fix the cells for more than 10 minutes; the cells were then washed in PBS (5 minutes x 3 times). Then, Antifade mounting medium was added, and the cells were imaged under a fluorescent microscope. RFP-GFP-LC3 double-labeled adenovirus was used for testing. GFP degrades in the acidic environment used. When red and green fluorescent images are merged, the yellow spots that appear indicate autophagosomes. Red spots indicate autophagic lysosomes. In normal phagosome-lysosome fusions, there will be more red fluorescence than yellow fluorescence. In impeded autophagy, where phagosome-lysosome fusion does not work, yellow fluorescence will be dominant.To 6-well plates , 2 mL of Lv-HO1-H9c2 cell suspension  and reactive nitrogen species (RNS) rapid oxidation. After binding nucleic acid oxidation products, the probe can produce large amounts of fluorescence by flow cytometry, thus, the changes of mitochondrial reactive oxygen species content may be reflected.Using flow cytometry to detect the 5,5',6,6'-tetrachloro-1,1',3,3'-. tetraethylbenzi-midazolylcarbocyanine iodide (JC-1) staining. After incubation with the JC-1 staining solution at 37\u00b0C in the cell incubator for 10 min, cells were washed with JC-1 staining buffer two times and then analyzed using a fluorescence microplate reader. In the normal mitochondria, JC-1 aggregate to form a polymer in the mitochondrial matrix, the polymer sends a strong red fluorescence ; While in the unhealthy mitochondrial, due to the decline or loss of the mitochondrial membrane potential, JC-1 monomers just can be present in the cytoplasm, resulting in a green fluorescence . Therefore, using flow cytometry to observe the color changes reflects very directly the early change of mitochondrial membrane potential.5 cells were harvested and placed in EP tubes. The cells were rinsed with 0.1 mmol/L PBS (pH = 7.4) and collected brief centrifugation with trypsin EDTA-free. After that, the cells were resuspended in 500 \u03bcL of binding buffer, mixed with 5 \u03bcL of Annexin V-FITC, and then kept in darkness for 10 min at room temperature. Then, 5 \u03bcL of propidium iodide (PI) was added and the samples were kept in darkness for 10 min at room temperature. Approximately 300 \u03bcL of binding buffer was added, and the samples were analyzed using flow cytometry within 1 h. The blank control was the cells of the control group without Annexin V-FITC and PI. The Annexin V-FITC single-labeled control consisted of cells labeled with Annexin V-FITC. The PI single-labeled control consisted of cells labeled with PI.After treatment, 1~5\u00d710The data are presented as means \u00b1 SEM. Statistical analysis was performed using the Mann-Whitney test for two-group comparisons. Significant differences between multiple treatments were calculated using one-way analysis of variance followed by the Bonferroni post hoc test when appropriate. Western blot densities were analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test. Probabilities of 0.05 or less were considered statistically significant.S1 Fig(A) Cell viability; (B) HO1 QPCR; (C)WB analysis.(RAR)Click here for additional data file.S2 Fig(A) Cell viability (CCK-8); (B) WB analysis; (C) Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (F) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S3 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S4 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file."}
+{"text": "Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation. In biological sciences development of new specific monoclonal antibodies (mAbs) is a pressing requirement for several aspects in the field: from basic research on protein function, to medical diagnosis, prophylaxis and therapy of several pathogenic conditions As tags are often short (6\u201315 amino acids in length), they are generally presumed to have no effect on the biological functions of the tagged proteins. However, if located in inappropriate positions, they might interfere with protein structure, function and interactions. In addition, not all mAb are suitable for every immunodetection method, as in the case of mAb specific for non-linear epitopes. For those reasons, it is useful to develop mAbs and epitope tags of different sequence characteristics  or that can be fused in different positions of the target protein to increase the chances of success in tagging applications.Here we describe and characterize a new 10 amino acids long epitope tag (roTag) derived from the sequence of the rotavirus (RV) non-structural protein 5 (NSP5). NSP5 has an essential role during the RV replication cycle, as it is essentially required for the assembly of viroplasms, the sites of viral genome replication and initial assembly of progeny virus ++-purified His-tagged NSP5 protein of the RV porcine OSU strain A panel of anti-NSP5 mAbs were generated from BALB/c mice immunized with a NiTo establish the epitope on NSP5 we took advantage of a series of NSP5 deletion mutants To further define the epitope, a series of progressive N-terminal deletion mutants, NSP5-\u0394N3, NSP5-\u0394N8, NSP5-\u0394N13, NSP5-\u0394N18, NSP5-\u0394N23 and NSP5-\u0394N28, were assayed with a polyclonal anti-NSP5 serum or with 1F2 by WB. As shown in PSISSSIFKNE)  suggesting involvement of S12 and S14. In fact, a tag initiating in S12 (tag 12\u201324) resulted in a pattern of bands equally recognized by anti-SV5 and 1F2, both in the intracellular and in the secreted material  and because of the relevant impact of P11 on O-glycosylation, we defined S12 as the N-terminal border of the epitope and termed roTag the peptide 12\u201321 and P-roTag the one starting in P11 (11\u201321). The C-terminal border of the anti-roTag/1F2 epitope was confirmed to be E21, as peptides 9\u201318, 9\u201319 and 9\u201320 were not detected by anti-roTag . All theper se sufficient to cause retention in the ER and to completely prevent O-glycosylation, according to three different criteria: unchanged mobility, lack of secretion and detection by mAb anti-roTag.O-glycosylation was confirmed for roTagO, P-roTag and OG-tag by treatment of supernatants containing the reporter protein (with either of the three tags) with a glycosidase cocktail. As shown in L-linker-VH) fused to the hinge-CH2-CH3 domains of the \u03b3H chain of murine IgG2b. The resulting recombinant antibodies  containing the scFv  the 10 amino acids long roTag (peptide 12\u201321) that can be efficiently used with different proteins, in N-terminal, middle and C-terminal positions and in different cellular compartments  , approved by the ICGEB Trieste Ethics Committee for Animal Experimentation. Animals were anesthetized (isoflurane) before venipuncture and sacrifice (asphyxiation in carbon dioxide). All efforts were made to minimize suffering.++-purified His-tagged NSP5 protein Balb/c mice have been immunized with NiNSP5 deletion mutants were previously described 5\u2032-GATATTGTGATGACCCAGTCTCCA-3\u2032, CK-2 3\u2032: 5\u2032-TGGATACAGTTGGTGCAGC-3\u2032, VHS-2 5\u2032: 5\u2032-TGTGCACTCYSAGGTSMARCT-3\u2032, CH\u03b3 3\u2032: 5\u2032-GGCCAGTGGATAGAC-3\u2032. From pUC18 VL and VH regions were amplified with primers 1F2-VL-for: 5\u2032TATGGTGCACTCTGATGTTGTGATGACCCAGACTCCA-3\u2032, 1F2-VL-rev: 5\u2032-TATAACTAGTGCTGCCTTTCAGCTCCAGCTTGGT-3\u2032, VH-for 5\u2032: 5\u2032-TCTCTCGAGCAAAGGTCAGGTCCAACTGCAGCAGTC-3\u2032, 1F2-VH rev: 5\u2032-AGTTCCGGAGGAGACGGTGACTGA-3\u2032 and cloned ApaLI/BspEI into pUT vector downstream of a leader peptide H2 e CH3 of IgG2b, which were previously amplified from murine splenocytes and inserted in pcDNA3 by BspEI/XbaI (primers MHG2B-1 5\u2032: 5\u2032-CCCTCCGGACCCATTTCAACAATCA-3\u2032 and MHG2B-2 3\u2032: 5\u2032-TCTTCTAGAGCTCATTTACCCGGAGA-3\u2032).After cDNA synthesis VL and VH were amplified and cloned in pUC18 using primers VKB-2 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAATCGFor mapping the anti-roTag epitope the following synthetic oligonucleotides encoding the different epitope tags were cloned as BamHI/EcoRI, downstream of SV5 in a plasmid containing scFv-SV5-BAP previously described 5\u2032-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATCTTCTTAAG-3\u2032, 9-24-2 3\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTGAGGGAAGG-3\u2032, 9-24G-1 5\u2032: 5\u2032-AATTCTTAAGAAGAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-21-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 9-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 12-24-1 5\u2032: 5\u2032-GATCCATTTCTTCTAGTATCTTTAAAAATGAATCGTCTTCTTAAG-3\u2032, 12-24-2 3\u2032: 5\u2032-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATG-3\u2032, 9-16-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTTAAG-3\u2032, 9-16-2 3\u2032: 5\u2032-AATTCTTAACTAGAAGAAATTGAGGGAAGG-3\u2032, 10-21-1 5\u2032: 5\u2032-GATCCGGTGGCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 10-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGGCCACCG-3\u2032, 11-21-1 5\u2032: 5\u2032-GATCCGGTGGCCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032, 11-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGGCCACCG-3\u2032, 12-21-1 5\u2032: 5\u2032-GATCCATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3\u2032 12-21-2 3\u2032: 5\u2032-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATG-3\u2032, 9-20-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATTAAG-3\u2032, 9-20-2 3\u2032: 5\u2032-AATTCTTAATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-19-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAATAAG-3\u2032, 9-19-2 3\u2032: 5\u2032-AATTCTTATTTAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, 9-18-1 5\u2032: 5\u2032-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTTAAG-3\u2032, 9-18-2 3\u2032: 5\u2032-AATTCTTAAAAGATACTAGAAGAAATTGAGGGAAGG-3\u2032, roTagG KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTAAGGATGAGCTTTAAG, roTagG KDEL-2: AATTCTTAAAGCTCATCCTTACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG, OG-Tag KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTGGAAAGGATGAGCTGTAAG, OG-Tag KDEL-2: AATTCTTACAGCTCATCCTTTCCAAAGATACTAGAAGAAATTGAGGGAAGG.TTCTTCTTAAG-3\u2032, 9-24G-2 3\u2032: 5 cells/well) by standard calcium phosphate technique Sp2/0 myeloma cells (ATCC CRL-1581) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS); hybridoma clones were grown in the same medium supplemented with 2% Hybridoma Media Supplement (Sigma-Aldrich) and 1 mM sodium pyruvate. MA104 and HEK 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS). Cells were co-transfected in 6-well plates  supplemented with protease inhibitors cocktail (Sigma-Aldrich). In the experiments shown in Immunofluorescence experiments were performed after cells were fixed in 3.7% paraformaldehyde in PBS for 10 min at room temperature. Coverslips were washed in PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min and incubated with mouse anti-NSP5 serum (1:500) in PBS\u20131% BSA, supernatants of anti-roTag hybridoma for 1 h at room temperature. After three washes in PBS, the slides were stained for 45 min with rhodamine isothiocyanate-conjugated secondary antibody (Sigma), washed, and mounted with ProLong mounting medium (Molecular Probes). Samples were analyzed by confocal microscopy (Zeiss LSM510).3-Na2CO3 50 mM, pH 9.5 (100 \u00b5l/well). After reaction with HRP-labeled goat anti-mouse gamma Fc (Jackson Immunoresearch) plates were developed with tetramethylbenzidine (TMB) reagent (Sigma) and blocked with H2SO4. O.D. at 450 nm was read on a BioRad microplate reader 550. ELISA comparison of relative affinity of recombinant anti-SV5 and anti-roTag was performed by capturing the double-tagged (SV5-roTag) reporter ELISA to determine relative antibody concentrations was performed by capturing supernatants containing Abs on polystyrene microplates (Nunc Maxisorp C96) coated with 0.1 \u00b5g/ml of goat anti-mouse gamma in buffer NaHCOFigure S1(A) Full gel, of non-infected and RV-infected MA104 cell extracts reacted with mAb 1F2. (B) Full gel of lanes shown in (TIF)Click here for additional data file."}
+{"text": "G\u03b1s, which transduces mitogenic signals, in 30% of the analyzed JGCTs.Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K\u2013AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays.We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays.AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols.Our study incriminates somatic mutations of   \u2022We detected duplications affecting the pleckstrin-homology domain of AKT1 in >\u00a060% of the Juvenile Granulosa cell tumors studied.\u2022The JGCTs without duplications carried point mutations affecting highly conserved residues.\u2022Mutated proteins with duplications displayed an enrichment at the plasma membrane and a striking phosphorylation level. Our study points to AKT1 as a major driver in the pathogenesis of JGCTs.Sex cord stromal tumors involve granulosa, theca or stromal cells, alone or combined. The most common ones are ovarian granulosa cell tumors (GCTs), which represent up to 5\u20138% of all ovarian tumors . Two disof AGCTs . This muof AGCTs . With reof AGCTs . In anote latter . FSHR sie latter  that phoe latter . Becausee latter , we hypo22.1This study involves a cohort of 16 histologically proven JGCTs, occurring in girls under 15\u00a0years of age, collected between 1994 and 2014 . The available clinical data are displayed in 2.2Twelve samples were formalin-fixed paraffin-embedded (FFPE) (T1\u2013T12). Four tumors were obtained as frozen samples (T13\u2013T16). We isolated genomic DNA and RNA from FFPE tumors using the AllPrep DNA/RNA FFPE Kit (Qiagen) and the frozen ones were processed using standard procedures. To assess the somatic status of the mutations, we extracted DNA from manually isolated peritumoral tissue fragments (only available for 8 samples). We also analyzed the COV434 cell line, supposed to derive from a JGCT .G\u03b1s gene, potentially harboring the activating mutations R201C and R201H, was performed as described , supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin .2.4AKT1RED\u2013EcoR1-F: 5\u2032AGCTTCGAATTCGCCACCATGAGCGACGTGGCTATTGTGAAGG3\u2032AKT1-F2: GTGGACCACTGTCATCG3\u2032AKT1RED\u2013BamH1-R: 5\u2032ACCGGTGGATCCCG GGCCGTGCCGCTGGCCGAGTAGGAGAAC3\u2032InsertionT3R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCAGCGGATGATGAAGG3\u2032InsertionT8R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCAGGCAGCGGATGATGAAGG3\u2032InsertionT5R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCGCAGCGGATGATGAAGGTGTTGG3\u2032InsertT11R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCAGGCAGCGGATGATGAAGG3\u2032InsertT12R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCGTCTTCATCAGCTGGCGGATGATGAAGGTGTTGGG3\u2032InsertT14R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCTGCAGGCAGCGGATGATGAAGG3\u2032InsertT15R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGATGATGAAGGTGTTGG3\u2032InsertT16R: 5\u2032CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTGGCAGCGGATGATGAAGG3\u2032E17K F: 5\u2032GCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGAAGTACATCAAGACCTGG3\u2032Q79K-F: 5\u2032CATCATCCGCTGCCTGAAGTGGACCACTGTCATCG3\u2032Q79K-R: 5\u2032CGATGACAGTGGTCCACTTCAGGCAGCGGATGATG3\u2032W80R-F: 5\u2032CATCATCCGCTGCCTGCAGAGGACCACTGTCATCG3\u2032W80R-R: 5\u2032CGATGACAGTGGTCCTCTGCAGGCAGCGGATGATG3\u2032Q79K\u2013W80R-F: 5\u2032CATCATCCGCTGCCTGAAGAGGACCACTGTCATCG3\u2032Q79K\u2013W80R-R: 5\u2032CGATGACAGTGGTCCTCTTCAGGCAGCGGATGATG3\u2032.The plasmids driving the expression of wild-type and mutated AKT1 fused to the mCherry protein were constructed by fusion PCR. Briefly, for the insertion mutations, two PCRs were performed to generate the 5\u2032 and 3\u2032 portions of the AKT1 coding sequence using, respectively, AKT1RED\u2013EcoR1-F primer and the corresponding mutagenic R primer and AKT1-F2 primer along with AKT1RED\u2013BamH1-R. After purification of the PCR products, they were quantified, mixed in similar amounts and allowed to undergo eight cycles of PCR in the absence of primers, to generate the full-length mutated coding regions. Then, a final PCR reaction was performed using the EcoR1\u2013BamHI primers. For E17K, we used the primers E17K F and AKT1RED\u2013BamH1-R to generate the amplicon in a single PCR. The amplified EcoR1\u2013BamHIs were cloned (EcoR1\u2013BamHI) into digested pDsRed vector to produce fusion proteins in frame with the mCherry. All constructs were sequenced to exclude the presence of PCR-induced mutations. The sequences of the primers used are the following:2.5HeLa cells were transfected with constructs driving the expression of AKT1 fused to the mCherry protein. Cells were seeded, in 24-well plates containing sterile coverslips, 16\u00a0h before transfection to be confluent at the time of transfection. Cells were transfected using the calcium-phosphate method with 250\u00a0ng of plasmid per well and rinsed 24\u00a0h after transfection. At this point, cells were serum-starved or not for 24\u00a0h. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline solution (PBS) and fixed for 15\u00a0min with paraformaldehyde (PFA 4%). They were washed three times in PBS and nuclei were stained with Hoechst 33342 . Coverslips were mounted on microscope slides using the fluorescence mounting medium DakoCytomaton . Cells were visualized with an epifluorescence microscope provided with an ApoTome module.For Western-blot studies, HeLa cells were transfected with the constructs driving the expression of wild-type or mutated AKT1 forms. One day after transfection cells were rinsed and serum-starved or not for 24\u00a0h before lysis. Electrophoresis and Western-blot were performed as previously described . The priDual-Luciferase Reporter Assays  involved the reporter promoter 4XDBE-luc which contains 4 copies of the FOXO response element (DAF-16 family member-binding element or DBE) upstream of a minimal promoter driving the expression of the firefly luciferase gene . Each ex33.1G\u03b1s altering protein position 201  samples, the allelic combinations (i.e. haplotypes) of the other co-occurring mutations could not be worked out, thus preventing their experimental exploration. In total, 14 out of 16 JGCTs harbor putative driver alterations of AKT1.Given the high degree of identity between and AKT2 . These m3.21H10, 3O96 and 4EJN3O964EJN). The tandem duplications described here involve the 6th beta strand, according to the ribbon model displayed in The tandem duplications described here alter the PHD of AKT1 . The PHDAKT1 in our experimental setting. The pervasive presence at the plasmalemma of the elongated AKT1 proteins suggested the existence of a high level of activation. To test this hypothesis, we analyzed the phosphorylation status of seven AKT1 variants carrying the in-frame duplications, which is supposed to reflect protein activation  or by the presence of a myristoylation signal, artificially introduced or provided by the viral Gag sequence fused to Akt in v-Akt . StructuAlthough JGCTs are of good prognosis, it would be interesting to study the properties of the filopodia-like processes induced by the mutated AKT1 carrying the duplications because of the known roles of filopodia/invadopodia in sensing, migration and intercellular interactions . These pAKT1 mutations and the involvement of the PI3K\u2013AKT pathway in regulating cellular proliferation and survival suggests that the molecular lesions reported here are driver events. Irrespective of the precise role of AKT1 in the origin and/or progression of the JGCTs, which is yet to be studied, the uncovering of the AKT1 insertions will facilitate the molecular diagnostics of JGCTs. Our findings open also targeted therapeutic perspectives because inhibitors of the PI3K\u2013AKT\u2013mTOR pathway(s) are being tested in clinical trials  partners. Finally, we cannot exclude that granulosa cells may need a strong level of AKT1 activation to become transformed, which would only be achieved with the observed duplications or with several AKT1 mutations per cell, such as Q79K and W80R. The fact that most of the JGCTs harbor l trials .GEFLUC (Groupement des Entreprises Fran\u00e7aises dans la Lutte contre le Cancer), the Centre National de la Recherche Scientifique (CNRS), the Universit\u00e9 Paris Diderot and the Agence Nationale pour la Recherche (Projet ICEBERG).This work was supported by LB, ALT, AA and RAV conceived and designed the study. LB, ALT, AA, LG, BL, DF and RAV developed the methodology. LB, ALT, AA, LG, BL and RAV acquired data. LB, ALT, AA, SS, DF, CS, NK, LG and RAV analyzed and/or interpreted data. All authors wrote and provided final approval of the manuscript.The authors declare no competing interests.Tumoroth\u00e8que Necker-Enfants Malades and Tumoroth\u00e8que Montpellier.This project was approved by the ethics committees of the tumor repositories The funding sources had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. The corresponding author had full access to all data and had the final responsibility for the decision to submit for publication."}
+{"text": "AbstractHeteranassa Smith , native to the southwestern United States and Mexico, includes two recognized species, namely Heteranassamima (Harvey) and Heteranassafraterna Smith. These are separated mainly by subtle differences in wing color and pattern, leading to speculation about the validity of the described species. This study examines variation in external and internal morphology across the geographic range of the genus, aiming to clarify species limits, describe morphology, and provide a comprehensive assessment of variation within the genus. Results indicate that Heteranassafraternasyn. n., is a junior synonym of Heteranassamima. Heteranassa Smith, 1899, is a genus of moths native to warmer desert regions of southwestern United States southward to southern Mexico, currently containing two valid species. Heteranassamima , described from Texas, and Heteranassafraterna Smith, 1899, described from Death Valley, California. PageBreakan additional species, Heteranassaminor Smith, 1899, with Heteranassafraterna. Heteranassa feed on mesquite (Prosopis sp.) and Acacia (Acacia sp.) (both Fabaceae) and are multivoltine , White Sands National Monument, Otero Co., New Mexico, , Cuatrocienagas Protected Area, Cuatroci\u00e9nagas, Coahuila, Mexico, , Pima Co., Arizona, (July 2012), and Socorro Co., New Mexico (October 2012). Specimens were collected with a sheet trap using 15W UV fluorescent lamp, 175W Mercury Vapor lamp, or a 175W, 6500K metal halide lamp. Death Valley specimens were collected at incandescent or fluorescent outdoor lighting at the Furnace Creek Ranch Hotel.Specimen loans were generously provided by the following institutions: University of Alberta Strickland Entomology Museum, Edmonton, Alberta (F.A.H. Sperling)UASM Essig Entomology Museum, University of California, Berkeley (J. Powell)EMEC Natural History Museum of Los Angeles County, Los Angeles, California (B. Brown)LACM University of Arizona Insect Collection, Tucson (W. Moore)UAIC Arizona State University Entomology Collection, Tempe (T. Dowling)ASUT Cornell University Insect Collection, Ithaca, New York (J. Liebherr)CUIC Kansas State Entomological Museum, Manhattan (G. Zolnerowich)KSUCUnited States National Museum  and the Lepidoptera and Biodiversity McGuire Center for . A complete list of specimens examined is included in Suppl. material Heteranassa. A list of dissected specimens is included in Suppl. material Specimens were also examined during visits to the PageBreakwatch glass with distilled water, and scales were removed with a fine brush. Once clear of scales, the integument of the abdomen was cut along the left pleural membrane, and the genital capsule removed. On male specimens setae were carefully removed from the membranous costal region of valves with a fine camel\u2019s hair brush. The aedeagus was separated from the valves by grasping the distal end with fine\u2013tipped forceps, and gently pulling to separate from the juxta. The ductus seminalis was then cut where it enters the side of the proximal part of the aedeagus. The vesica was then carefully teased out of the aedeagus with a #20 minuten with the tip bent to a right angle, held in a standard pin vice, and with water pressure from a syringe. A syringe with a modified 30 gauge needle . Line drawings were made in Adobe Illustrator with a Wacom Intuos 4 drawing tablet from photographs or sketches made with a drawing tube attached to a Wild M5 stereomicroscope.Photographs of dissected specimens, genitalia, and adults were made using a Visionary Digital imaging system (Prosopisglandulosa Torr.) foliage and bark. Larvae were reared on Prosopisglandulosa foliage.Eggs were obtained from gravid females collected in Box Canyon, Pima Co., AZ (18 July 2012). The females were placed in brown paper bags with Honey Mesquite , Toxonprucha species and Coxina species are frequently misidentified as Heteranassa. Of these, Acritogrammametaleuca is the most similar to Heteranassa Homopteramima Harvey, 1876: 155\u2013156.Eubolinamima ; Campometramima ; Elousamima ; Draudt and Gaede (in Seitz) 1923: 478.Heteranassamima ; Campometrafraternasyn. n.;  Smith, 1899: 104, Heteranassafraterna ; Elousafraterna ; Draudt and Gaede (in Seitz) 1923: 478.Campometraminor Smith, 1899: 104\u2013105; Elousaminor : Draudt & Gaede (in Seitz) 1923: 478.Heteranassaminorsyn. n.;  , PageBreakThis is the only species in the genus and can be diagnosed with the generic combination (see above).Heteranassamima . Holotype,  labeled: \u201cHomoptera mima, type, Harvey, Holotype, 15/9, 73.\u201d The specimen and associated labels were examined through high-resolution photographs provided by the BMNH. Type locality: Texas [USA]pe, Fig.  \u2640 in theHeteranassafraterna . Lectotype . Lectotype,  barcodes from California, Arizona, New Mexico, Texas, and northern Mexico showed a maximum divergence of less than 0.8%. One haplotype* dominated the sample, representing more than half of the specimens; the other barcodes included 36 haplotypes that had no more than two base-pair differences from each other. One haplotype, restricted to central and southern Texas, departed from this pattern in being 0.8% different from those from farther west. This is most probably the haplotype that should be associated with the name Heteranassamima, it being described from this part of Texas. However, this \u201ceastern\u201d haplotype is found with \u201cwestern\u201d haplotypes in central Texas and there is no indication in genital structural characters, or wing color or pattern, that Heteranassa includes more than a single species. The barcodes of Heteranassa are so divergent that they give no indication of a close relationship to any other erebid genus, other than belonging in the subfamily Erebinae, tribe Omopterini. Heteranassa specimens from Texas and Mexico are frequently confused with some species associated with the genus Coxina Guen\u00e9e, which can have a similar superficial pattern, but the barcodes are more than 10% different and the two genera do not appear to be closely related. (D. Lafontaine pers. comm.).Barcode variation in *CNCNoctuoidea13382 PageBreakTATTATAATTTTCTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAGTCCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTCTGATTATTACCCCCATCTTTAACTCTTTTAATCTCAAGAAGAATCGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAACATTGCTCATAGAGGAAGATCAGTAGATTTAGCAATTTTCTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACTACTATTATCAATATACGATTAAATAGATTAATATTTGACCAAATACCTTTATTTGTTTGAGCTGTTGGTATTACTGCTTTTTTACTATTATTATCTTTACCTGTTTTAGCTGGAGCTATTACTATACTCTTAACAGATCGAAATTTAAATACTTCCTTTTTTGATCCTGCTGGAGGAGGAGATCCTATTCTTTACCAACATCTATTTAACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTTTATTAATTCGTGCTGAATTAGGAAACCCTGGTTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTLACM.Warm, arid habitats from California to Texas, northward to Oklahoma, and south as far as Oaxaca, Mexico Fig. . A singlHeteranassa wing pattern and coloration is continuous, with many specimens appearing intermediate to the phenotypes described by Heteranassa contains a single, highly variable species, Heteranassamima. Studies of another erebine genus, Catocala, have shown that pressure from avian predators may drive high levels of polymorphism in forewing pattern and coloration  than they are in Elousaschausi  and the other Coxina species we have dissected. We have examined 10 species in these genera from the Caribbean and South America. Specimens belonging to this group that we collected in the Nicaraguan highlands appear more similar to Elousaschausi specimens from Argentina and Coxina specimens from Mexico, south Texas, and Florida, than they do to Caribbean Elousa or North American Heteranassa. Future research could test the monophyly of Coxina and Elousa with respect to Heteranassa, and how these genera speciated in North and South America and the Caribbean.During the course of this research, we became aware of potential taxonomic affinities with the neotropical genera PageBreak"}
+{"text": "Staphylococcus aureus and vancomycin-resistant Enterococcus, there is not much information describing its activity against gram-negative bacteria. In this study, we report the effect of methylglyoxal against multidrug-resistant Pseudomonas aeruginosa (MDRP) using 53 clinically isolated strains. We also assessed the effect of deleting the five multidrug efflux systems in P. aeruginosa, as well as the efflux systems in Escherichia coli and Salmonella enterica serovar Typhimurium, on MICs of methylglyoxal. Our results indicate that methylglyoxal inhibits the growth of MDRP at concentrations of 128\u2013512 \u03bcg/ml (1.7\u20137.1 mM) and is not recognized by drug efflux systems.Honey has a complex chemistry, and its broad-spectrum antimicrobial activity varies with floral source, climate, and harvesting conditions. Methylglyoxal was identified as the dominant antibacterial component of manuka honey. Although it has been known that methylglyoxal has antibacterial activity against gram-positive bacteria, including methicillin-resistant  Pseudomonas aeruginosa is endemic among critically ill patients, and multidrug-resistant strains are increasingly being isolated in intensive care units  has been defined as P. aeruginosa resistant to imipenem, amikacin, and ciprofloxacin  foraging on a shrub known as manuka (Leptospermum scoparium) that is indigenous to New Zealand. Manuka honey is broad in spectrum, able to inhibit a diverse range of bacterial and yeast pathogens, and equally effective against multidrug-resistant bacteria  from wounds  reported to be <3% (v/v)    and NKS233 (\u0394tolC) were constructed as described previously  and NKE95 (\u0394tolC), we performed gene disruption using procedures described previously ; acrB-P2 (GAACAGTCCAAGTCTTAACTTAAACAGGAGCCGTTAAGACCATATGAATATCCTCCTTAG); acrD-P1 (TGAAAAAGGCGACACATTGGCATGTCGCCTTTTTTATTGCGTGTAGGCTGGAGCTGCTTC); acrD-P2 (AAGCCTACAACGATACGCAGAAACACGAGGTCCTCTTTTACATATGAATATCCTCCTTAG); mdtA-P1 (ATCATTCCGCGAAACGTTTCAGGAAGAGAAACTCTTAACGGTGTAGGCTGGAGCTGCTTC); mdtC-P2 (GAGATACACCACCGGCGTGGTATACAGCGTAAGGAGCTGGCATATGAATATCCTCCTTAG); mdtE-P1 (TTAAAGAACCGTTATTTCTCAAGAATTTTCAGGGACTAAAGTGTAGGCTGGAGCTGCTTC); mdtF-P2 (AGGCTGAACCTTCATGTTCAACCTTACTCTCATTTACACGCATATGAATATCCTCCTTAG); acrE-P1 (TTGGGTAAATAACGCGCTTTTGGTTTTTTGAGGAATAGTAGTGTAGGCTGGAGCTGCTTC); acrF-P2 (AAATAATAAAGGCACCCGAAAGCGCCTTTATGTTTCTGATCATATGAATATCCTCCTTAG); tolC-P1 (ACTGGTGCCGGGCTATCAGGCGCATAACCATCAGCAATAGGTGTAGGCTGGAGCTGCTTC); and tolC-P2 (TTACAGTTTGATCGCGCTAAATACTGCTTCACCACAAGGACATATGAATATCCTCCTTAG). The chloramphenicol resistance gene cat or the kanamycin resistance gene aph, flanked by Flp recognition sites, was amplified by PCR using the primers listed above. The resulting PCR products were used to transform E. coli MG1655, which harbors plasmid pKD46, expressing Red recombinase. The chromosomal structures of the mutated loci were verified by PCR; cat and aph were further eliminated using the plasmid pCP20, as described previously . The inoculated agar plates were examined after incubation at 37\u00b0C for 16 h. MIC was the lowest concentration of a compound that inhibited cell growth.Antibacterial activities were determined on Muller Hinton II agar  plates containing methylglyoxal (32\u20132048 \u03bcg/ml), imipenem (0.0625\u20132048 \u03bcg/ml), amikacin (0.125\u20134096 \u03bcg/ml), or ciprofloxacin (0.0078\u20132048 \u03bcg/ml) . Agar plates were prepared using the two-fold agar dilution technique. Bacteria were grown at 37\u00b0C overnight and then tested at a final inoculum volume of 1 \u00d7 10E. coli  and S. enterica  strains were grown in Luria\u2013Bertani broth , and P. aeruginosa (PAO1 and PMX52) strains were grown in Muller Hinton II (MHII) broth . Bacterial cells were cultured overnight at 37\u00b0C, and then 100 \u03bcl of cell cultures were diluted in 5 ml of the same medium. The diluted bacterial cells were incubated at 37\u00b0C until OD600 reached 0.5. Then, the bacterial cells were diluted in the same medium to an OD600 of 0.05. This diluted bacterial cells were incubated in NUNC Edge 96-well plates  with shaking at 37\u00b0C for 7 h. Bacterial growth was monitored using an Infinite M200 Pro plate reader .P. aeruginosa resistant to imipenem , amikacin (\u226532 \u03bcg/ml), and ciprofloxacin (\u22654 \u03bcg/ml) . Although PMX52 was more susceptible to amikacin and ciprofloxacin than PAO1, MIC of methylglyoxal for these strains was the same. This suggests that methylglyoxal is not recognized by drug efflux systems in P. aeruginosa. To confirm whether same phenomenon could be observed in other gram-negative bacteria, we determined MICs of methylglyoxal for the efflux-deficient mutants of E. coli and S. enterica serovar Typhimurium. There are five RND-type drug efflux systems  in E. coli, and all of them require the TolC outer membrane channel for their function , NKE1329 (\u0394acrB acrD mdtABC mdtEF acrEF), and NKE95 (\u0394tolC) strains. The susceptibility of NKE1329 and NKE95 to methylglyoxal was same as that of the wild-type strain, although they were more susceptible to ciprofloxacin than the wild-type strain. S. enterica serovar Typhimurium harbors at least nine drug efflux systems belonging to RND, multidrug and toxic compound extrusion, and ATP-binding cassette (ABC) superfamilies , NKS196 (\u0394acrAB acrEF acrD mdtABC mdsABC emrAB mdfA mdtK macAB), and NKS233 (\u0394tolC) strains. Although NKS196 and NKS233 were more sensitive to ciprofloxacin than the wild-type strain ATCC14028s, MICs of methylglyoxal for ATCC14028s, NKS196, and NKS233 were the same. In addition to MIC determination using agar plates, we tested the effect of methylglyoxal on bacterial growth in liquid medium. The growth of E. coli  and Salmonella  strains was inhibited by methylglyoxal at a concentration of 256 \u03bcg/ml, and the growth of P. aeruginosa (PAO1 and PMX52) strains was inhibited at 512 \u03bcg/ml, which is consistent with MICs determined . Methylglyoxal is a key antimicrobial component of manuka honey, and manuka honey has previously been suggested as a topical treatment option for burn patients infected with P. aeruginosa  (Mavric et al., P. aeruginosa, E. coli, and S. enterica.In this study, we showed that methylglyoxal equally inhibits drug-susceptible The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The correct sentence is: Then an exchange of ADP with ATP in ATP domains of MutS takes place  shown in the \u201cSynthetic DNA Fragments\u201d subsection of the Materials and Methods. Please see the correct structures here.GGGTGCC I (G/C)CAAGCCTATGCCCTCAGCACCCACCCACGGGTTCGGATACGGGAGTCGTGGGTGGGTGCC II (G/T)CAAGCCTATGCCCTCAGCACCCATCCACGGGTTCGGATACGGGAGTCGTGGGTG-GGTGCC III (G/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGTA-GGTGCC IV (A/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGT"}
+{"text": "Correction: Genome Biol 19, 205 (2018)https://doi.org/10.1186/s13059-018-1581-3Following the publication of the original paper , the autATCTTGTGGAAAGGACGAAACAThe sequence for Primer 1: SAMlibrary-HiSeq_50bp-F1 has two extra bases inserted (in bold) ACACTCTTTCCCTACACGACGCTCTTCCGATCTThe correct sequence should be:ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA"}
+{"text": "Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-\u03b1, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-\u03b1 and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-\u03b1-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-\u03b1 expression increased the expression T-cell markers. Both TNF-\u03b1 and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-\u03b1, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology. Accordingly, anti-VEGF treatment has emerged as standard-of-care for wet AMD5 and DME6. Another common hallmark of many retinopathies is inflammation: proinflammatory cytokines such as TNF-\u03b1 as well as IL-6 proteins are upregulated in the vitreous of DR8 and uveitis patients10.Retinopathies such as diabetic retinopathy (DR), age-related macular degeneration (AMD), uveitis or retinopathy of prematurity (ROP) display complex pathologies in patients including vasculopathies, inflammation, neurodegeneration, and fibrosis, ultimately leading to blindness. A number of molecular changes have been associated with these retinal pathologies, for example changes in expression of Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor alpha (TNF-\u03b1), or Interleukin-6 (IL-6). VEGF causes vascular leakage and pathological neovascularization, and VEGF protein has been shown to be upregulated in wet AMD12. Transgenic mice expressing VEGF15, intraocular injection of recombinant VEGF protein18 or recombinant adeno-associated viruses (AAVs) expressing VEGF23 further demonstrated that VEGF is not only necessary but also sufficient to cause vasculopathies. Accordingly, anti-VEGF treatment prevents neovascularization in the OIR model24 and these preclinical studies paved the way for modern anti-VEGF therapies. Furthermore, inflammatory processes observed in patients of retinopathies can also be modeled in rodents, for example by uveitis mouse models such as endotoxin- or antigen-induced uveitis27 or transgenic mice lacking the Aire gene29. Similarly, recombinant proteins or AAV-mediated expression of TNF-\u03b1 and IL-6 induces inflammation in the rodent eye, although the direct function of IL-6 is more controverse32. Again, anti-TNF-\u03b1 or anti-IL-6 treatment improves the induced pathologies in diverse uveitis models35.Various pre-clinical rodent models have directly or indirectly shed light on the function of VEGF, TNF-\u03b1, or IL-6 in retinopathies. One frequently used animal model displaying vascular pathologies, similar to those observed in proliferative retinopathies such as wet AMD or ROP, is the oxygen induced retinopathy (OIR) model19. We further demonstrated that AAV-mediated expression of human VEGF, TNF-\u03b1, and IL-6 in the murine eye leads to pathway-specific, human-relevant retinal pathologies. In brief, AAV-driven expression of VEGF induced vascular leakage and neovascularization. On the other hand, pro-inflammatory cytokines TNF-\u03b1 and IL-6 both activated immune cells. TNF-\u03b1 additionally led to vasculitis, fibrosis, and development of fibrotic epiretinal membranes.In the context of pre-clinical research, AAV mediated gene transfer has recently emerged as a powerful method to create novel animal models. As a main benefit, AAVs allow for long-term expression of a transgene in a tissue- and cell-type-specific manner. Previously, we have shown that AAV-mediated expression leads to constant and long-term expression of human transgenes at 1, 3 and 6\u00a0weeks after IVT-injection38. Similarly, the transcriptome of multiple rodent models of retinopathies has been sequenced and analyzed42. For the OIR model, several studies have identified time point-dependent gene expression changes related to hypoxia, angiogenesis, and inflammation47. However, comparisons with other mouse models of retinopathies are still missing. Additionally, modern single cell RNA-sequencing (scRNA-Seq) approaches gave unprecedented insights into the cellular organization of healthy and diseased mammalian ocular tissues54. In more detail, Heng et al. demonstrated by single cell sequencing of Aire-/- mice, a spontaneous uveoretinitis model, that a very diverse population of immune cells invades the retina of Aire-/- mice and that Th1 cells represent the main effector T cells in this model, highlighting the great potential of such single cell sequencing approaches. As the number of available bulk and single-cell datasets in the context of retinopathies increases, so does the potential to integrate data from various retinopathy models and compare them on a molecular level.In the recent years, next generation sequencing techniques allowed for a detailed investigation of the molecular changes in retinopathies and brought about a better understanding of disease progression in AMD and DR patientsIn this study, we provide transcriptomic analysis of intravitreally injected recombinant AAVs expressing human VEGF, TNF-\u03b1, or IL-6 in mice. We observed that AAV-mediated expression of human transgenes introduced distinct transcriptomic responses: While TNF-\u03b1 and IL6 displayed overlapping gene expression changes, VEGF overexpression led to a more distinct response compared to TNF-\u03b1 and IL-6. In more detail, investigating pathways affected by each experimental condition, VEGF led to a specific regulation of extracellular matrix (ECM)-related genes, while TNF-\u03b1 and IL-6-induced changes in interleukin signaling. We further identified specific gene expression changes associated with TNF-\u03b1 that included cellular adhesion molecules, such as Madcam1 and we further demonstrated a conserved regulation of MAdCAM-1 by TNF-\u03b1 in human retinal endothelial cells. By integration of single cell RNA-Sequencing data, we could show an increase of T-cell-specific genes following TNF-\u03b1 expression and, indeed, TNF-\u03b1 mediated T-cell invasion was validated by immunofluorescence. In addition to assessing transcriptome changes in the various transgenic mice models, we generated a detailed time-course of transcriptome changes in the established OIR model. Comparing transcriptome changes in AAV overexpression models and OIR mice, we observed the largest overlap between AAV-VEGF treated eyes and the late response (P16) in the OIR model, most prominently including changes in the ECM pathway and endothelial cell specific marker genes. Prominently, OIR-specific changes in gene regulation included neuronal signaling pathways which were not observed after AAV-VEGF transgene expression. In conclusion, our study suggests that each animal model produces distinct gene expression profiles and a careful selection of models according to the requirements of different research questions is necessary.8\u00a0VG/eye), while AAV-TNF-\u03b1 and AAV-IL-6 were injected at 1\u2009\u00d7\u2009109\u00a0VG/eye. Eyes of all animals were imaged in vivo directly before tissue dissection and RNA sequencing to validate expected pathologies and grade phenotype severity. In line with our previously published study19, AAV-stuffer injections did not induce obvious retinal pathologies at both concentrations based on Blue Autofluorescence (BAF) imaging . Since no mRNA staining was observed in AAV-stuffer control treated eyes , choroid, sclera and the ciliary body. Apart from two samples that were excluded due to a low RNA integrity number (RIN), six biological replicates per group were sequenced. Sequencing libraries were sequenced at an average depth of 31.3 million reads, with 80.1% mapping to mRNA transcripts and low ribosomal content (2.1%), suggesting overall good quality of the RNA sequencing data. As expected, human transgenes showed strong expression in their respective treatment groups  indicated the largest variance in gene expression between retina and eye cup samples Fig.\u00a0d. The reVcam1 and Madcam1 and chemokines such as Ccl2 were among the strongest deregulated genes, supporting the well-known function of TNF-\u03b1 in regulating immune cell adhesion to endothelial cells56. To confirm the RNA sequencing results, Ccl2 expression was validated by qRT-PCR, which demonstrated similar expression patterns with the highest expression of Ccl2 found in AAV-TNF-\u03b1 treated eyes  genes in the three treatment groups AAV-VEGF, TNF-\u03b1, and AAV-IL-6 in comparison to their respective AAV-stuffer control, as well as AAV-stuffer compared to non-injected controls  by AAV-VEGF, AAV-TNF-\u03b1 or AAV-IL-6 treatment. Only 1 pathway was enriched in the retina of all three treatment groups Fig.\u00a0a, namelySince only very few genes were significantly differentially regulated in AAV-VEGF transduced eye cup tissue, only AAV-TNF-\u03b1 and AAV-IL-6 treatment was compared in the eye cup tissue  of endothelial and perivascular cell-specific marker genes after AAV-VEGF treatment was observed  were mostly positive , P13, P14, P15 and P16. PCA demonstrated a clear separation between the OIR model and controls  model, presenting both neovascularization and neurodegeneration. Neonatal mice are exposed to 75% oxygen from postnatal day 7 to 12, and thereafter return to room air (21% oxygen) until day 17. At day 7, the retinal vasculature of mouse pups is still immature and vulnerable to high oxygen conditions. Loss of capillaries in the center of the retina leads to the formation of an avascular area. Upon return to room air at day 12 the area becomes hypoxic and Hif1\u03b1 dependent VEGF expression has been shown to trigger angiogenesis, peaking at P17ols Fig.\u00a0. At late54 demonstrated that, among others, also vascular cell-specific marker genes were enriched in the OIR data set  in OIR retina compared to those identified in the AAV-VEGF model Fig.\u00a0c,d. The Finally, we aimed to understand which pathways were affected in the AAV-VEGF driven retinopathy models compared to the OIR model  into mouse eyes with a biologically inactive sequence, produced very few gene expression changes. AAV-mediated expression of hTNF-\u03b1, hIL-6, or hVEGF, led to high expression of these human transgenes, which in turn produced strong and distinct transcriptome changes. Although measurement errors in RNA-Seq caused by the simultaneous presence of human and murine versions of the AAV-expressed transgene are theoretically possible, we expect no large mis-quantification, since human transgenes are codon optimized and possess a V5 tag, hence they are sufficiently dissimilar to their endogenous transcript sequence. In addition, we did not observe any reduction in the percentage of uniquely mapped reads in the presence of AAV expressed transgenes (data not shown).AAVs have emerged as a useful tool to quickly generate novel animal models that allow a high degree of flexibility regarding timing and tissue-specific expression. In our study, we chose the ShH10 capsid for expression of the three human transgenes. Even on a global scale, measured via a high-throughput method such as RNA-Seq, injection of the control capsid  secondary to AMD72. In the past, transgenic animals modifying the expression of complement genes, or mice expressing human variants of complement-related genes, have proven extremely useful to understand their contribution to disease progression78. To further develop novel and effective therapies, animal models with robust activation of the complement pathway are needed. In LPS-induced uveitis model, complement-related genes including C3 are upregulated39, suggesting complement activation. Similarly, experimental autoimmune uveitis (EAU) leads to activation of the complement system and blockage of the complement system ameliorates disease pathology79. Activation of the complement system is also visible in the laser-induced choroidal neovascularization (CNV) model, and inhibition of various complement factors had beneficial effects82. However, both the experimental uveitis models and laser CNV model are transient and only allow for short-term investigations. In contrast, in the presented AAV-TNF-\u03b1 model, C3 showed strong and sustained upregulation at 3 and 6\u00a0weeks after IVT injection of AAV-TNF-\u03b1, thus, allowing long-term studies of the complement system. In addition, as a proof-of-mechanism we showed that treatment with the neutralizing TNF-\u03b1 antibody golimumab reduces C3 expression, demonstrating that complement activation is reversible in our model. In summary, the AAV-TNF-\u03b1 induced mouse model provides a valuable tool to study the role of the complement system in disease progression, featuring long-term TNF-\u03b1 and C3 expression, which is comparable to the human pathology that develop over decades.In our analysis, significantly changing genes present in the complement cascade were strongly and specifically upregulated in eyes injected with AAV-TNF-\u03b1. Among them, the complement pathway factor C3 was one of the strongest upregulated genes in AAV-TNF-\u03b1 injected eyes. C3 plays a central role in the activation of the classical and alternative complement pathway and mutations in C3 and other members of the complement cascade correlate with higher risk of AMDIcam1, Vcam1 and Madcam1 were highly upregulated in AAV-TNF-\u03b1 injected eyes, strengthening the previous observation of immune cell infiltration and vasculitis in AAV-TNF-\u03b1 injected eyes by histology19. MAdCAM-1 is induced by TNF-\u03b1 in diverse endothelial cells and recruits T-cells to inflamed tissues85 and, therefore, has recently been identified as an interesting target to treat inflammatory bowel diseases86. In addition to Madcam1 upregulation, we observed T-cell infiltration in our AAV-TNF-\u03b1 driven uveitis-like model, in line with well-described role for T-cells in uveitis patients and uveitis rodent models88. Although there is general interest in the role of TNF-\u03b1 and CAMs in the context of retinopathies, to our knowledge there exists only one recent study by Peng et al. demonstrating a direct function for murine Madcam1 in retinal degeneration83. In line with Peng et al., we demonstrated upregulation of Madcam1 in AAV-TNF-\u03b1 injected eyes correlating with an invasion of T-cells. Expanding on this observation, we demonstrated that MAdCAM-1 is also upregulated in TNF-\u03b1 stimulated HRMECs, suggesting that the TNF-\u03b1 induced regulation of MAdCAM-1 may also be relevant in human retinal cells.In addition to C3, cellular adhesion molecules (CAMs), such as 12. To capture transient expression dynamics present in the OIR model, we collected mouse eyes at 5 subsequent time points after hyperoxic treatment. Indeed, other authors have performed analysis of OIR gene expression, however, many studies relied on microarray data only assessing part of the transcriptome in contrast to the whole transcriptome analysis presented here. On the other hand, OIR RNA-Seq studies included less dense sampling of timepoints compared to our study, in which we provide more detailed insights into time-dependent gene expression changes. The study of Yang et al.46 focused on gene expression changes during and shortly after hyperoxic treatment, while our study was mostly interested in gene expression changes after the return to normoxic conditions and a detailed comparison to AAV induced gene expression changes. Nevertheless, we observed shared gene expression signatures of key genes between the two studies, for example VEGF and Edn2 gene were both upregulated at P13.In order to better understand the differences between various retinopathy disease models, we compared gene expression profiles of the generated AAV-driven retinopathy models to RNASeq data of the frequently used OIR model24. Thus, as expected, DE genes in the AAV-VEGF model showed the largest overlap with those identified in the OIR model. By integration of single-cell RNA-Seq expression data, we showed that within the time-course measured for the OIR model, endothelial cell-specific genes were first down-regulated and then up-regulated starting from P15. Also other studies have found downregulation of angiogenesis-related genes such as CD34 in the OIR model at P12, while endothelial cell-specific genes such as Esm1 or Edn2 were upregulated at P1789. Esm1 is a well-known gene induced by VEGF in tip cells with a crucial role in angiogenesis and its blockage inhibits neovascularization of diverse rodent models of neovascularization including the OIR, laser CNV and the rho/VEGF transgenic mouse91. Accordingly, we also observed upregulation of the Esm1 gene in both the OIR dataset but also the AAV-VEGF driven model, resembling very well the observed neovascularization phenotype. Since we provide a high time-resolution of gene expression changes in the OIR model, we were able to pinpoint the switch between down- and up-regulation of endothelial cell-specific genes to P14. These gene expression changes fit very well with the phenotypic changes present in the OIR model, where at P12 the development of the vasculature is impaired and avascular areas are present, and later on, neovascularization peaks at around P1793.It is well known that VEGF is a major driver of the OIR model and anti-VEGF treatment prevents neovascularization in the OIR model95. Furthermore, neuronal death and decreased retinal function has been observed in the OIR model97, probably due to ischemia and malnutrition induced by hypoxia. Accordingly, the OIR model has been discussed as the more appropriate model for retinopathy of prematurity (ROP)93, a disease affecting human preterm infants. While very young mouse pups are needed for the OIR model, old or diabetic mice may be combined with AAV overexpression of human transgenes, to mirror more closely age-related or diabetes-associated human retinopathies such as AMD or DR in the future.Although many pathways including ECM-related pathways and endothelial cell-specific responses were similar between the OIR model and AAV-VEGF treated mice, we also observed differences between these disease models: For example, in the OIR model, but not AAV-VEGF, neuronal signaling pathways and synapse-related genes were significantly enriched for DE genes. In line with this observation, we observed a significant overlap of neuronal cell-specific genes  and DE genes identified in the OIR model. For the OIR model pups at age P12 to P17 are used, a timepoint which corresponds to a time-point where vascular and neuronal networks are still developing in the murine retinaDespite our efforts to ensure optimal experimental design, certain limitations apply, most prominently that AAV and OIR sequencing data sets were generated in independent experiments. Furthermore, also from a biological perspective, the models differ, since the OIR model uses very young mouse pups during development, in contrast to our AAV-induced models that were developed using adult mice. While in the OIR study 3 or 4 samples where included at each time-point, the AAV study was performed with 5 or 6 replicates per group. In addition, library preparation and sequencing was dependent on different protocols. To alleviate these limitations, our analysis is based on relative fold changes between treatment and respective controls, rather than on absolute expression values, thereby accounting for differences in library preparation.Altogether, here we characterized the gene expression profiles of AAV-VEGF, TNF-\u03b1, or IL-6 driven retinopathy mouse models, and combined these measurements with publicly available singe-cell RNA expression data. Our study gives unique insights into the molecular and cell-specific changes leading to retinal pathologies, hereby uncovering novel potential treatment options for retinal diseases, and at the same time offering the means to test them in a stable disease model comparable to the human pathology. By further measuring gene expression changes in the well-established OIR model, we compared new and established animal models for retinopathies and provide researchers with important information guiding the decision of which animal model best suits the need of a given pre-clinical research project.C57BL/6\u00a0J mice were purchased from Charles River . 6\u20138-week-old male mice were used for the AAV study. Pregnant females were purchased for the OIR study and male and female pups at P12 were used for the OIR experiments. Mice were housed in individually ventilated cages in groups of 2\u20135, constant temperature and a 12-h light/dark cycle. Mice had ad libitum access to standard rodent chow and water. Animal experiments were performed in accordance with the German animal welfare act, the guidelines of the Federation of the European Laboratory Animal Science Association (FELASA), the ARRIVE guidelines and were reviewed and approved by the governmental body responsible for animal welfare in the state of Baden W\u00fcrttemberg .19.The AAV-stuffer control construct includes a non-coding sequence from the 3\u2019 UTR of the UBE3A gene and has been described elsewhere (Strobel 2015). AAVs used in this study were packaged into the ShH10 capsid (Klimczak 2009). AAV production and quantification by quantitative PCR was done according to previously published protocols (Strobel 2019). The following human codon-optimized sequences were included in each of the recombinant AAVs:Plasmids with the ubiquitous CAG promoter were generated encoding for three human, codon optimized transgenes  that have a V5-tag sequence at the 3\u2019 end of the gene were generated previouslyAAV-hIL6\u2014ATGAACAGCTTCAGCACCAGCGCCTTCGGACCTGTGGCTTTTTCTCTGGGACTGCTGCTGGTCCTGCCTGCCGCTTTTCCAGCTCCTGTTCCTCCTGGCGAGGACAGCAAAGATGTGGCCGCTCCTCATAGACAGCCTCTGACCAGCTCCGAGCGGATCGATAAGCAGATCCGGTACATCCTGGATGGCATCAGCGCCCTGCGGAAAGAGACATGCAACAAGAGCAACATGTGCGAGAGCAGCAAAGAGGCCCTGGCCGAGAACAACCTGAACCTGCCTAAGATGGCCGAAAAGGACGGCTGCTTCCAGAGCGGCTTCAACGAGGAAACCTGCCTGGTCAAGATCATCACCGGCCTGCTGGAATTCGAGGTGTACCTGGAATACCTGCAGAACAGATTCGAGTCCAGCGAAGAACAGGCCAGAGCCGTGCAGATGAGCACCAAGGTGCTGATCCAGTTCCTGCAGAAGAAGGCCAAGAACCTGGACGCCATCACCACACCTGATCCTACCACAAATGCCAGCCTGCTGACAAAGCTGCAGGCCCAGAATCAGTGGCTGCAGGACATGACAACCCACCTGATTCTGCGGAGCTTCAAAGAGTTTCTGCAGAGCAGCCTGCGGGCCCTGAGACAAATGGGAGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.AAV-hTNFa\u2014ATGAGCACCGAGAGCATGATCAGAGATGTGGAACTGGCCGAGGAAGCCCTGCCTAAGAAAACAGGCGGACCTCAGGGCAGCAGAAGATGCCTGTTTCTGAGCCTGTTCAGCTTCCTGATCGTGGCAGGCGCCACCACACTGTTCTGTCTGCTGCACTTTGGAGTGATCGGCCCTCAGAGAGAGGAATTCCCCAGAGATCTGTCCCTGATCTCTCCACTGGCTCAGGCTGTGCGGAGCAGCTCTAGAACACCTAGCGATAAGCCTGTGGCTCACGTGGTGGCCAATCCTCAGGCTGAAGGACAGCTGCAGTGGCTGAATAGAAGGGCCAACGCTCTGCTGGCCAACGGCGTGGAACTGAGAGATAATCAGCTGGTGGTGCCCAGCGAGGGCCTGTACCTGATCTATAGCCAGGTGCTGTTCAAAGGCCAGGGCTGCCCTTCTACACACGTGCTGCTGACCCACACCATCAGCAGAATCGCCGTGTCCTACCAGACCAAAGTGAACCTGCTGAGCGCCATCAAGAGCCCCTGTCAGAGAGAAACACCTGAGGGCGCCGAAGCCAAGCCTTGGTACGAACCTATCTATCTCGGCGGCGTGTTCCAGCTCGAGAAGGGCGATAGACTGAGCGCCGAGATCAACAGACCCGACTACCTGGATTTTGCCGAGAGCGGCCAGGTGTACTTCGGCATTATTGCTCTCGGAGGCGGAGGAAGTGGTGGCGGAGGATCTGGCAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACTCCACCTGATGATAA.AAV-hVEGF\u2014ATGAACTTCCTGCTGAGCTGGGTGCACTGGTCACTGGCTCTGCTGCTGTATCTGCACCACGCCAAATGGTCACAGGCCGCTCCTATGGCTGAAGGCGGAGGACAGAATCACCACGAGGTGGTCAAGTTCATGGACGTGTACCAGCGGAGCTACTGTCACCCCATCGAGACACTGGTGGACATCTTCCAAGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCTAGCTGCGTGCCCCTGATGAGATGCGGCGGCTGTTGTAACGATGAAGGCCTGGAATGCGTGCCCACCGAGGAATCCAACATCACCATGCAGATCATGCGGATCAAGCCCCACCAGGGCCAGCATATCGGCGAGATGTCTTTCCTGCAGCACAACAAGTGCGAGTGCAGACCCAAGAAGGACCGGGCCAGACAAGAGAATCCTTGCGGCCCTTGCAGCGAGCGGAGAAAGCACCTGTTTGTGCAGGACCCTCAGACCTGCAAGTGCTCCTGCAAGAACACCGACAGCAGATGCAAGGCCCGGCAGCTGGAACTGAACGAGAGAACCTGCAGATGCGACAAGCCTAGAAGAGGTGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.8\u00a0viral genomes (VG)/eye (low dose) or 1\u2009\u00d7\u2009109\u00a0VG/eye (high dose) in 1\u00a0\u00b5L AAV buffer. Note that AAV-VEGF was injected at a concentration of 1\u2009\u00d7\u2009108\u00a0VG/eye and AAV-TNF-\u03b1 and AAV-IL-6 at 1\u2009\u00d7\u2009109\u00a0VG/eye with matching AAV-stuffer controls. In an independent mouse cohort, 2\u00a0weeks after AAV-TNF-\u03b1 treatment, eyes were IVT injected with 1\u00a0\u00b5L of vehicle or neutralizing anti-TNF-\u03b1 Golimumab . If IVT injection failed (e.g. due to damage of a major blood vessel or the lens), the eye was excluded from analysis.Mice were intravitreally injected with the different AAVs under isoflurane anesthesia and after local anesthetic eye drops were applied . Both eyes of 6 mice per group were injected with either 1\u2009\u00d7\u20091019. In brief, mice were anesthetized by intraperitoneal injection with 60\u201390\u00a0mg/kg ketamine (Medistar) and 6\u20138\u00a0mg/kg xylazine . Pupils were dilated with 5\u00a0mg/mL tropicamide  and phenylephrine . A Spectralis HRA/OCT device (Heidelberg Engineering) equipped with a 55\u00b0 widefield lens was used for recording of Optical Coherence Tomography (OCT) pictures and Autofluorescence (AF) images. For Fundus Fluorescein Angiography (FFA), 200\u00a0\u00b5L of a 0.2% fluorescein solution (Alcon) were injected subcutaneously and recorded 90\u00a0s after injection with the Spectralis HRA/OCT (Heidelberg Engineering). Mice were euthanized by cervical dislocation. The retina and the eye cup  were dissected, and flash frozen in liquid nitrogen.In vivo imaging was done as described previously11). In brief, 7-days-old male and female pups with their mothers were transferred to a hyperoxic chamber (75% oxygen). At P12, oxygen concentration was slowly reduced back to normoxic conditions (21% oxygen) within approximately 3\u00a0h. Control mice remained at normoxic conditions for the whole experiment. Mice were euthanized at P12, P13, P14, P15 and P16 by cervical dislocation. Dissected retinae were stored in RNAlater (Invitrogen).The OIR experiments were performed according to previously published protocols  were homogenized in 700\u00a0\u00b5L Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Retina samples were homogenized by ceramic beads MPbio, 6913-500) and eye cup samples by metal beads  and processed in 4 separate batches. Total RNA was extracted using the miRNeasy Micro Kit  according to the manufacturer\u2019s tissue protocol including on-column DNase treatment . Total RNA samples  were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher Scientific) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 48.7\u00a0ng/\u00b5L while RIN ranged from 5.2 to 9.6, with a median at 9.2. Two samples with RIN below 6 were excluded from downstream processing: AAV-stuffer (high) eye cup replicate 4 and AAV-VEGF eye cup replicate 5. See Table 3-500 andRetinae were dissected from 12 to 16-day postnatal C57BL/6\u00a0J mice (n\u2009=\u200932). RNAlater-preserved tissues were disrupted and homogenized in CK14 tubes  with RLT buffer using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was then extracted using the MagMAX-96 total RNA isolation kit  including a DNase digestion prior to final elution . Total RNA samples were quantitatively and qualitatively assessed on a Synergy microplate reader with a Gen5 Take3 module (BioTek), and the Eukaryote total RNA 6000 Nano microfluidic chip  on a 2100 Bioanalyzer system (Agilent), respectively. Concentrations averaged at 136.3\u00a0ng/\u00b5L while RIN of selected samples were above 8.4. All samples were further processed for library preparation. See Table 36.70 retina- and eye cup-derived RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 250\u00a0ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream . The only deviation from the manufacturer\u2019s protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. The index PCR was performed with 12 cycles, while the final library was eluted in 35\u00a0\u00b5L. mRNA libraries were then quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 149\u00a0nM. mRNA libraries were also assessed for size distribution  and adapter dimer presence (<\u20090.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). All 70 sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered on an S4 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2\u2009\u00d7\u2009100\u00a0bp length , reaching an average depth of 31.3 million Pass-Filter reads per sample (7.0% CV). The description of RNA library preparation and sequencing closely aligns with the one given in Becker et al.Total RNA input of 100\u00a0ng was used for library construction with the TruSeq Stranded mRNA LT kit\u2014Set A , together with the SuperScript II Reverse Transcriptase , according to the manufacturer\u2019s protocol. The quality of mRNA libraries was assessed for adapter and heterodimer presence using the DNA 1000 microfluidic chip , while library molarity was measured using the Quant-iT PicoGreen dsDNA Assay Kit . All 32 sequencing libraries were then normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered using the TruSeq SR Cluster Kit v3 , and sequenced with TruSeq SBS Kit v3 reagents  on a HiSeq 2000 Sequencing System (Illumina) with single index, single-read reads at 1\u2009\u00d7\u200951\u00a0bp length , reaching an average depth of 24.9 million Pass-Filter reads per sample (13.1% CV).2. The LUNA-FL cell counter (LogosBio) with Acridine Orange dye  was used for cell quantification before seeding. HRMECs were stimulated with 10\u00a0ng/mL recombinant human TNF-\u03b1  for 24\u00a0h at 37\u00a0\u00b0C. Cells were washed with PBS and lysed in RLT buffer provided by Qiagen and RNA extracted using the RNeasy Mini Kit  according to the manufacturer\u2019s manual.Primary human retinal microvascular endothelial cells  were cultivated in Endothelial Cell Growth Medium  on plates pre-coated with 0.1% gelatin . Cells were cultivated in a humidified chamber at 37\u00a0\u00b0C and 5% COcDNA synthesis was done using the high-capacity cDNA Kit  and qRT-PCR was performed with the Taqman Universal PCR Master Mix  on a QuantStudio 6 Real-Time PCR system (Applied Biosystems). Relative expression (fold induction) was calculated using the \u0394\u0394CT method and the 18S (for mouse tissue) and POLR2A (for HRMECs) genes were used as a normalization controls. The following Taqman probes were used in this study: 18S (Hs99999901_s1), POLR2A (Hs00172187_m1), Madcam1 (Hs00369968_m1), CCL2 (Mm00441242_m1) and a custom-made Taqman probe detecting human codon-optimized VEGF with a V5 tag .57. In brief, 2\u00a0weeks after IVT injection of AAV-TNF-\u03b1, 100\u00a0\u00b5g golimumab (Simponi) were injected intravitreally. 6\u00a0weeks after AAV injection, whole eyes were enucleated, flash frozen in liquid nitrogen, and homogenized in lysis buffer  supplemented with proteinase inhibitor Pefabloc SC  with metal beads  in a Precellys Evolution tissue homogenizer (Bertin Technologies). C3 expression was measured with the Mouse C3 SimpleStep ELISA Kit  according to the manufacturer\u2019s protocol and measured with a SpectraMax Plus 384 plate reader (Molecular Devices).Eyes for validation of C3 expression were generated from an independent mouse cohort that is published elsewhere19. Eyes were enucleated 3\u20136\u00a0weeks after IVT injection and directly fixed in 4% paraformaldehyde  for 48\u00a0h at 4\u00a0\u00b0C. Eyes were dehydrated, incubated in xylol, and infiltrated with paraffin using a tissue processing machine . Immunohistochemical stainings were performed on 3\u00a0\u00b5m sections with the Opal Multiplex IHC Kit (Akoya Biosciences) and carried out on the automated Leica Bond platform . Antigen retrieval was done with the BOND Epitope Retrieval Solution 1  at 95\u00a0\u00b0C and pH 6.0 for 20\u00a0min. Polyclonal rabbit anti-CD3 antibody  was diluted 1:200 and used as a pan-T-cell marker. OPAL polymer anti-rabbit-HRP secondary antibody  and OPAL 570 reagent  were used to develop the fluorescent signal. Nuclei were stained with spectral DAPI  and slides were mounted with ProLong Antifade Mounting Medium . Immunofluorescence stainings were imaged using a laser-scanning microscope LSM700 . RNAscope in situ hybridizations for human VEGF, TNF-\u03b1 and IL-6 were performed externally at ACDBio/Biotechne using the RNAscope 2.5 LSx Red assay. Custom-made probes were designed based on the codon-optimized, V5-tagged human transgenes expressed by each AAV. As technical controls, ACD Positive Control  and ACD Negative Control  probes were used. Epitope retrieval was done for 15\u00a0min at 88\u00a0\u00b0C and proteolysis was performed with Protease III for 15\u00a0min at 40\u00a0\u00b0C at ACDBio. Slides were imaged using an AxioScan.Z1 slide scanner .Eyes for histological analysis were generated in an independent experiment that was published elsewhereIn case of the OIR dataset GRCm38.96 was used as the reference genome. For the AAV study, a custom genome was created by merging codon optimized sequences for hIL6, hTNFa, and hVEGF into the mouse genome. Generation of genomic indices and read alignment was carried out via STAR (version 2.5.2b). Quantification on gene level was performed using FeatureCounts (version 1.5.1) and RSEM (version 1.3.0), while discarding multimapping reads. Quality control metrics were obtained using MultiQC (version 1.0), with information assembled from STAR, picardmetrics (version 0.2.4), and fastQC (version 0.11.5), among other sources.Genes with a read count value of below 10 across all samples in either the AAV or OIR study were removed from our analysis. We normalized counts expression values using the voom function provided by limma (version 3.44.3). In the AAV study we corrected for an identified batch effect associated with the RNA extraction batch (variable: rna_extraction_batch) using the ComBat function (sva package version 3.40.0). Principal component analysis was performed using the pcaMethods R package (version 1.80.0) selecting for the top 1000 most variable genes.Differential gene expression was calculated using the lmFit function provided by the limma R package. Significantly changing genes were selected based on a BH-adjusted p-value\u2009<\u20090.05. Pathway information was retrieved using the misgdbr R package (version 7.1.1). Gene set enrichment analysis was performed using the over-representation analysis (ORA) function provided by Clusterprofiler version 4.0.2. Direct comparisons between gene sets derived from the AAV experiment and the OIR study were carried out using a hypergeometric test provided by the stats R package (version 4.1.0). Venn diagrams were plotted using ggvenn (version 0.1.9).http://feb2021.archive.ensembl.org/. Raw counts were further processed using the Seurat R package (version 4.0.3). Cell specific genes were identified for each individual dataset and cell type via the FindMarkers function. Genes were defined as cell specific if the BH-adjusted p-value\u2009<\u20090.01 and the gene was not identified as specific to any other cell type (unique genes only). As above, the overlap between cell specific marker genes and genesets derived from the AAV or OIR studies were quantified by the ORA function part of clusterProfiler.Single cell RNA-Seq datasets were obtained from GSE1352229, GSE130636, and GSE135922. If required, human ensembl IDs were mapped to mouse homolog using the biomaRt R package (2.48.2), with host parameter set to Supplementary Figure 1.Supplementary Table 2.Supplementary Table 3."}
+{"text": "E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53\u00a0kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52\u00a0kDa Pro-macrobody and with a 13\u00a0kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7\u00a0\u00c5. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two\u00a0decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The  Membranes of cells compartmentalize metabolic processes and present a selective barrier for permeation. To preserve the characteristic intracellular milieu, membrane transporters with specialized functions have evolved to maintain the nutrient homeostasis of cells . Many ofE. coli genome codes for four different POTs named Di- and tripeptide permease A-D (DtpA-D), also known as YdgR (=DtpA), YhiP (=DtpB), YjdL (=DtpC) and YbgH (=DtpD). They cluster in pairs, DtpA and B (sequence identity 51%), and DtpC and D (sequence identity 56%) with around 25% identity between them. DtpA and B exhibit a prototypical substrate preference similar to the human PepT1 transporter  fused the transporter to split-sfGFP , ii) rai. This resulted in a 52\u00a0kDa Pro-macrobody (short Mb26), and we expected it to bind to the periplasmic side of the transporter as seen in other MFS transporter-Nb complexes , or ten residues (split sfGFP-DtpC1-470) of the transporter were deleted. We then assessed proper folding and complementation by monitoring the fluorescence of the chromophore on an HPLC system (FL) and lowest in the most truncated version (split sfGFP-DtpC1-470). In order to extend this observation to other MFS transporters, we repeated this experiment with the human POT homologue PepT1, and noticed a similar trend upon shortening of the termini. Yet, since the decrease of fluorescence was only minor in split sfGFP-DtpC1-475 in comparison to split sfGFP-DtpCFL, we proceeded to imaging with the shorter construct in the presence of Nb26.Since MFS transporters typically lack additional domains outside their transport unit, which is a major impediment for accurate particle alignment in single particle cryo-EM approaches, we assessed three fiducial marker strategies introducing additional density outside of detergent micelles containing DtpC, by analyzing the quality of 2D class averages . To obtaomplexes . In a thC system . All con(1\u2013475)-Nb26 sample allowed clear visualization of the transmembrane helices after clustering a small subset of particles, but the majority of particles clustered in classes with blurry density for the split sfGFP fiducial, or with the two complementary parts \u03b21-6 and \u03b27-11 not assembled   . HoweverAs we obtained the best 2D class averages for DtpC with the fiducial marker Nb26, we proceeded to a large data collection  and coul\u03b1-atoms). The peptide binding site of DtpC is exposed to the cytoplasmic side  organized in two helical bundles and additional two\u00a0TMs specific for the POT family (known as HA and HB domains). It is highly similar compared to the previously determined DtpD structure  with an mic side , 4. Almomic side .Shewanella oneidensis (PepTSo), where the inter-bundle periplasmic salt bridge is formed between TM5 and TM7, the IF state is in all other analyzed structures stabilized by a salt bridge on the tip of TM2 and TM7  and R294  and hydrogen bonds between H37  and D293 (TM7) as well as R28 (TM1) and N421  . We also and TM7 . Additio1XXE2R motif on TM1 involved in proton coupling and ligand binding, and ii) the ability to accommodate dipeptides, tripeptides, and peptidomimetics, which relies on a set of conserved residues located in the central binding cavity. In DtpC, the E1XXE2R motif, has evolved to Q1XXE2Y . In all high resolution X-ray structures of canonical POTs, R is in salt-bridge distance to E2 and the C-terminus of substrate peptides. Mutation of either E1 or E2 in the conventional E1XXE2R motif to glutamine residues abolishes uptake  the presence of the Es uptake . A revereases it . In addiproposed . This bi1XXE2Y motif instead of E1XXE2R.In DtpC, we now observe that the side chain pocket has a different architecture and characteristic in comparison with the one of canonical POTs. It displays an overall more acidic groove caused by the presence of the aspartate residue 392, conserved among atypical POTs. This residue has been predicted to be involved in substrate coordination and mutation of this residues in DtpC and homologues DtpD (corresponding residue is D395) abolished transport activity . CanonicE. coli POTs structures. Lastly, it displays some of the challenges related to high resolution cryo-EM structure determination of MFS transporters devoid of soluble domains, and manifests once again, the benefit of fiducial markers in overcoming those.In summary, our work provides new insights into promiscuous versus selective substrate recognition in POTs and constitutes a step forward towards completing the family of Escherichia coli genome, and cloned into a pNIC-CTHF vector by ligation-independent cloning (LIC). This vector contains a C-terminal His-Tag and a Tobacco Etch virus (TEV) cleavage site and a kanamycin resistance gene as selectable marker. The first 6\u00a0N-terminal beta strands of sfGFP were fused to the N-terminus of DtpC, and the beta strands 7 to 11 fused to the C-terminus. We named this construct split sfGFP-DtpCFL. Two additional constructs were cloned with truncations of 5 (split sfGFP-DtpC1-475), and 10 residues (split sfGFP-DtpC1-470), on the C-terminal side of DtpC.The full-length cDNA of DtpC wild type (WT) was amplified from the HsPepT1 was previously cloned into a pXLG vector containing an expression cassette composed of an N-terminal Twin-Streptavidin tag followed by the HRV-3C protease recognition sequence  a C-terminal truncation of 36 residues (split sfGFP-HsPepT11-672), and ii) a C-terminal truncation of 36 residues and a N-terminal truncation of 10 residues (split sfGFP-HsPepT110-672) were cloned.sequence . SimilarE. coli C41(DE3) cells grown in terrific broth (TB) media supplemented with 30\u00a0\u03bcg/ml kanamycin according to established procedures , and the pellet was stored at -20\u00b0C until further use. Cell pellets were resuspended in lysis buffer  glycerol, 15\u00a0mM imidazole, with 3\u00a0ml of lysis buffer per Gram of wet weight pellet), supplemented with lysozyme, DNase and 0.5\u00a0mM tris(2-carboxyethyl)phosphine (TCEP). The cells were lysed by three cycles using an Avestin Emulsiflex homogenizer at 10,000\u201315,000 psi. Recovered material was centrifuged to remove non-lysed cells  and the supernatant was subjected to ultracentrifugation to separate the membrane fraction . Membranes were resuspended in lysis buffer supplemented with cOmplete EDTA-free protease inhibitors (Roche), and solubilized by adding 1% n-Dodecyl-\u03b2-D-Maltoside (DDM) detergent (Anatrace). The sample was centrifuged for 50\u00a0min at 90,000 \u00d7 g, and the supernatant was applied to Ni-NTA beads for immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in lysis buffer and incubated with the solubilized membrane proteins for one hour at 4\u00b0C on a rotating wheel. Loaded beads were washed with buffer with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration  and combined with 1\u00a0mg of TEV protease to perform the His-tag cleavage during dialysis overnight at 4\u00b0C. The dialysis buffer contained 20\u00a0mM HEPES at pH 7.5, 150\u00a0mM NaCl, 5% glycerol, 0.5\u00a0mM TCEP, 0.03% DDM. The cleaved protein was recovered by negative IMAC, concentrated to 4\u00a0ml using a 50\u00a0kDa concentrator (Corning\u00ae Spin-X\u00ae UF concentrators) and run on an \u00c4KTA Pure system , using a HiLoad 16/ 600 Superdex 200 column for DtpC, and a Superdex 200 Increase 10/300 column for the split sfGFP-DtpC constructs. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80\u00b0C until further use.Recombinant DtpC, and the three split sfGFP-DtpC constructs were expressed in ocedures . CultureHsPepT1 constructs, expression was done in mammalian cells as described previously . After 20\u00a0min of incubation on a rotating wheel, the suspension was transferred to a gravity column. Following two wash steps with 300\u00a0mM NaCl, 20\u00a0mM HEPES (pH 7.5), 0.03% DDM, and 0.003% CHS, split sfGFP-HsPepT1comnstructs were eluted with 0.03% DDM, 0.003% CHS, 150\u00a0mM NaCl, 20\u00a0mM HEPES (pH 7.5), and 10\u00a0mM desthiobiotin (Sigma-Aldrich).For the split sfGFP-eviously . BrieflyTo generate DtpC specific nanobodies, two non-inbred llamas were injected six times at weekly intervals with a mixture of 94 different proteins including DtpC purified in the detergent DDM (50\u00a0\u00b5g of each antigen weekly). After 6\u00a0weeks of immunization, two separate phage display libraries were constructed, one from each animal, in the pMESy2 vector, which is a derivative of pMESy4 that contain a C-terminal EPEA-tag for affinity purification. After pooling both libraries, nanobodies were selected against individual antigens in two rounds of parallel panning in 96-well plates containing one immobilized antigen in each well. After two selection rounds on DtpC, 60 clones were picked for sequence analysis, 13 clones encoded antigen-specific nanobodies as tested in ELISA, grouping them in 5 different sequence families. A nanobody family is defined as a group of nanobodies with a high similarity in their CDR3 sequence . Nanobodies from the same family derive from the same B-cell lineage and likely bind to the same epitope on the target. Immunizations, library construction, selection by panning and nanobody characterization were performed according to standard procedures . Five naE. coli WK6 cells and purified following standard procedures. Specifically, the cell pellet was resuspended in TES buffer  supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20\u00a0min at 10,000 \u00d7 g and additionally for 30\u00a0min at 100,000 \u00d7 g. The supernatant was applied to CaptureSelect beads (Thermo Fisher Scientific), which were equilibrated with wash buffer . After three column volumes of washing, the nanobody was eluted with 20\u00a0mM HEPES, pH 7.5, 1.5\u00a0M MgCl2. The nanobodies were further purified on a HiLoad 16/600 Superdex 75\u00a0pg column in 20\u00a0mM HEPES, pH 7.5, 150\u00a0mM NaCl, 5% glycerol, concentrated with a 5\u00a0kDa cut-off concentrator, flash-frozen and stored at -80\u00b0C until further use.The nanobodies were expressed in E. coli WK6 cells as above. The cell pellet was resuspended in TES buffer  supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20\u00a0min at 10,000 \u00d7 g and additionally for 30\u00a0min at 142,000 \u00d7 g. The supernatant was further purified by immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in 20\u00a0mM NaPi at pH 7.5, 300\u00a0mM NaCl, 5% glycerol, 15-30\u00a0mM imidazole, 0.5\u00a0mM TCEP and incubated. Loaded beads were washed with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration  and combined with 1\u00a0mg of 3C protease to perform the His-tag cleavage. The cleaved protein was recovered by negative IMAC, concentrated to 0.5\u00a0ml using a 30\u00a0kDa concentrator (Corning\u00ae Spin-X\u00ae UF concentrators) and run on an \u00c4KTA Pure system , using a Superdex 75 Increase 10/300 column. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80\u00b0C until further use.The nanobody 26 (Nb26) was first inserted into a pBXNPH3 vector containing a C-terminal penta-histidine tag preceded of a HRV-3C protease recognition sequence. The maltose binding protein (MBP) was then inserted in frame with the 3\u2019 end of the nanobody, with two proline residues as a linker between the two genes similar as described in . The resulting Pro-macrobody 26 (Mb26) was expressed in 1-475-Nb26 with a Prometheus NT.48 device . The purified proteins were diluted to 16 \u00b5M, and the complexes were formed using a 1:1.5\u00a0M ratio of membrane protein: fiducial. The fluorescence at 330 and 350\u00a0nm was recorded over a temperature gradient scan from 15\u00b0 to 95\u00b0C and processed in GraphPad Prism 9.0 (GraphPad Software).The differential scanning fluorimetry method was used to follow the thermal unfolding event  of Nb17,Structures with the following sequences were used as input for AlphaFold2 structure prediction , and AMBMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDGGGGGNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.One hour before vitrification, the purified protein complexes were thawed on ice and run on a Superdex Increase 200 5/150 column in 0.015% DDM, 100\u00a0mM NaCl, 10\u00a0mM HEPES (pH 7.5), 0.5\u00a0mM TCEP in order to remove the excess of empty detergent micelles earlier generated upon sample concentration. The top fraction reached a concentration ranging between 3 and 6\u00a0mg/ml, and for each sample, 3.6\u00a0\u03bcl were applied to glow-discharged gold holey carbon 2/1 300-mesh grids (Quantifoil). Grids were blotted for 4\u00a0s at 0 force and 1-s wait time before being vitrified in liquid propane using a Mark IV Vitrobot (Thermo Fisher Scientific). The blotting chamber was maintained at 4\u00b0C and 100% humidity during freezing.All movies were collected using a Titan Krios (Thermo Fisher Scientific) outfitted with a K3 camera and BioQuantum energy filter (Gatan) set to 10\u00a0eV. Automated data acquisitions were set using EPU (Thermo Fisher Scientific). The applied defocus ranged between -0.9\u00a0\u00b5m and -1.8\u00a0\u00b5m in all datasets.2 was used with 2.8\u00a0s exposure time, fractionated in 50 frames. For split sfGFP-DtpC1-475-Nb26, movies were collected at a nominal magnification of \u00d7,130,000 and a physical pixel size of 0.67\u00a0\u00c5, with a 50-\u03bcm C2 aperture and 100-\u03bcm objective aperture at a dose rate of 19.0 e\u2212/pixel per second. A total dose of 57 e\u2212/\u00c52 was used with 3\u00a0s exposure time fractionated in 40 frames.For DtpC-Nb26 and DtpC-Mb26, movies were collected at a nominal magnification of \u00d7105,000 and a physical pixel size of 0.85\u00a0\u00c5, with a 70-\u03bcm C2 aperture and 100-\u03bcm objective aperture at a dose rate of 19.5 e\u2212/pixel per second. A total dose of 75 e\u2212/\u00c5All movies were motion-corrected using Relion-3.1  own impl1-475-Nb26, 7602 movies were collected, 1,049,399 coordinates were picked and used for 2D averaging and clustering. For DtpC-Nb26, 24,333 movies were collected, 6,464,070 coordinates were picked and used for 2D averaging and clustering, and 878,428 particles were used in the final 3D reconstruction. Briefly, DtpC-Nb26 dimeric population was clustered using 3D class averaging in Relion3.1  were collected on the EMBL P12  beamlineGraphs were generated using GraphPad Prism 9.0 (GraphPad Software). Molecular graphics and analyses performed with UCSF ChimeraX-1.2.5 . Figures"}
+{"text": "Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals. Neural stem cells in modern humans have a longer metaphase and fewer chromosome errors compared with Neanderthals and chimpanzees. FOXP2  Kinesin 8], KNL1 (a.k.a. CASC5), and SPAG5 (a.k.a. astrin), which are highly expressed in the germinal zones of the developing neocortex and are associated with mitotic spindle, kinetochore, and chromosome segregation functions. The kinetochore is a complex, three-dimensional (3D), multiprotein structure mediating the attachment of chromosome centromeres with the ends of kinetochore microtubules  that carry the ancestral variants of KIF18a and KNL1 are used to generate cerebral organoids, shorter metaphases, less SAC-positive kinetochores, and more chromosome segregation defects are observed in APs. Together, our data suggest that the three amino acid substitutions in KIF18a and KNL1 cause fewer chromosome inheritance errors to occur in APs of modern humans than in archaic humans and apes.P > 0.05; P > 0.05; fig. S1, A, B, and D). Organoids can therefore be used to study the number of SAC-positive kinetochores in cerebral tissue APs.An active SAC, triggered, e.g., by one or more kinetochores not being properly attached to kinetochore microtubules, delays anaphase onset and therefore prolongs metaphase  in the fetal human neocortex and human organoid APs, respectively, than in chimpanzee organoid APs . Thus, human metaphase APs have more SAC-positive kinetochores than chimpanzee APs.To investigate whether the previously described metaphase prolongation in human relative to chimpanzee APs  mice; P < 0.001; P < 0.001) that did not differ (P > 0.05) from the hKIF18a-hKNL1-hSPAG5 mice with the six amino acid substitutions , two in KNL1 (H159R and G1086S), and three in SPAG5 , albeit not significantly so  versus 4.6 min (hKNL1 substitution 2) versus 4.6 min (wt), respectively; fig. S4, see also In contrast, in hSPAG5 mice, the length of the AP metaphase was 4.4 min and hence similar to wt mice , indicatNotably, the durations of prometaphase, which precedes metaphase, and of anaphase, which follows metaphase, did not show any significant changes in any of the mouse lines analyzed .We also explored whether the modern human-specific changes in KIF18a and KNL1 may influence general aspects of cortical development. However, we did not observe any effects on the overall morphology, thickness, or perimeter of the neocortex in adult mice humanized for KIF18a and KNL1 . Consistent with this, the adult mice did not show any change in the distribution or numbers of Ctip2KIF18a and the two codons in KNL1 back to the ancestral, Neanderthal-like state in the H9 ESC line. Individual cells subjected to the relevant RNA guides and donor DNAs were expanded to cell lines, among which we selected two independent ancestralized lines [aKif18a-aKNL1 lines 1 (L1) and 2 (L2)] and two lines where none of the three codons had been changed  at the midpoint of the dark period . After positive plug detection in the morning, the cumulus complexes were isolated and zygotes were removed with a treatment of hyaluronidase [final concentration of 0.1% (801 U/ml)]. CRISPR-Cas9 solutions were injected into the male pronucleus of fertilized zygotes by using a motor-driven manipulator-based microinjection stage. About 2 hours after injections, the surviving embryos were transferred into Crl:CD1(ICR) pseudopregnant recipient female mice (ca. 20 embryos per recipient).The recipient mice were mated with vasectomized males Crl:CD1 (ICR). After detection of a copulation plug in the morning of the transfer day, pseudopregnant mice were used for unilateral surgical embryo transfer into the oviduct as described  with 2 \u03bcl of genomic tail DNA, 0.25% dimethyl sulfoxide, and 0.5 \u03bcM forward and reverse primer. Primer sequences (target-seq-primer) and PCR conditions are in table S1. Products were Sanger sequenced either with the forward and reverse PCR primers or if required with internal sequencing primers. Primary candidates have been identified by sequence alignments of potentially modified PCR fragments against the wt and modified reference sequence. The products from putatively edited mice were cloned (TOPO) and verified by Sanger sequencing of 10 to 20 randomly selected clones. Founders carrying the respective genome modification either homo- or heterozygously were propagated and genotyped by Sanger sequencing of PCR fragments. From the second generation onward, allele-specific PCRs were performed on 2 \u03bcl of crude buccal DNA in a total of 10 \u03bcl using the REDExtract-N-Amp PCR ready Mix (Sigma-Aldrich) according to the manufacturer\u2019s protocol using 0.5 \u03bcM forward, reverse, and mutant primer. The readout is done on standard agarose gels. Primers and PCR conditions are in table S1.Kif18a, Knl1, and Spag5 in the wt and humanized embryonic mouse telencephalon was confirmed by PCR, using generated cDNA as templates, followed by Sanger sequencing of the PCR products. Wt and gene-edited sequences were mapped to the Kif18a, Knl1, and Spag5 genes, respectively, using Geneious Prime (version 2020.2.4). Data showed the expression of only the wt Kif18a, Knl1, and Spag5 in E11.5 wt embryonic mouse telencephalon, and the expression of only the gene-edited hKif18a, hKnl1, and hSpag5 in E11.5 humanized hKIF18a-hKNL1-hSPAG5  and LightCycler 96 Instrument (Roche). Gene expression analyses by qPCR showed that the relative mRNA levels of Kif18a, Knl1, and Spag5 were not significantly different between E11.5 wt and humanized embryonic mouse telencephalon .For Sanger sequencing and real-time qPCR, total RNA was isolated from E11.5 wt and humanized mouse dorsolateral telencephalon using the RNAeasy Micro Plus Kit (Qiagen) according to the manufacturer\u2019s instructions. cDNA was synthesized using the Maxima First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The expression of PAG5 see  embryoniThe following primers were used for PCR:Forward primer for Kif18a (5\u2032-AGAAAAGGCGGTGCAGTTCT-3\u2032),Reverse primer for Kif18a (5\u2032- GGAATCTTCCCGGACAGCAA-3\u2032);Forward primer for Knl1 H159R (5\u2032-CCCCAGACAAGTCAAGCAGAA-3\u2032),Reverse primer for Knl1 H159R (5\u2032-TCAACTCCATACACTCATTGCC-3\u2032);Forward primer for Knl1 G1086S (5\u2032-TGGATATCACCAAGAGTTGCAC-3\u2032),Reverse primer for Knl1 G1086S (5\u2032-CAAAACTGAAGCCCTTTCTGTC-3\u2032);Forward primer for Spag5 D410H and E162G (5\u2032-TGAATCTCGGTTTGTCGCCT-3\u2032),Reverse primer for Spag5 D410H and E162G (5\u2032-TTCCTCCCCTGGATCGACAT \u22123\u2032);Forward primer for Spag5 P43S (5\u2032-GTTCAAATAGAGGCGGCGGG-3\u2032),Reverse primer for Spag5 P43S (5\u2032-GTTCTTTCCCACCAGCTACAAG-3\u2032).The following primers were used for qPCR:Forward primer for Kif18a (5\u2032-CAAACTCAGGACCACTTGCTGT-3\u2032),Reverse primer for Kif18a (5\u2032-ATGGGAACGAGAAGACACTGC-3\u2032);Forward primer for Knl1 (5\u2032-CCTCTGGGGGAGATGGCTACAT-3\u2032),Reverse primer for Knl1 (5\u2032-GATGGACTTTGTTGGGCTGAGA-3\u2032);Forward primer for Spag5 (5\u2032-GTCTCACCCTCTTCTTACAGGC-3\u2032),Reverse primer for Spat5 (5\u2032-GCTGGTTCTGGCACTTCATCTA-3\u2032);Forward primer for Actb (5\u2032-CGGGACCTGACAGACTACCTC-3\u2032),Reverse primer for Actb (5\u2032-GGTGGTGAAGCTGTAGCCACG-3\u2032).AAVS1 locus from the H9 human ESC line  that we generated as previously described :KIF18A_sg1: 5\u2032-TGTTATAAAGAAACAAAATAKIF18A_sg2: 5\u2032-GATTTGTAGTTTTCTTTCCAKNL1_H109R_sg1: 5\u2032-ATTTGCATGTTTCCTTTCACKNL1_G1086S_sg1: 5\u2032-TGAATGAACCTCTATCAAGCAssDNA KIF18A_K67R: 5\u2032-AGTTGACGTTTCATCAAAAACAGCATCAAATACAAATTTAAGATCCTTGTTTTGTCTCTTTATAACATTTTGATTTGTAGTTTTCTTTCCATGGAAAAAACTGACTssDNA KNL1_H159R: 5\u2032-TGTTGTAAAATGCCTTTTTAAAAGTTTGCTTTTGTTCTGATCTCTTATATTTTGCTTTCATTATAGTTTTCAATTATAGAACATACCCATGAAAGGAAACATGCAAATGACCAGACAGTCATTTTTTssDNA KNL1_G1086S: 5\u2032-TCTTGTCATTTTTTAGCTTAAGGCTTTTTCTTCTCTGACTTTTGCCTGATAGAGGTTCGTTCAGAAATCCAGGACTTTGTACATCTTTGATo derive colonies from single cells, cells were incubated with StemFlex with supplement  and CloneR  for 1 day and then sorted with a Cytena cell printer.Genomic DNA of each colony was isolated, a region of ~200 base pairs (bp) around the cut site was amplified and sequenced, and from sequences, the editing state was evaluated as described  amplification included 1\u00d7 ddPCR Supermix for probes , 0.2 \u03bcM of each primer and 0.2 \u03bcM probe (both IDT) for target and reference, together with 1 \u03bcl genomic DNA in QuickExtract DNA Extraction Solution (Lucigen). After droplet generation with the QX200 Droplet generator (Bio-Rad), the PCR was for 5 min at 95\u00b0C, followed by 42 cycles of 35 s at 95\u00b0C (at a ramp rate of 1.5\u00b0C/s) and 65 s at 54\u00b0C for KIF18A and KNL1_G1086S or 65 s at 56\u00b0C for KNL1_H109R (at a ramp rate of 1.5\u00b0C/s), and 5 min at 98\u00b0C. Readout was in a QX200 Droplet reader (Bio-Rad), and allele copy numbers were determined relative to the FOXP2 reference and unedited controls.Deletions bigger than ~200 bp and therefore not detected by the amplicon sequencing KNL1_G10862_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCAATCCCACACCTGACTKNL1_G10862_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTCGTCCTCTAACACATCCAKNL1_G10862_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCCTGGAGGATAAAGAGGAKNL1_G10862_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAATCGGTGACTTCCAGATCAKNL1_H159R KNL1_H159R_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGACCCTTCAGAATCCTACCCKNL1_H159R_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTATCCTCTGTGTGGCTGAAKNL1_H159R_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAGAGCCTGTCAAATCCTTKNL1_H159R_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGGTGTCGGTTAAGCTGTKIF18A KIF18A_upSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGGTAAGGGAGTGGGAATKIF18A_upSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTCAGAGCCTGCACTCTTKIF18A_downSNP_F: 5\u2032-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAACCGTTTTAGCCAAGATKIF18A_downSNP_R: 5\u2032-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCCCGAATGTCTTTCTCATo find large-scale chromosomal deletions, duplications, or karyotype abnormalities, we performed \u201cshallow\u201d whole genome sequencing to an average genome coverage 0.10 to 0.15 of edited and nonedited colonies as well as the cell line from which the colonies were derived, as described (KIF18a and KNL1 in edited and nonedited cell lines as well as their \u201cmother\u201d cell line was measured with CellsDirect One-Step qRT-PCR Kit (Invitrogen) using TaqMan probes for KIF18a (FAM Hs0105428_m1), KNL1 (FAM Hs00538241_m1), and a \u201chousekeeping gene\u201d PPIB (VIC Hs00168719_m1). The producer\u2019s protocol was followed to set up reactions. For a subset of reactions, reverse transcriptase was inactivated by 15-min incubation at 70\u00b0C. The cycler program for RT-qPCR was 50\u00b0C for 10 min, 95\u00b0C for 2 min, then 50 cycles of 95\u00b0C for 3 s, 53\u00b0C for 15 s, and 60\u00b0C for 30 s.Expression of Organoids were generated from the H9 ESCs and cultured as described  and immunohistofluorescence (fixed). Mouse neocortex was fixed, embedded, cryosectioned, and prepared for immunohistofluorescence as described  for multiphoton excitation, and using 63\u00d7 Plan-Apochromat 1.4 numerical aperture (NA) oil or 40\u00d7 C-Apochromat 1.2 NA W objectives .http://imagej.nih.gov/ij/). Brightness and contrast were recorded and adjusted linearly. Measurements of mitotic phases were performed as described . Because at least some datasets in most experiments did not pass normality tests (Shapiro-Wilk and Kolmogorov-Smirnov tests), nonparametric Mann-Whitney"}
+{"text": "The structure of mammalian V-ATPase with mEAK-7 shows how a TLDc domain-containing protein can bind the proton pump to form an activity-sensitive interaction. V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in \u223c3% of complexes, whereas \u223c1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7\u2019s TLDc domain interacts with V-ATPase\u2019s stator, whereas its C-terminal \u03b1 helix binds V-ATPase\u2019s rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme\u2019s activity. ATP hydrolysis in the A3B3 subcomplex of the V1 region induces rotation of the rotor subcomplex, which contains subunits D, F, d, and a ring of membrane-embedded c subunits  are large, membrane-embedded protein complexes that function as proton pumps in eukaryotic cells. V-ATPase activity is essential for acidification of numerous intracellular compartments including endosomes, lysosomes, and secretory vesicles . In somesubunits . In the the ring . Rotatiothe ring , station regions , with su1 region  and subuociation .V-ATPase\u2013driven acidification is necessary for targeting and post-translational modification of proteins in the Golgi , degradaV-ATPases interact with other proteins in the cell . In partAnalysis of V-ATPase from kidney tissue identified ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27, and nine subunits of the CCT complex as V-ATPase associated proteins . Two of 1 and VO regions .To identify low abundance complexes between mammalian V-ATPase and its binding partners, we first determined the subunit composition and structure of V-ATPase prepared from porcine kidney. We then subjected images of this complex to an exhaustive 3D classification strategy designed to detect low-population structures. This procedure revealed a small population of V-ATPase complexes with super-stoichiometric occupancy of the C subunit, and a second small population of complexes with an additional density bound to the catalytic Aignaling  and an mmEAK-7 has been proposed to activate the mTOR pathway but was not previously suggested to interact with V-ATPase , 2019. T1 region, and subunits a1, c, c\u2033, d1, e2, RNAseK, ATP6AP1/Ac45, and ATP6AP2/PRR in the VO region Fig 1A). O region . AlthougO region . DespiteO region , the speMass spectrometry analysis of trypsin digested V-ATPase sample.Table S1 Mass spectrometry quantification of V-ATPase subunits.Table S2 left and 1 regions at 3.6\u20134.0 \u00c5 resolution and the VO regions at 3.7\u20135.8 \u00c5 resolution . This density appears to correspond to an additional copy of subunit C . This density abuts subunit B, subunit A, and peripheral stalk 3. The shape of the density does not correspond to any of the known subunits of V-ATPase. It is not clear if the fraction of particle images with this additional density represents the true fraction of V-ATPase complexes in cells with this protein bound, or if the interaction is more common but is disrupted during purification of V-ATPase. The list of proteins identified by mass spectrometry of the preparation (Table S1) was inspected to identify candidate proteins that could explain the density. After excluding V-ATPase subunits, the most abundant \u223c50-kD proteins based on iBAQ scores (intensity-based absolute quantification) were the \u03b1 and \u03b2 subunits of mitochondrial ATP synthase followed by mEAK-7. Structures are known for both subunit \u03b1 and \u03b2 from ATP synthase , which regulates dauer formation in nematodes , followed by a region resembling an EF-hand domain (cyan), a TLDc domain , and a C-terminal \u03b1 helix that is separated from the rest of the protein by an extended linker region (red)  . The N tleft) as well as a B1 subunit (right), facilitated mainly by hydrophobic interactions. This \u03b1 helix is formed from sequence in the protein that was previously proposed to bind mTOR . Binding involving subunit A occurs through apparent ionic interactions between Thr354 and Gln317 from mEAK-7 with backbone atoms from residues Arg553 and Thr557 from subunit A, respectively, and hydrophobic interactions between Pro316 from mEAK-7 and Ala559 from subunit A (middle). Residues Gly276 and His277 from the TLDc domain of mEAK-7 interact with backbone atoms from Phe481 and Pro482 of subunit B1 (right). Together the TLDc domain and C-terminal \u03b1 helix form a pincer-like grip around B1  confirmed the lysosomal localization of the protein, as shown previously (lower). Endogenous mEAK-7 expression in HEK293T cells is nearly undetectable . Whereas 3D classification identified a population comprising \u223c25% of particle images where the density for mEAK-7\u2019s N-terminal domain and TLDc domain are weak and poorly resolved, suggesting that they are loosely attached via the TLDc domain, density for mEAK-7\u2019s C-terminal \u03b1 helix remains strong and well resolved even in this 3D class . In contrast, when the C-terminal \u03b1 helix is truncated most V-ATPase complexes in rotational State 2 do not bind mEAK-7 (upper middle). Only \u223c22% of particle images showed any density for the N-terminal domain and TLDc domain of mEAK-7 when the C-terminal \u03b1 helix was truncated, and even this density was weak and poorly resolved (lower middle). These experiments indicate that mEAK-7\u2019s C-terminal \u03b1 helix is important for the tight binding of V-ATPase by mEAK-7.To understand the relative importance of mEAK-7\u2019s TLDc domain and C-terminal \u03b1 helix for V-ATPase binding, we prepared a construct of mEAK-7 with the C-terminal \u03b1 helix removed by truncation at residue Ser415. This construct was expressed heterologously in is state , left. Wupper left). In contrast, with ATP added a population of particles appeared with mEAK-7 entirely absent in rotational State 2 (upper right). In this state, even density for the C-terminal \u03b1 helix is missing . Thus, it appears that rotation of the rotor, driven by ATP hydrolysis can result in the displacement of mEAK-7 from V-ATPase and disruption of the cross-link between the enzyme\u2019s rotor and stator. Despite several biochemical experiments, we were unable to demonstrate that mEAK-7 can be fully dissociated from V-ATPase by the addition of ATP to the complex, including experiments where we washed immobilized V-ATPase:mEAK-7 extensively with a buffer containing ATP before elution of V-ATPase. These experiments suggest that mEAK-7 can remain associated to V-ATPase, either by its TLDc domain or its C-terminal \u03b1 helix, during ATP hydrolysis.The observation that mEAK-7, which crosslinks the rotor and stator of V-ATPase in the structure, does not affect the enzyme\u2019s activity in vitro or in cells suggests that the crosslink is broken during rotary catalysis. To determine the effect of ATP hydrolysis on mEAK-7 crosslinking of the rotor and the stator, V-ATPase was mixed with a \u223c20\u00d7 molar excess of mEAK-7, ATP was added to 10 mM and mixed, and cryoEM grids were frozen within 5 s. Given the concentration of V-ATPase (20 mg/ml) and the enzyme\u2019s specific ATPase activity (2.9 \u00b1 0.72 \u03bcmol ATP/min/mg), these conditions ensure that the grids were frozen before the supply of ATP was consumed. In the absence of ATP, it was impossible to find a 3D class in rotational State 2 that lacked density for mEAK-7 completely and density for mEAK-7\u2019s C-terminal \u03b1 helix was always clear , an article was published showing that the yeast TLDc protein Oxr1p binds to the yeast V1 complex, releasing subunit H and promoting dissociation of V1 from VO (1 complex (1:Oxr1p is inhibited even though subunit H is absent (left) and mEAK-7 (middle) shows notable differences in their interactions with V-ATPase: Oxr1p binds the enzyme in rotational State 1, whereas mEAK-7 binds it in rotational State 2. Furthermore, Oxr1p attaches to peripheral stalk 2, whereas mEAK-7 attaches to peripheral stalk 3. Most notably, Oxr1p binding forces the peripheral stalk 2 into a highly strained conformation (left) that is not seen in any of the peripheral stalks in V-ATPase with mEAK-7 (center). This strained peripheral stalk explains why Oxr1p binding promotes dissociation of V1 from VO, whereas mEAK-7 binding does not. Recent structures of yeast V1 and V1\u2206C complexes show a sharp bending of peripheral stalk 2 induced by interaction with subunit H (right). The interaction of subunit H with peripheral stalk 2 and the resulting bending of the peripheral stalk was proposed to be responsible for inhibiting ATP hydrolysis of V1, as disruption of the interaction prevents subunit H from inhibiting ATP hydrolysis  was added to the membrane fraction at 8 ml/g and resuspended with a Dounce homogenizer. Approximately 6 g of membranes were used for each preparation. The detergent glycol diosgenin (GDN) was added to 1% (wt/vol) and incubated with gentle mixing overnight. After solubilization, V-ATPase purification and assay was performed with no added lipids at 37\u00b0C as described previously . All subsequent steps were performed at 4\u00b0C. Each kidney was dissected to separate the softer cortex and medulla tissue from the more rigid pelvis tissue. The cortex and medulla tissue were blended with homogenization buffer for 1 min in a total volume of \u223c500 ml for four kidneys at high speed followed by a 10-s pause and an additional 1 min of blending. Cell debris was removed by centrifugation at 800eviously , except eviously . BrieflySus scrofa (UniProt ID: A0A4X1T484) was synthesized in a pET28a(+) vector with an N-terminal 6\u00d7 His tag (GenScript). The codon optimized sequence for mEAK-7 was:Recombinant mEAK-7 from GGGAATTCAAAAAGTAGGTCTGGACAAGGTCTTTGCAGCCGTTTCCTGCCGGAGGAACAAGCGGAAGTGGACGGTCTGTTCGATGCACTCAGCTCTGAGAAGCTGTCCTCACGGACCAGCCCGCGTAGCTTTAGCCTGCAAGCGCTGAAATCTCACGTAGGCGAAGCGTTGCCACCGGAGATGGTTACGCGTCTGTTCGAGGGTATGCGTCGTGCGGATCCGACAGGCAAGGCGACCGGTCCGAGCGCACGCATCAGCCAGGAGCAATTTACCTTGAGTATGAGCCATTTGCTGCGCGGCAGCAGCGAGGAAAAATCCTTGGTTATTCTGGCAATGGCTGCCGCTACCGATGGCCCAGCGGAAGCCCGTGAGGTTCTGCGCTTCACGGAAGACCTGGTGGGCAGCGTCGTGCATGTCTTACACTACCGCCAAGAGCTGCGCGGTTGGACCCAGAAACAGGCCTCTGGTTCCCCGCCTCGTGTTCAGGCGTTGGCGGCACAATTATTCTCCGAGCTGAAGCTGCAGGACGGCGAGAAGCTGCCGGGTCCGCAGCGTCTGGACTGCGATTGTGATCGTGCAGTCGTGGAAGCGTGGCTGTTCCGCGCTCCGCATGTTGCAACCTTTCTGTCCGTGGTGATTCATCAGGGGTTTCGCTTGCTGCGCTCCAGCCTGGACTTGGCGACTCTGCTGCCAGAACGTCAAGTTGACCGAGGCCGTGAATTTGCGTCGCTGCTGGACGTGCTGAGCGTTGCCTATATCAACAGTCACCTGCCGCGTGATCTCCGTCATCGTTGGCGTCTCCTTTTCGCCACGGCTCTGCACGGTCACAGCTTTGCTCAATTGTGCGGTCGTATCACCCAGCGCGGCCCGTGCGTGGTGCTGTTGGAAGATCAGGATGGCCACGTTTTTGGCGGCTTCGCGTCTTGTAGCTGGGAAGTGAAACCGCAGTTTCAGGGTGACAGCAAGTGCTTTCTTTTCTCGATCTGCCCGGCTATGGCGGTTTACACCTGCACCGGGTATAACGATCATTACATGTATCTGAATCATGGTCAGCAGACCATTCCGAATGGTCTGGGTATGGGTGGTCAACACAACTACTTCGGTCTGTGGGTTGACGTTGATTTTGGTAAAGGTCACTCCAAAGCAAAACCGACCTGTACCACGTACAGCAGCCCACAACTGTCGGCTCAAGAGGACTTCCGCTTCGAAAAGATGGAAGTTTGGGCAGTGGGCGACCCGTCTGTCACTCAACCGGCGAAAAGCAGCAAGTCCATCCTGGACGGCGACCCGGAGGCGCAAATTCTGCTTGAGGCGAGCGGCAGAAGCCGTCACTCCGAAGGTTTGCGTGCGGTGCCGGAGGACGATTAA.\u22121 kanamycin (Sigma-Aldrich) and grown overnight at 37\u00b0C. A transformed colony was used to inoculate a starter culture containing 10 ml of 2xYT medium supplemented with 50 \u03bcg ml\u22121 kanamycin. The starter culture was grown overnight at 37\u00b0C with shaking at 240 rpm, and 10 ml of this culture was used to inoculate 1 liter of 2xTY medium supplemented with 50 \u03bcg ml\u22121 kanamycin. The culture was grown at 37\u00b0C with shaking at 240 rpm to an OD of \u223c0.8, with the temperature then reduced to 7\u00b0C and protein expression was induced overnight by addition of 1 mM isopropyl \u03b2-D-1-thiogalactopyranoside (IPTG) (Fisher). All further steps were performed at 4\u00b0C. Cells were harvested by centrifugation at 4,000g for 15 min and the cell pellet was resuspended in PBS and centrifuged again at 4,000g for 30 min. The cell pellet was then resuspended in lysis buffer  at \u223c8 ml/g of wet cell pellet. The resuspended cells were lysed by sonication (Q Sonica Q500) with 2 s on, 2 s off at 30% amplitude for 10 min, and cell debris was removed by centrifugation at 130,000g for 45 min with a Type 70 Ti Rotor (Beckman Coulter). The supernatant was filtered through a 0.22 \u03bcm filter (Sigma-Aldrich) and loaded onto a HisTrap HP 5 ml column previously equilibrated with HisTrap buffer . The column was washed with 10 column volumes (CV) of HisTrap buffer followed by elution with five CV of HisTrap buffer with imidazole added to 300 mM. Eluted protein was concentrated with a 10-kD MWCO concentrator (Amicon) to \u223c500 \u03bcl before two rounds of gel filtration, first with a Superose 6 Increase 10/300 column and then a Superdex 75 10/300 column. The purified protein was pooled, flash frozen, and stored at \u221280\u00b0C for subsequent use.ArcticExpress competent cells (Agilent) were transformed with the plasmid and plated onto Luria broth (LB) agar supplemented with 50 \u03bcg ml2 at 50 mM (0.4 \u03bcl) was applied on the grid, and immediately before plunge freezing, 1.6 \u03bcl of V-ATPase:mEAK-7 mixture was added and mixed by pipetting for 2 s. This procedure results in a final concentration of 10 mM ATP and requires a total of 5 s from mixing to plunge freezing.Purified V-ATPase was concentrated to \u223c20 mg/ml and 1.2 \u03bcl was applied onto the copper side of nanofabricated gold grids  in the e\u2212/\u00c52, were collected. For V-ATPase with recombinant mEAK-7, mEAK-7\u0394Cterm, mEAK-7 plus ATP, and mEAK-7 plus EDTA with EGTA, cryoEM data were acquired with the same electron microscope. However, for these datasets, a lower magnification was used with a calibrated pixel size of 1.3225 \u00c5/pixel, and 5,142, 2,013, 5,498, and 2,468 movies consisting of 29 exposure fractions each with total exposures of 36, 37, 44, and 41 e\u2212/\u00c52, respectively, were collected. For the V-ATPase with mEAK-7 and calcium, a Thermo Fisher Scientific Glacios electron microscope and Falcon 4 camera with Selectris X energy filter was used. The slit width was 10 eV, and aberration-free image shift was used during data collection. The calibrated pixel size was 0.895 \u00c5/pixel and 3,209 movies with 1,288 frames in EER mode .For V-ATPase alone, movies were aligned with UCSF MotionCor2  through elion v3  and proceference . 2D claseference . This coolishing  and tranRelion signal subtraction and focused classification strategy  and was applied to the map. Map-to-model FSC was calculated with proc3d in EMAN  (All raw (WIFF and WIFF.SCAN) files were saved in ProHits . mzXML fiz.org/) .5 cells/ml and were transfected with FuGENE 6 (Promega) transfection reagent 24 h later. RAW264.7 cells were seeded on 18 mm glass coverslips at a density of 5 \u00d7 105 cells/ml and transfected 24 h later with FuGENE HD. Cells were transfected with 3 \u03bcl of FuGENE per 1 \u03bcg of total plasmid. Both cell types were imaged 18\u201324 h post-transfection.The sequence for mEAK-7 was amplified from the pET28a(+) vector from GenScript for Gibson assembly (New England Biolabs), cloning into the pmCherry-N1 (Clonetech) expression vector for mammalian cells with the forward primer GTCGCCACCATGgggaattcaaaaagtaggtctggacaaggtctttgcagccgtttc and the reverse primer CTCGCCCTTGCTCACatcgtcctccggcaccgcacgcaaaccttcggagtgacggcttct, which maintains the C-terminal mCherry tag. The reverse primer CTCGCCCTTGCTCACttaatcgtcctccggcaccgcacgcaaaccttcggagtgacggct was used to create a plasmid with a stop codon before the mCherry tag for expression of mEAK-7 without the tag. HeLa and HEK293T cells were seeded on 18 mm glass coverslips at a density of 3 \u00d7 10To visualize lysosomal compartments, HeLa cells transiently expressing mEAK-7-mCherry were incubated with 250 \u03bcg/ml fluorescein isothiocyanate (FITC)-conjugated 10 kD-dextran at the time of transfection and chased with complete medium 1 h before imaging. HEK293T cells were transiently transfected with mEAK7-mCherry and LAMP1-GFP to label the lysosomes. Coverslips with cells were mounted in a Chamlide magnetic chamber, incubated at 37\u00b0C in HBSS medium, and visualized with a Zeiss Axiovert 200 M confocal microscope operated by Volocity v6.3 software. Images were acquired using a 63\u00d7/1.4 NA oil objective (Zeiss) with an additional 1.5\u00d7 magnifying lens and processed using Volocity v6.3 and Adobe Illustrator.+ solutions  at different pH values , containing 10 \u03bcM nigericin and 5 \u03bcM monensin. Cells were imaged 5 min after the addition of each solution to obtain the 490 nm/440 nm fluorescence ratio corresponding to each pH standard. A calibration curve was prepared from a least-squares fit of mean background-subtracted fluorescence ratios as a function of pH. The pH measurements were compared with an unpaired two-tailed t test.To measure pH of lysosomes, HEK293T cells transiently transfected with unlabelled mEAK-7 and PLC\u03b4-PH-RFP  were incubated with 250 \u03bcg/ml FITC-conjugated 10-kD dextran at the time of transfection and chased with complete medium 1 h before imaging to visualize lysosomal compartments. RAW264.7 cells co-transfected with unlabelled mEAK-7 and PLC\u03b4-PH-RFP were incubated with FITC-conjugated zymosan particles, which were taken up into phagosomes, 1 h before imaging and were chased with complete medium 30 min before imaging to wash away unbound particles. Steady-state lysosomal and phagosomal pH were determined by exciting FITC labelled lysosome/phagosomes at 481 \u00b1 15 nm and 436 \u00b1 20 nm, respectively, and collecting emitted light at 520 \u00b1 35 nm. The pH-dependent fluorescence intensity of FITC when excited at \u223c490 nm was used as described below to determine the steady-state pH of lysosomes. The relatively pH-insensitive fluorescence intensity of FITC when excited at \u223c440 nm was used to control for photobleaching during image acquisition. Multiple fields of cells were imaged for each condition, and the data were processed with Volocity v6.3. To convert fluorescence ratios to pH values, cells were sequentially subjected to isotonic KEMD-26385, EMD-26386, EMD-26387, and EMD-26388. Models have been deposited into the PDB with accession codes 7U8O, 7U8P, 7U8Q, and 7U8R. Mass spectrometry data for proteins that bind mEAK-7 were deposited into the ProteomeXchange with accession code PXD034953, through partner MassIVE with accession code MSV000089750.CryoEM maps have been deposited into the EMDB with accession codes"}
+{"text": "The second near-infrared (NIR-II) window is a fundamental modality for deep-tissue in vivo imaging. However, it is challenging to synthesize NIR-II probes with high quantum yields (QYs), good biocompatibility, satisfactory pharmacokinetics, and tunable biological properties. Conventional long-wavelength probes, such as inorganic probes  and organic dyes (which contain large \u03c0-conjugated groups), exhibit poor biosafety, low QYs, and/or uncontrollable pharmacokinetic properties. Herein, we present a bioengineering strategy that can replace the conventional chemical synthesis methods for generating NIR-II contrast agents. We use a genetic engineering technique to obtain a series of albumin fragments and recombinant proteins containing one or multiple domains that form covalent bonds with chloro-containing cyanine dyes. These albumin variants protect the inserted dyes and remarkably enhance their brightness. The albumin variants can also be genetically edited to develop size-tunable complexes with precisely tailored pharmacokinetics. The proteins can also be conjugated to biofunctional molecules without impacting the complexed dyes. This combination of albumin mutants and clinically-used cyanine dyes can help widen the clinical application prospects of NIR-II fluorophores. It is currently difficult to synthesise NIR-II probes with good quantum yields, biocompatibility and pharmacokinetics. Here the authors report a strategy to alter these properties by modifying the protein coatings with biofunctional molecules, and generate long-wavelength fluorophores for in vivo imaging. The development of high-quality NIR-II fluorophores is essential to realizing deep bioimaging11 and facilitating clinical translation. For example, clinically used cyanine dyes14 were discovered to possess a tail peak in the NIR-II window when viewed using an InGaAs camera20. However, the majority of organic NIR-II fluorophores exhibit very low fluorescence quantum yields (QYs)19 and/or poor pharmacokinetic properties. Moreover, multiple and complex synthetic steps are followed to synthesize these fluorophores22. The photoluminescence of small molecules is controlled by their energy gap, which depends on their chemical structure through \u03c0-conjugated groups23. Hence, long-wavelength fluorophores bear large \u03c0-conjugated groups 26. However, fluorophores with large \u03c0-conjugated groups usually have inherently low QYs and poor pharmacokinetics18.Near-infrared II (NIR-II) fluorescence imaging is preferrable to near-infrared I (NIR-I) imaging as this window provides reduced tissue scattering and autofluorescence, thus achieving deeper tissue penetration with superior contrast and resolution3. Additionally, large conjugated fluorophores are usually hydrophobic and do not exhibit satisfying pharmacokinetic properties22. In recent years, efforts have been made to synthesize rigid structures bearing electron-withdrawing groups at the center of the scaffold and/or to introduce shielding groups to reduce collisional quenching29. For example, rigid cyclohexanol structures have been introduced at the center of cyanine/polymethine dyes to produce improved dye variants. Researchers introduced a rigid alkyl thiophene moiety to increase the dihedral angle in a donor-acceptor-donor (D-A-D) dye to improve QYs19. Shielding units containing long polyethylene glycol (PEG) groups have also been used to develop good-performance D-A-D dyes and reduce collisional quenching. Some researchers have introduced dialkoxy-substituted benzene- and/or fluorene-based groups as the shielding units  to develop S-D-A-D-S fluorophores that exhibit relatively high QYs. By modifying the PEG and S groups, the dispersion of these molecules in solution can be improved and nonradiative decay caused by collisional quenching/\u03c0-\u03c0 stacking can be reduced31.Large \u03c0-bridges can potentially induce nonradiative decay through vibrational relaxation, collisional quenching, and/or \u03c0-\u03c0 stacking-induced quenching. Molecular simulation studies revealed that the low QYs exhibited by fluorophores containing large \u03c0-bridges could be attributed to the properties of the excited states of the \u03c0-bridges. The excited states can be directly attacked and subsequently quenched by water molecules and they are also prone to oxidation7 that can potentially influence the QY of the dyes. Moreover, some cyanine/polymethine dyes, such as the clinically used indocyanine green (ICG), do not possess modifiable units in their scaffolds and so cannot be easily conjugated to other moieties. Most importantly, achieving a good-performance fluorophore with high QY, biosafety, clinical translation potential, and satisfying pharmacokinetic properties as well as terrific biofunction is extremely challenging. We have previously reported that cyanine dyes can \u201chitchhike\u201d with serum albumin in vivo to produce ultra-bright NIR-II fluorophores with improved pharmacokinetics16. The cyanine dyes inserted into a hydrophobic pocket of albumin, which stabilized the dyes and so reduced nonradiative decay and synergistically increased the extent of \u03c0-conjugation. The protein shell also protected the dyes from collisional quenching and provided biocompatibility. Inspired by this work, here we screen high-affinity recombinant subunits of albumin that could be used to generate a series of editable protein shells to chaperone dyes. Using yeast to express fragments of albumin, we successfully identify the exact binding domain for cyanine dyes. This allows us to re-engineer a series of minimal subunit-incorporating albumin variants. We use this genetic engineering technique to tune the size and brightness of cyanine/polymethine dye@protein complexes  to identify the exact binding domain for cyanine dyes. HSA consists of three independent subdomains that contain highly homologous sequences: domain I (DI), domain II (DII), and domain III (DIII)Kd\u2009=\u20090.57\u2009nM) than HSA (Kd\u2009=\u20091.7\u2009nM)  and slower dissociation (Koff\u2009=\u20090.1\u2009s\u22121) with DIII compared to HSA   Fig.\u00a0. IR-783 PBS Fig.\u00a0. We furtPBS Fig.\u00a0. The resThe binding behavior and loading capacity of cyanine dyes and albumin were further studied by ultra-high performance liquid chromatography\u2013mass spectrometry (UHPLC-MS). Each DIII molecule bound only one IR-783 molecule . The results indicated that the dye-labeled peptide sequence was C(dye)C(carbamidomethylation)TESLVNR. The b and y ion series were further analyzed to identify the exact binding residue. The full mass (MS1) of the peptide was calculated to be 1770.749\u2009m/z (measurement accuracy\u2009<\u20095 ppm). The measurement accuracy for the fragments was\u2009<\u200910 ppm . The results revealed that the Cys476 residue was the potential binding site  and Cl\u2013C groups and to understand the reaction mechanism , prostate-specific membrane antigen-617 (PSMA-617) and cyclic Arg-Gly-Asp (cRGD), which specifically bind to somatostatin receptor (SSTR), PSMA and integrin \u03b1V\u03b23, respectively. SH-modified TATE, PSMA-617 and cRGD were first reacted with maleimide-NHS ester. Following this, the purified products were conjugated to DIII Fig.\u00a0. The reaPichia pastoris with their 3D structures retained  in DIII that binds cyanine dyes, resulting in increased brightness. Using this information, the independent DIIIa and DIIIb subunits were expressed by recombinant ned Fig.\u00a041\u201343. Thnts Fig.\u00a0. To idennts Fig.\u00a0. The brints Fig.\u00a0. Thus, tSince DIIIa is a fragment of DIII with approximately half its molecule weight, we hypothesized that the rate of renal excretion of IR-783@DIIIa would be higher than that of IR-783@DIII. The pharmacokinetic properties of IR-783@DIIIa were evaluated in healthy mice Fig.\u00a0. As expe32. The three domains of albumin  are homologous  of endogenous albumin if the DI, DII, or DIII units are substituted with each other. Hence, we re-engineered a recombinant HSA plasmid with 3 copies of the DIII sequence (triple DIII or TDIII)  and (glycine-glycine-glycine-glycine-serine)2 (-[Gly-Gly-Gly-Gly-Ser]2-). After the recombinant Pichia pastoris clones were identified by polymerase chain reaction (PCR), TDIII was expressed in Pichia pastoris and verified by SDS-PAGE  without altering the inserted cyanine dyes. However, the ideal pharmacokinetics of a fluorophore depend on the imaging application. For example, renally cleared probes are preferrable for molecular imaging due to their safety, while long-circulating probes are useful for vascular or lymphatic imaging. Long-circulating probes are also preferred for imaging-guided surgery. As dye@DIII/DIIIa are rapidly cleared through the renal system, they cannot function as long-circulating probes. IR-783@albumin was shown to be a long-circulating probe with a circulation time of 19 days, comparable to that of endogenous albuminII) Fig.\u00a0. To retaAGE Fig.\u00a0. And alsAGE Fig.\u00a0.Fig. 6Dyn\u2009=\u20095). The dye-to-protein molar ratios investigated were 0.25:1, 0.5:1, 0.75:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1 and 16:1. The samples were then exposed to a xenon lamp (780\u2009nm) and the fluorescence intensity of each sample was detected by NIR-I (700\u2013900\u2009nm) and NIR-II (1100\u20131300\u2009nm) detectors  were mixed with various molar concentrations of IR-783  NIR-II imaging was performed in mice with shaved heads, and vessels underneath the skull and skin were imaged with high resolution  and imaged lymphatic vessels. The ultra-bright IR-783@TDIII was able to illuminate the tiny lymphatic vessels with a resolution of 87.8 \u03bcm per pixel Fig.\u00a0. NIR-II 16. Thus, dye@TDIII could be excellent candidates for imaging-guided surgery with translational potential.Intraoperative navigation technique is widely used in the field of medicine. Currently, various cyanine dyes are used in the clinic, but these exhibit rapid photobleaching and clearance. We hypothesized that dye@TDIII could address the existing problems in the field. We first compared IR-783@TDIII to the clinically used ICG to study the optical properties of the probes  for binding cyanine dyes. To our surprise, we observed that Cl-containing cyanine dyes formed covalent bonds with endogenous or mutant albumin, while Cl-free dyes formed non-covalent bonds. Some researchers have reported that Cys34 on albumin could covalently bind with IR-78351. However, the binding site was speculated on Cys34 since it is the only free cystine on albumin. In this study, we systematically studied and discovered a binding site (Cys476) on subdomain IIIa which could also covalently bond with Cl-containing cyanine dyes, and this finding has never been reported before. To confirm our finding, we used domain III or subdomain IIIa (without Cys34) instead of full albumin to react with Cl-containing dyes, and systematically studied the nucleophilic substitution of Cys476 and Cl-containing dyes. Based on this finding, we designed a series of recombinant albumin variants to chaperone Cl-containing dyes for in vivo NIR-II bioimaging. Furthermore, we determined that the interaction between cyanine dyes and albumin occurs in two independent stages and studied the mechanism of brightness enhancement. In the first stage, dyes bind to albumin or albumin mutants through non-covalent bonds, resulting in increased brightness though strengthened TICT. In the second stage, a \u201cclasp\u201d formed by the SH\u2013 group of Cys476 and the Cl\u2013C group of Cl-containing dyes stabilizes the dyes through nucleophilic substitution. One group has observed some absorption spectra shifts  but with no explanation52, while, according to this phenomenon, another group provided with a possible explanation that a noncovalent adduct would be slowly transformed covalent51. However, they did not provide with more evidence for the speculation.The discovery of an albumin chaperone that functions as a brightness amplifierThis discovery provides a platform for the development of dyes that can attach themselves to serum albumin for improved pharmacokinetics. Cyanine dyes protected with a shell of albumin mutant or variant were shown to have enhanced optical properties. We also showed that the shells can be modified by fusion with a biofunctional peptide or conjugation with a small molecule. Additionally, the sizes of the bioengineered shells can be tuned to tailor the pharmacokinetic properties and clearance pathway. These excellent properties make the molecules suitable candidates for use in the fields of molecular imaging and imaging navigated surgery. In this study, we presented several applications in biofunctional imaging and imaging-guided surgery using complexes composed of bioengineered albumin fragments of various sizes with tailored pharmacokinetics and clearance pathways. We also designed an albumin mimic composed of three tandem DIII, which showed the highest brightness enhancement and generated dye complexes with albumin-like in vivo behavior.In conclusion, we demonstrated an alternative strategy to conventional chemical modification techniques to develop targeted NIR-II probes. We developed a facile strategy to design bright and editable NIR-II fluorophores that exhibit tailored pharmacokinetic properties to address the problems of existing NIR-II probes. Our innovation uses existing cyanine dyes with peak emission at ~800\u2009nm, which could achieve full color imaging in both the NIR-I and NIR-II windows to provide accurate diagnosis and guide surgery. We believe that development of cyanine dyes with peak emission at 1000\u20131500\u2009nm will further promote our genetic engineering strategy. More importantly, this genetic engineering strategy would provide us with a platform for modifying NIR-II dyes and exhibit an additional perspective to design dyes. Our strategy can help the design and development of NIR-II probes with clinical translation potential in the near future.The investigators adhered fully to the \u201cGuide for the Care and Use of Animals\u201d by the NIH Clinical Center Animal Care and Use Committee (NIHACUC), and all animal work was conducted in compliance with protocols approved by the NIHACUC.The aimed sequences of DIIIa and DIIIb were designed as follows. To make sure the connection part of DIIIa and DIIIb including, we designed an overlay segment with several amino acid residues (highlighted by blue color) on each of them.The amino acid sequence of DIIIa (381\u2013494):LEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSThe amino acid sequence of DIIIb (490\u2013585):KTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKATo make sure the subdomains were successfully expressed and purified , we used two strategies to achieve the small proteins: His tag and GST tag. Typically, a thrombin cleavage site  was insert between tag and the targeting protein, respectively.GST tag sequence:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDHence, the GST-LVPRGS-DIIIa/DIIIb:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS*MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPEFPGRLERPHRDLVPRGSKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*After achieved the above corresponding pPIC9K recombinant plasmid, we transformed them into GS115 strain. Specifically, the pPIC9K-DIIIa/DIIIb plasmid was linearized with XhoI and EcoR1, respectively. Then, GS115 strain was transformed by electroporation. Recombinant Pichia clones were isolated on RD plate. After the clones appeared in the plate, washed the clones with sterile water, and put them onto YPD\u2009+\u2009GS115 (1000\u2009\u00b5g/ml) plate. Recombinant Pichia clones were identified by PCR.PCR primes, suggest by Invitrogen yeast protocol, are documented as below:5\u2032AOX: 5\u2032 ctgctgatagcctaacgttc 3\u20323\u2032AOX: 5\u2032 gctgatcaggagcaagctcg 3\u2032\u22121 in 25\u2009mM sodium phosphate, 100\u2009mM sodium sulphate, 0.05 % (w / v) sodium azide, pH 7.0. The column was TSK G3000SWXL (Tosoh Bioscience), and the eluted proteins were detected by UV at 280\u2009nm compared to an HSA standard. Finally, proteins were analyzed using SDS-PAGE in PhastGelTM electrophoresis system and appropriate media.Then, yeast (1\u2009mL) was inoculated into 10\u2009ml BMMD medium (2 % w / v glucose), and grown for 48\u2009h on a rotary shaker of 250 r.p.m. at 30\u2009\u00b0C. After that, 4\u2009ml of each culture was inoculated in 2\u2009\u00d7\u2009200\u2009ml BMMD media to grow for 120\u2009h on a rotary shaker of 250\u2009r.p.m at 30\u2009\u00b0C. Proteins produced by recombinant Pichia were purified using the AlbuPure matrix (ProMetic BioSciences). Briefly, the supernatant was collected after centrifugation and filtered with 0.2\u2009\u00b5m vacuum filter membranes (Millipore). Subsequently, a Pall Filtron LV system equipped with an Omega 10\u2009kDa filter  was employed to further concentrate the filtered supernatant. The column was equilibrated with 50\u2009mM sodium acetate pH 5.3 and loaded with the concentrated supernatant. After that, the column was washed with 10 column volume (CV) of equilibration buffer and 50\u2009mM ammonium acetate pH 8.0 (10 CV) to elute the unbound fraction. The bound protein was eluted with a series of buffers, 50\u2009mM ammonium acetate, 10\u2009mM octanoate pH 8.0, 50\u2009mM ammonium acetate, 30\u2009mM sodium octanoate pH 8.0 or 200\u2009mM potassium thiocyanate, respectively. Then, the eluted protein was concentrated and filtered against 10 CV of 50\u2009mM NaCl by Vivaspin20 10\u2009kDa PES (Sartorius). GP- HPLC was performed to quantify the HSA variants. Samples were chromatographed at a flow rate of 1\u2009ml\u2009minThe DNA sequence of TDIII:ATGAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGACGAGTTCAAGCCATTGGTTGAGGAACCACAGAACCTGATCAAGCAGAACTGTGAGTTGTTCGAGCAGCTGGGTGAGTACAAGTTCCAGAACGCTTTGTTGGTCAGATACACCAAGAAGGTCCCACAGGTTTCCACTCCAACCTTGGTTGAAGTCTCCAGAAACCTTGGTAAGGTCGGTTCCAAGTGTTGTAAGCACCCTGAGGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTCTTGAACCAGTTGTGCGTCTTGCACGAAAAGACTCCAGTTTCCGACAGAGTTACCAAGTGCTGTACTGAGTCCTTGGTCAACAGACGTCCATGTTTCTCTGCTTTGGAGGTCGACGAAACCTACGTGCCAAAAGAGTTCAACGCTGAGACTTTCACTTTCCACGCTGACATCTGTACCCTGTCCGAAAAAGAGAGACAGATCAAGAAGCAGACTGCCTTGGTCGAGTTGGTTAAGCACAAGCCAAAGGCTACCAAAGAGCAGTTGAAGGCTGTTATGGATGACTTCGCTGCCTTCGTTGAGAAGTGTTGCAAGGCTTTGGAAAAGTGTTGCGCAGCTGCAGATCCTCATGAGTGTTACGCCAAAGTCTTTGATGAGTTTAAGCCCCTGGTCGAAGAACCCCAAAACTTGATTAAGCAAAACTGCGAACTGTTTGAGCAATTGGGCGAGTACAAATTTCAAAACGCCCTGCTGGTTAGGTACACTAAGAAAGTTCCTCAGGTGTCTACCCCAACTTTGGTCGAGGTTTCTAGGAACTTGGGTAAAGTGGGTTCTAAGTGCTGCAAACATCCAGAGGCCAAAAGAATGCCTTGCGCAGAGGACTACTTGTCTGTTGTTCTGAACCAGCTTTGTGTGCTGCACGAGAAAACCCCAGTCTCTGATAGAGTCACCAAATGTTGCACCGAGTCTCTGGTTAACCGTAGACCATGTTTTTCCGCCTTGGAAGTGGATGAGACTTACGTCCCTAAAGAGTTTAACGCCGAAACCTTTACCTTTCACGCCGATATCTGCACTTTGTCTGAGAAAGAGCGTCAGATTAAGAAACAAACCGCTCTGGTCGAACTTGTCAAGCACAAACCTAAAGCCACAAAAGAACAACTGAAGGCCGTCATGGACGATTTTGCCGCTTTTGTTGAGAAATGCTGTAAGGCCCTTGAAAAGTGCTGTGCCGCAGCCGATCCACATGAATGCTATGCTAAAGTGTTCGATGAGTTCAAACCACTTGTGGAAGAACCTCAGAATCTTATCAAACAAAATTGCGAGCTTTTCGAACAGTTGGGAGAGTATAAGTTTCAAAATGCCTTGTTGGTGCGTTACACAAAAAAGGTGCCTCAAGTCTCCACTCCTACTCTGGTTGAGGTTTCCCGTAACCTGGGAAAAGTTGGTAGCAAATGCTGCAAGCACCCCGAAGCTAAACGTATGCCTTGTGCCGAGGATTATCTGAGCGTTGTCTTGAATCAGCTGTGTGTCCTTCATGAGAAAACTCCCGTTTCTGACCGTGTCACTAAGTGTTGTACCGAAAGCTTGGTGAACAGAAGGCCTTGCTTTTCTGCTCTGGAAGTTGACGAGACATATGTTCCCAAAGAGTTCAACGCAGAAACATTCACATTTCATGCAGACATCTGCACACTTAGCGAGAAAGAAAGGCAAATCAAAAAGCAAACAGCCCTGGTTGAGCTGGTCAAACATAAGCCCAAGGCCACAAAAGAGCAGCTTAAAGCAGTAATGGACGATTTCGCTGCATTTGTCGAAAAGTGTTGTAAAGCCTAAGCGGCCGCThe amino acid sequence of TDIII MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA*The His tag was used in this sequence design since the TDIII is a big protein with molecule weight similarly equal to wild type albumin.The amino acid sequence fused with cleavage site for thrombin and His tag (TDIII-LVPRGS-His tag).MKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKALVPRGSHHHHHH.Most of the cyanine dyes were purchased from Sigma-Aldrich. IR-12N3 was purchased from Nirmidas Biotech Co. Indocyanine Green  for human injection was purchased from Dandong Yichuang Pharmaceutical Co., LTD. PBS was purchased from HyClone. Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) were purchased from Sigma-Aldrich. Fetal Bovine Serum (FBS) was purchased from Neuromics. Sucrose was purchased from Sigma-Aldrich.Binding affinity of cyanine dyes with albumin variants was determined by biolayer interferometry (BLI) by interaction using an Octet Red96 system (fort\u00e9Bio), respectively. Because of the same protocol, binding affinity of IR-783 with HSA was taken as an example. IR-783 powder was freshly dissolved in anhydrous DMSO (26.7\u2009mM). Then, IR-783 is serially diluted into different concentrations  by PBS. The assay protocol was briefly described as follows. After washing with 1\u2009\u00d7\u2009PBS for 60\u2009s, biotinylated HSA (1\u2009\u03bcg/mL) was loaded to the biosensor for 600\u2009s, and after another 60\u2009s washing, quenching with biocytin was performed for 180\u2009s. Followed by another 60\u2009s washing, association for 600\u2009s and dissociation for another 600\u2009s were performed in turn. Data was calculated and analyzed using Octet Analysis software v 7.0.Synthesis of dye@HSA and dye@HSA-domains/variants complex shared the same protocol and IR-783-DIII complex was taken as an example. DIII powder was dissolved in 1XPBS with a concentration of 20\u2009mg/mL (855\u2009\u00b5M); IR-783 powder was freshly dissolved in anhydrous DMSO (26.7\u2009mM). 500\u2009\u00b5L DIII PBS solution was first added in 500\u2009\u00b5L PBS under slight vortexing. Then, 8, 16, 32, 48, 96 \u03bcL\u00a0of\u00a0the IR-783 (26.7\u2009mM) was added into the DIII solutions, respectively . Finally, the suspension was vortexed for 30\u2009s and heated at 60\u2009\u00b0C for 10\u2009min.Since we do not know if the conjugation of TATE/PSMA-617 would impact the binding of IR-783, we attempted to add IR-783 before or after the conjugation of TATE/PSMA. Results indicated that both routes did not lead to obvious fluorescence diminishing; hence, we conclude that tailoring DIII does not impact the in vitro and in vivo behavior of the fluorophores. IR-783@DIII or DIIIa (TDIII) was reacted with maleimide-PEG-NHS ester (Sigma) at RT. for 4\u2009h under shaker. Then products  were filtered with 10 k filter against PBS for 5 times to move unreacted maleimide-PEG-NHS ester. The maleimide-PEG-NHS labeled compounds were incubated with PSMA-SH and TATE-SH peptides (molar ratio of protein and peptide was 1:5), as described in our previous paper. After that, the conjugates were washed with 10\u2009K filter against PBS for 5 times.UV-Vis-NIR spectrophotometer (Cary 6000i) with background correction was employed to measure the optical absorption spectrum. Fluorescence spectrophotometry was carried out on a Hitachi F-7000 fluorescence spectrophotometer. Fluorescence microscopy was performed on an Olympus fluorescence microscope.DIII-cRGD was mixed with HCCA (\u03b1-cyano-4-hydroxycinnamic acid) in a 1:1 ratio, 2\u2009\u03bcL of sample and 2\u2009\u03bcL of HCCA. Sample matrix was mixed and loaded onto a MALDI target plate (MTP) 384 by direct droplet method. It was allowed to dry for a few minutes. MALDI-TOF mass spectrometry was performed on Autoflex maX (Bruker) instrument. Finally, the data was plotted using Graphpad 8.0.Mice were shaved using Nair hair removal cream and anesthetized using isoflurane before placing them for injection of imaging agents. For each imaging experiment, at least 3 mice were used as a parallel group cohort. All NIR-II images were collected on a Princeton InGaAs array. The excitation laser was an 808\u2009nm laser set-up. Emission was typically collected with different long pass filters. A lens set was used for obtaining tunable magnifications, ranging from 1\u00d7 (whole body) to 2.5\u00d7 (high magnification) by changing the relative position of two NIR achromats . NIR-II microscopy was built based on the previous report, and the excitation laser was a 785\u2009nm laser set-up.2, 37\u2009\u00b0C). All cells were tested to be free of mycoplasma.AR42J (from ATCC) were cultured in F-12k medium with 20% heat-inactivated FBS and 1% penicillin and streptomycin. PC3 (from ATCC), PC3-PIP (from NIBIB at NIH) and 4T1-fluc cell lines (from ATCC) were cultured in RPMI-1640 medium with 10% heat-inactivated FBS and 1% penicillin and streptomycin. Cells were grown in a humidified atmosphere (5% CO6) were inoculated to nude mice to grow subcutaneous tumor, respectively. 4T1-fluc cells (1.0\u2009\u00d7\u2009105) were inoculated in the mammary fat pad to establish the orthotopic mammary tumor model/metastatic tumor model.Nude mice , C57BL/6j mice  and balb/c mice  were purchased from Jackson\u2019s Laboratory . Bedding, nesting material, food, and water were provided ad libitum. Ambient temperature was controlled at 20 to 22\u2009\u00b0C with 12\u2009h light/12\u2009h dark cycles. AR42J or PC3 cells  were intravenously injected with PBS, DIII, IR-783@DIII, IR-783@HSA, IR-783@DIIIa, and IR-783@TDIII, respectively. Peripheral blood was collected to analyze subtypes of lymphatic cells. Briefly, blood was collected from the treated mice on day 3, day 7 and day 14. Blood cells were enriched by centrifugation. Red blood cells were lysed using ACK lysis buffer for 10\u2009min at room temperature. Then, cells were washed twice in PBS and stained with the according antibodies for 15\u2009min. After that, cells were washed with FCS buffer (PBS buffer with 0.1% FBS), and resuspended for flow cytometric analysis. Flow cytometry was conducted on a Beckman CytoFlex S flow cytometer. Data were analyzed using FlowJo V10 version.The ground state (S0) geometries of IR-783 and ICG molecules were optimized using the B3LYP-GD3GJ/6-31\u2009G(d) method dispersion correction. The excited state (S1) properties were calculated using the time-dependent (TD) LC-BLYP*/6-31\u2009G(d) method with the polarizable continuum model (PCM). The HOMO and LUMO distributions were displayed using VMD code. All the (TD)DFT calculations were performed using Gaussian 16 software.Further information on research design is available in the\u00a0Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSuppl Movie 1Suppl Movie 2Suppl Movie 3"}
+{"text": "Caenorhabditis elegans. We observed that both the catalytic (KIN-3::V5) and regulatory (KIN-10::2xMyc) subunits of the Casein Kinase II (CK2) holoenzyme complex are associated with meiotic DNA, enriched in the midvalent rings during meiotic divisions in fertilizedC. elegans oocytes.By using CRISPR/Cas9 genome-editing, we have generated epitope-tagged KIN-3 and KIN-10 expressing strains at the endogenous C-terminal loci in C. eleganscatalytic and regulatory subunits are encoded bykin-3andkin-10, respectively . We used CRISPR/Cas9 genome-editing to generate epitope-tagged KIN-3::V5 and KIN-10::2xMyc expressing strains at the endogenous C-terminal loci. By staining early embryos with commercially available epitope antibodies, we observed the localization of KIN-3::V5 and KIN-10::2xMyc in fertilizedC. elegansoocytes. During meiosis in fertilizedC. elegansoocytes, both the catalytic (KIN-3) and regulatory (KIN-10) subunits of the CK2 holoenzyme are associated with meiotic DNA, enriched around the center of the bivalent, referred to as the ring complex . It has been shown that AIR-2/Aurora B and KLP-19 localize to the ring complexes associated with meiosis I bivalent and meiosis II chromosomes. Both AIR-2 and KLP-19 are required for proper chromosome segregation duringC. elegansmeiosis . The close association of KIN-3 and KIN-10 with meiotic DNA suggests that CK2 kinase activity might influence chromosome organization and segregation during meiotic divisions in theC. elegans oocyte. In support of this, previous work has reported that depletion of CK2 results in polar body extrusion failure and extra DNA, likely due to meiotic errors in fertilizedC. elegansoocytes . A study in porcine oocytes has also shown that CK2 localizes to meiotic chromosomes and that CK2 activity is required for normal meiotic progression . Thus, CK2 function during meiotic division appears to be evolutionarily conserved.The kinase Casein Kinase II (CK2), a tetrameric holoenzyme, consists of two catalytic (CK2\u03b1) and two regulatory (CK2\u03b2) subunits . TheC. elegansCulture:All strains were derived from the wild-type Bristol N2 strain and maintained on MYOB plates seeded withEscherichia coliOP50 at 20\u00b0C.Immunostaining and Confocal Microscopy: Immunofluorescence and confocal microscopy were performed as described . For immunostaining, the following primary and secondary antibodies were used at 1:3000 dilutions: \u03b1-Myc , \u03b1-V5 , and Alexa Fluor 488 and 568 secondary antibodies . Confocal microscopy was performed using a Nikon Eclipse Ti-U microscope equipped with a Plan Apo 60\u00d71.4 NA lens, a Spinning Disk Confocal (CSU X1), and a Photometrics Evolve 512 camera. MetaMorph software  was used for image acquisition and Adobe Photoshop/Illustrator 2022 for image processing.CRISPR/Cas9 Genome Editing: For genome editing, we used the co-CRISPR technique described previously . To design crRNA, we used the CRISPOR webserver . Animals were microinjected with a mixture of commercially available SpCas9  and custom-designed oligonucleotides  including crRNAs at 0.4\u20130.8 \u00b5g/ml  tracrRNA at 12 \u00b5g/ml, and single-stranded DNA oligonucleotides at 25\u2013100 ng/ml. After injection, we screened fordpy-10(cn64) II/+rollers in F1 progeny and genotyped F2 for the epitope-tag insertion. The genome editing was verified by Sanger Sequencing . All theC. elegansstrains generated in this study produce nearly 100% viable progeny.Single-stranded DNA oligonucleotides homologous repair templates  for genome editing were as follows.GGTAAGCCTATCCCAAATCCTTTGTTGGGTCTGGACTCCACGTAAAATTTCTTTCTATTTTTTTTTTAATTTTCCTGKIN-3::V5 tag at the C-terminus (5'-3'): CATCGAATTCCGCTTCTTCTCAATCCTCCGATGCTAAAATTGACGGCGCTGGAGGTTCCGGTGGTTCTGGTGGATCCGAACAAAAACTGATATCTGAAGAAGACCTTGAGCAGAAGTTGATTAGTGAGGAGGATCTTTGAGCCACTTTCTTCCTTATTTTTGTTTTGATTTCKIN-10::2xMyc tag at the C-terminus (5'-3'): CAAAACAACACGACTCCAGCCGGGCAACAATCTGGCGGCCAGTTCAACAACTATGGTCTCGGTGGCTCTGGTGGAAGTGGAGGCTCAN2: wild-type (CGC), MTU137:kin-3(mhs464[KIN-3::V5]) I (This study), MTU598:kin-10(mhs688[KIN-10::2xMyc]) I (This study)"}
+{"text": "S-adenosyl-L-methionine (SAM) L-tyrosine lyase and catalyzes the L-tyrosine C\u03b1\u2013C\u03b2 bond break to produce dehydroglycine and p-cresol while the radical SAM L-tryptophan lyase NosL cleaves the L-tryptophan C\u03b1\u2013C bond to produce 3-methylindole-2-carboxylic acid. It has been difficult to understand the features that condition one C\u2013C bond break over the other one because the two enzymes display significant primary structure similarities and presumably similar substrate-binding modes. Here, we report the crystal structure of L-tyrosine bound ThiH from Thermosinus carboxydivorans revealing an unusual protonation state of L-tyrosine upon binding. Structural comparison of ThiH with NosL and computational studies of the respective reactions they catalyze show that substrate activation is eased by tunneling effect and that subtle structural changes between the two enzymes affect, in particular, the hydrogen-atom abstraction by the 5\u00b4-deoxyadenosyl radical species, driving the difference in reaction specificity.2-iminoacetate synthase ThiH is a radical  ThiH is a radical SAM L-tyrosine lyase involved in the biosynthesis of the thiazole ring of vitamin B1. Here, the authors report the crystal structure of ThiH in complex with its L-tyrosine substrate, revealing an unexpected protonation state and tunneling effect that lowers the reaction energy barrier. It enables, for example, the formation of acetyl coenzyme A by pyruvate dehydrogenase3 and pyruvate: ferredoxin oxidoreductase4 as well as the transfer of a glycoaldehyde from a ketosugar to an aldosugar by transketolase5. Dehydroglycine (DHG), also termed 2-iminoacetate, is a key precursor in the bacterial biosynthesis of the thiazole ring that constitutes the core of TPP. In aerobic prokaryotes, such as the extensively studied Bacillus subtilis, DHG is produced by the oxygen-dependent glycine oxidase7. Such glycine oxidation is not possible under anaerobic conditions and anaerobes, including the facultative bacterium Escherichia coli, use instead the 2-iminoacetate synthase ThiH that catalyzes DHG formation from L-tyrosine processing , derived from vitamin B1, is a key ubiquitous cofactorS-adenosyl-L-methionine (SAM) superfamily13 and contains one [Fe4S4] cluster that reductively cleaves SAM to form both methionine and a highly reactive 5\u00b4-deoxyadenosyl radical (5\u00b4-dA\u2022) species. Subsequently, 5\u00b4-dA\u2022 abstracts from L-tyrosine, a hydrogen atom, long proposed to be the phenolic hydrogen atom10. By analogy, the same reaction step would be catalyzed by the radical SAM tyrosine lyase HydG involved in the assembly of the [FeFe]-hydrogenase active site15. ThiH and HydG are closely related members of the radical SAM superfamily sharing 27% sequence identity16. Both enzymes catalyze the cleavage of L-tyrosine C\u03b1\u2013C\u03b2 bond to generate p-cresol16 and DHG17. The latter is used by HydG at a second active site of the enzyme to synthesize the CO and CN\u2013 diatomic ligands20 that are part of the so-called [FeFe]-hydrogenase H-cluster22. Conversely, ThiH transfers DHG to ThiG, where it is further processed to produce the thiazole ring of thiamine11.ThiH is a member of the radical 23, involved in the synthesis of the thiopeptide antibiotic nosiheptide24. NosL shares about 23% primary structure identity with them25. But, whereas ThiH and HydG cleave the L-tyrosine C\u03b1\u2013C\u03b2 bond17, NosL cleaves the L-tryptophan C\u03b1-C bond instead and produces a 3-methylindole-2-carboxylic acid (MIA)27 , an observation supported by a subsequent biochemical study28. In addition, structure-based sequence comparisons of NosL with tyrosine lyases ThiH and HydG show that, in the latter enzymes, the corresponding [L-Tyr-NH\u2022] would be formed25. Using electron paramagnetic resonance spectroscopy (EPR), a methylene-centered p-cresyl radical intermediate (p-cresyl\u2022) was trapped during L-tyrosine scission by HydG15 and, more recently, the 5\u00b4-dA\u2022 was trapped using p-coumaric acid instead of L-tyrosine29.These two tyrosine lyases are often compared to the radical SAM tryptophan lyase NosLThermosinus carboxydivorans (Tc) in complex with its L-tyrosine substrate. In this study, an unexpected substrate protonation state, supported by its interactions in our X-ray model, has been confirmed by molecular dynamics (MD) simulations. More specifically, deprotonation of the L-tyrosine hydroxyl moiety is in agreement with the very recent report that, in HydG, the ensuing p-cresyl\u2022 intermediate is most probably deprotonated as well, and corresponds to a 4-oxidobenzyl radical instead30. The description of the mechanism of direct hydrogen atom abstraction by 5\u00b4-dA\u2022 at the amino nitrogen position of L-tyrosine, obtained by using hybrid quantum mechanical / molecular mechanical (QM/MM) methods, supports a highly controlled and tight local environment, inducing a tunneling effect during hydrogen transfer that we have estimated analytically to be about 50% of the reaction barrier. Additional calculations performed on both ThiH and NosL show that this step corresponding to the radical formation [L-Tyr-NH\u2022] ([L-Trp-NH\u2022] in NosL) is crucial for the orientation of the subsequent bond cleavage step . In addition, comparison of ThiH with HydG and NosL has revealed unique structural motions, most likely responsible for specific substrate access and product release sites, showing that these enzymes work as assembly lines. Thus, by combining X-ray crystallography and computational chemistry, we provide a rarely achieved mechanistic insight into the hydrogen-atom abstraction step catalyzed by radical SAM enzymes and underscore how subtle changes at the active site afford both substrate selectivity and chemical regiospecificity.Here, we report the crystal structure of ThiH from TcThiH was incubated with its substrate L-tyrosine, 5\u00b4-deoxyadenosine (5\u00b4-dA) and methionine as mimics of the SAM cleavage products prior to crystallization. Brownish prism-shaped crystals that diffracted to 1.27\u2009\u00c5 resolution were obtained. In this crystal form, two independent ThiH molecules termed chains A and B were present in the asymmetric unit. Surface interaction calculations using the PDBePISA server31 supported the fact that the protein does not form quaternary structures in the crystals and would likely correspond to monomers in solution, in agreement with our previous observation when performing size exclusion chromatography. The refined model . We have also performed a 250-ns molecular dynamics (MD) simulation of L-Tyr bound TcThiH (COO\u2212)  Fig.\u00a0.TcThiH numbering), all the key residues involved in L-tyrosine binding are strictly conserved in both proteins  and 0.25\u2009\u00c5 (molecule B), in the same range derived from comparing molecules A and B of the same crystal (see above). The Fo-Fc residual electron density map at the substrate-binding site indicates features that do not correspond to L-tyrosine, best modeled as two water molecules and a sulfate ion from the crystallization condition 25, in order to identify what makes such difference in substrate specificity. TcThiH and SaNosL share about 23% identity mainly located at key positions either to stabilize the tridimensional architecture or because they are important for their function. Yet, the two structures are very similar and display a RMSD of 2.1\u2009\u00c5 for 281 C\u03b1-atoms superimposed. The main structural differences lay in the N-terminal \u03b1-domain. As can be seen in Fig.\u00a0TcThiH and SaNosL, respectively). Their respective dihedral angles are also the same. Strikingly, the main significant difference corresponds to a one-residue insertion at the C-terminal end of strand \u03b28  and the L-tryptophan bound SaNosL structure we had previously solved25, both crystallized with 5\u00b4-dA, we first focused on the 5\u00b4-dA\u2022 attack on their respective substrates. Since the attack occurs for both models at the substrates\u2019 amino group  for [5\u00b4-dA\u2022\u2009+\u2009L-Tyr]-TcThiH and [5\u00b4-dA\u2022 + L-Trp]-SaNosL, respectively  at the start of the scan. By contrast, for NosL, the C5\u00b4 and adenine parts are energetically crossing, i.e. the characters of LUMO and LUMO\u2009+\u20091 are exchanged and their corresponding energies remain close within ~0.2\u2009eV range. This means that 5\u00b4-dA\u2022 is influenced by subtle changes in its environment. This results in an inflection of the energy increase  in addition to the first lowest point (\u03a6\u2009=\u2009\u221247.9\u00b0) mirroring that of ThiH. As it turns out, this energy scan inflection for NosL opens an alternative path leading ultimately to the C\u03b1\u2013C bond scission whereas the absolute minimum common to both ThiH and NosL would lead for NosL to the non-productive C\u03b1\u2013C\u03b2 bond scission . Hence, we decided to further investigate the impact of a hydrogen tunneling effect for the reaction to proceed  and 25\u2009\u00b0C (SaNosL), corresponding to the optimal growth of T. carboxidivorans and S. actuosus, respectively.The transition state geometries for these reactions were validated by frequency calculations. Strikingly, the unique imaginary frequency exhibits a large absolute value greater than 2000 cmect Fig.\u00a038. In adp-cresyl\u2022 and DHG  to extract a sampling of active site conformations corresponding to normal structural \u201cbreathing\u201d in order to determine whether some of them could separate the products further. As a reference, we first scanned the C\u03b1\u2013C\u03b2 bond break using a reduced quantum model consisting of about 300 atoms extracted from our QM/MM model of [L-Tyr-NH\u2022], and observed that the energy reaches a minimum when C\u03b1 and C\u03b2 are about 2.7\u2009\u00c5 apart but goes back up at greater scanned distances  are necessary for product release.However, a product escape route is observed through a gate defined by residues Y76 and Y181 Fig.\u00a0, left anydG Fig.\u00a0, center;osL Fig.\u00a0, right. TcThiH has shown that the substrate acquires an unusual protonation state upon its binding at the active site. Indeed, the phenol functional group of L-tyrosine is deprotonated and the resulting phenolate stabilized by four anion-dipole interactions with strictly conserved residues in ThiH but also in the tyrosine lyase HydG  between the NcoI and BamHI restriction sites, leading to a protein containing an N-ter Strep-tag and corresponding to the pTcThiH construct. The corresponding DNA sequence is as follow:The synthetic gene of ThiH from CCATGGGACATCATCATCATCATCACTCCGGCACGTTCTATGATGTGATTGAAGATTACCGCCACTTTGATTTCGCAGCGTATTTCGCGAAAGTTACCGATTCTGACGTCCGTCGCATCCTGCGTCAGGATCGCCTGTCAGCCCTGGACTTTCTGACGCTGCTGTCGCCGCAAGCGGAAGCCTATCTGGAAGAAATGGCACAGAAAGCTCATCGTCTGACCGTTCAACACTTTGGCCGCACCATGCTGCTGTATACGCCGCTGTACCTGGCGAACTATTGCGTGAATCAGTGCGTTTACTGTGGTTTCCAACTGAAAAACAAACTGGAACGTAAAAAACTGACCCTGGCGGAAGTGGAACAGGAAGCCCAACTGATTGCGGCCACCGGCCTGAAACATATTCTGATCCTGACGGGTGAAAGTCGCCAGCACTCACCGGTCTCGTATATCAAAGATTGTGTGAACATCCTGAAAAAATACTTTAGCTCTATCAGCATCGAAATTTATCCGCTGACCCAGGAAGAATACGCAGAACTGATCGGCGCTGGTGTTGACGGCCTGACGATTTACCAGGAAGTGTATAACGAAGAAGTTTATGCGGAAATGCATCCGGCCGGCCCGAAACGTAATTACCGTTTCCGCCTGGAAGCACCGGAACGTGCATGCCAGGCCGGTATGCGTACCGTGAACATCGGCGCTCTGCTGGGTCTGAATGATTGGCGCCAGGAAGCATTTTTCACGGGTCTGCATGCTGATTATCTGCAACGTCGCTTTCCGGACGTGGAAGTTAGTATTTCCCCGCCGCGTATGCGTCCGCACCTGGGCGGTTTCCCGCCGCGTGTGGTTGTCAGCGATCAGAACCTGGTCCAATATGTGCTGGCATTTCGTCTGTTCATGCCGCGTAGTGGTATCACCCTGTCCACGCGTGAAAATGGTCGTCTGCGTGACGCGATGGTCCGTCTGGGCGTGACCAAAATGAGCGCAGGTTCTTGTACGGCTGTTGGCGGTCGTAGCGATCAGGAAGCCGTCGGCCAGTTCCAAATTTCTGACGAACGCACCGTTGCGGAAGTCGCAGCTATGCTGTACGCCCAGGGTTATCAACCGGTGTACAAAGATTGGCAGGCACTGAGGATCCwhich leads to the following protein sequence:MWSHPQFEKASGTFYDVIEDYRHFDFAAYFAKVTDSDVRRILRQDRLSALDFLTLLSPQAEAYLEEMAQKAHRLTVQHFGRTMLLYTPLYLANYCVNQCVYCGFQLKNKLERKKLTLAEVEQEAQLIAATGLKHILILTGESRQHSPVSYIKDCVNILKKYFSSISIEIYPLTQEEYAELIGAGVDGLTIYQEVYNEEVYAEMHPAGPKRNYRFRLEAPERACQAGMRTVNIGALLGLNDWRQEAFFTGLHADYLQRRFPDVEVSISPPRMRPHLGGFPPRVVVSDQNLVQYVLAFRLFMPRSGITLSTRENGRLRDAMVRLGVTKMSAGSCTAVGGRSDQEAVGQFQISDERTVAEVAAMLYAQGYQPVYKDWQAL.thiH gene was coexpressed with the isc operon and the SAM synthase gene coding metK from E. coli using an E. coli BL21(DE3) strain containing both pTcThiH and our home-made pRSF-ISC-MetK plasmids25. Cells were grown aerobically at 37\u2009\u00b0C under agitation in 4\u2009L of TB medium supplemented with 50\u2009mM MOPS/KOH, pH 7.1, 0.5% D-glucose, ampicillin (100\u2009\u00b5g.ml\u22121) and kanamycine (50\u2009\u00b5g.ml\u22121) up to an OD600 of about 0.6. They were subsequently transferred in 2\u2009L sealed bottles into a glove box with an anaerobic atmosphere containing less than 5\u2009ppm O2 under constant stirring at 20\u2009\u00b0C. After about 15\u2009min, the bottles were opened to equilibrate the medium with the atmosphere of the glove box and the medium was supplemented with 10\u2009mL MEM Vitamin solution 100X (SIGMA M6895); 4\u2009g fumarate; 500\u2009\u00b5L 0.5\u2009M cysteine and 750\u2009\u00b5L 0.2 M L-methionine per L of culture. Fifteen more minutes later, the medium was enriched with 250\u2009\u00b5L 1\u2009M ammonium iron(III) citrate. Protein expression was induced when the OD600 reached 1 with addition of 1\u2009mM final concentration isopropyl-\u03b2-D-1-thiogalactopyranoside and cells were further grown overnight before being harvested and frozen in liquid nitrogen. All the purification steps were performed under anaerobic conditions. Cell pellets were resuspended in the glove box in buffer A  supplemented with EDTA-free protease inhibitor cocktail. They were subsequently disrupted by sonication and the crude-extract was cleared by centrifugation in sealed tubes at 15,000\u2009\u00d7\u2009rpm during 30\u2009min at 4\u2009\u00b0C. The clarified supernatant was filtered, and loaded onto a streptavidin-agarose column (Iba\u00ae) equilibrated with anaerobic cleared lysate was injected into a Strep-tactin column equilibrated with buffer A. After an extensive wash step, the protein was eluted with a 1\u2009mM desthiobiotin solution in buffer A. The iron-sulfur cluster was subsequently reconstituted. The protein was incubated 15\u2009min with 5\u2009mM DTT prior to the addition of 200\u2009nM NifS from Azotobacter vinelandii. 5 excess FeCl3 and L-cysteine were then added and the solution was kept under mild stirring overnight at 20\u2009\u00b0C. The protein was further purified to homogeneity by gel filtration using a GE-healthcare highload 16/600 S200 prep-grade column equilibrated with buffer B (50\u2009mM Tris pH 8 and 100\u2009mM NaCl). The protein eluted as two peaks corresponding to a dimer and a monomer, respectively. Only the fractions corresponding to the monomer (major peak) were pooled, concentrated to 10\u2009mg/mL and stored in liquid nitrogen. Iron content was determined using the method of Fish48 and indicated over 3.9 iron equivalents per ThiH molecule.The recombinant 4)2SO4, 100\u2009mM MES buffer pH 6.5, 15% dioxane and 10\u2009mg/mL TcThiH. The protein was preincubated with 1\u2009mM 5\u00b4-dA and 1 mM L-methionine. When required, 5-excess L-tyrosine was added to the crystallization drop. Crystals were cryoprotected using 25% glycerol in addition to the crystallization condition and were subsequently mounted in cryoloops before flash cooling in liquid propane in the glove box49. Data were collected at SOLEIL at beamlines PROXIMA-1 and PROXIMA-2A and were processed using the XDS package50  as a search model. The initial model was manually corrected using COOT52 and refined using PHENIX53 to an Rwork\u2009=\u20090.153 and Rfree\u2009=\u20090.174 with good geometry and over 98% residues in the most allowed region of the Ramachandran plot54. This model was subsequently used to solve the ligand-free TcThiH crystal structure following the same procedure, leading to a model with good geometry. The corresponding statistics are reported in Supplementary Table\u00a0Crystals were obtained using the vapor diffusion method. Initial conditions were determined by screening 1248 conditions using a Gryphon robot  setup in a glove box and were subsequently manually optimized. Crystals suitable for X-ray diffraction experiments were obtained with 1.4\u20131.6\u2009M (NH55.Details of all calculations are given in the Supplementary Information. All MD simulations, QM and QM/MM calculations were performed within the Schr\u00f6dinger suiteFurther information on research design is available in the\u00a0Supplementary information filePeer Review FileReporting Summary"}
+{"text": "We found that LTBR and CREB1 exhibited a significant upregulation in lungs of mouse model of BPD. LTBR and CREB1 expression were also increased by hyperoxia in A549 and ATII cells. According to results of cell counting kit-8 assay and flow cytometry analysis, silencing of LTBR rescued the suppressive effect of hyperoxia on cell viability and its promotive effect on cell apoptosis of A549 and ATII cells. Bioinformatics revealed CREB1 as a transcriptional factor for LTBR, and the luciferase reporter assay and ChIP assay subsequently confirmed it. The NF-\u03baB pathway was regulated by LTBR. CREB1 induced LTBR expression at the transcriptional level to regulate NF-\u03baB pathway and further modulate A549 and ATII cells viability and apoptosis. In conclusion, this study revealed the CREB1/LTBR/NF-\u03baB pathway in BPD and supported the beneficial role of LTBR silence in BPD by promoting viability and decreasing apoptosis of lung epithelial cells.Bronchopulmonary dysplasia (BPD) is a prevalent chronic pediatric lung disease. Aberrant proliferation and apoptosis of lung epithelial cells are important in the pathogenesis of BPD. Lymphotoxin beta receptor (LTBR) is expressed in lung epithelial cells. Blocking LTBR induces regeneration of lung tissue and reverts airway fibrosis in young and aged mice. This study is aimed at revealing the role of LTBR in BPD. A mouse model of BPD and two  A review research concludes that BPD may be caused by various factors like premature birth, fetal growth restriction, mechanical ventilation, oxygen toxicity, inflammation, and genetic susceptibility [Bronchopulmonary dysplasia (BPD), initially described by Northway et al., is caused by oxygen supply and mechanical ventilation in premature infants with severe respiratory distress syndrome . Accorditibility . Despitetibility , pulmonatibility  were devtibility . Thus, iBPD is a chronic disease featured by disrupting the alveolar together with microvascular development of the peripheral lung. The regeneration or induced growth of type II alveolar epithelial (ATII) cells has been the focus feature of lung regeneration research . Thus, e\u03b2 receptor (LTBR) signaling in ATII cells induces kinase NIK through nonclassical nuclear factor-kappaB (NF-\u03baB) signaling. Blocking LTBR signaling limits broncho-associated lymphoid tissue formation and reduces alveolar epithelial cell apoptosis, thereby driving alveolar regeneration [Activation of the LTneration . LTBR sineration . LTBR mRneration . LTBR exneration , a contiTranscription factors respond to stimuli of the external environment or signals at different stages of development, activate or inhibit gene transcription, and thus control the expression of different genes. It is very important to identify key transcription factors in BPD. cAMP responsive element binding protein 1 (CREB1) regulates late-stage lung development in mammals . CREB1 i\u03baB signaling plays a crucial role in cell survival, differentiation, and innate immunity [\u03baB dimers are not active in the cytoplasm of cells related to a suppressive protein, I\u03baB. Under the conditions of cytokines, growth factors, or bacterial products, I\u03baB kinases IKK-\u03b1 and -\u03b2 activate NF-\u03baB, leading to I\u03baB phosphorylation and degradation. Clearance of I\u03baB allows translocation of NF-\u03baB complexes into the nucleus and modulates a variety of downstream targets that affect inflammation, cell adhesion, and cell survival [\u03baB-mediated gene modulation is implicated in the pathogenesis of BPD [The NF-immunity . NF-\u03baB dsurvival . Many sts of BPD .in vivo and its role in the apoptosis of lung epithelial cells in vitro. Hyperoxia-stimulated A549 and ATII cells were used as the in vitro model of BPD [\u03baB pathway was assessed, providing new ideas for understanding the pathogenesis of BPD.This study is aimed at evaluating the expression of LTBR in the BPD lung injury mouse model l of BPD , 19. Then = 8) or the control group (n = 8). To establish the BPD model, mice were exposed to 100% O2 in a medium-sized airtight polypropylene chamber on postnatal days 1-4, followed by recovering for 10 days [Neonatal C57BL/6 mice of both sexes  were used for establishing the model of BPD. Animal studies were conducted in compliance with the ARRIVE guidelines under the approval of the animal care and ethic committee of First Affiliated Hospital of Nanjing Medical University. Mice were housed under controlled standard conditions . Neonatal mice in the normoxia group were exposed to room air and fed by lactating mice. Mice were randomly divided into the BPD model group  and dissolved in RNase-free water. Reverse transcription of total RNA into cDNA was performed using a High-Capacity cDNA Reverse Transcription Kit . Next, the QuantiFast SYBR\u00ae Green PCR Kit (QIAGEN) was used for a rapid and specific quantitative detection of target cDNA. PCR analysis was performed on a LightCycer\u00ae480II Instrument, 96-well block . LTBR or CREB1 expression was calculated using the 2t method  and was Mouse LTBR, forward, 5\u2032-TGAACACTGGAACCATCTC-3\u2032; reverse, 5\u2032-ACACTCATTGTCCAGATACAC-3\u2032.Human LTBR, forward, 5\u2032-GAAGGGTAACAACCACTGC-3\u2032; reverse, 5\u2032-CTTGGTTCTCACACCTGGT-3\u2032.Mouse CREB1, forward, 5\u2032-GAAGAGGAGACTTCAGCCC-3\u2032; reverse, 5\u2032-TAATGGCAATGTACTGCCCA-3\u2032.Human CREB1, forward, 5\u2032-ATGCAGCTGTAACAGAAGC-3\u2032; reverse, 5\u2032-CATAGATACCTGGGCTAATGTG-3\u2032.Mouse GAPDH, forward, 5\u2032-ACTCTTCCACCTTCGATGC-3\u2032; reverse, 5\u2032-CCGTATTCATTGTCATACCAGG-3\u2032.Human GAPDH, forward, 5\u2032-TCAAGATCATCAGCAATGCC-3\u2032; reverse, 5\u2032-CGATACCAAAGTTGTCATGGA-3\u2032.The radial alveolar count (RAC) determines alveolar septation and is a test of alveologenesis. Through images at 100\u00d7 magnification, a perpendicular line was made from a respiratory bronchiole to the nearest pleural edge or fibrovascular septum. Airspaces or saccules through this line were counted . Scion I2. A549 and ATII cells were exposed to different concentrations of O2  in a standard modular chamber  at 37\u00b0C to assess the effects of hyperoxia on LTBR and CREB1 expression. The normoxia condition contains 21% O2. Based on the results of the preliminary assays, A549 and ATII cells were exposed to 85% O2 for 48\u2009h to mimic the in vitro model of BPD. Si-NC, si-LTBR, si-CREB1, empty pcDNA 3.1, pcDNA 3.1-LTBR, and pcDNA 3.1-CREB1 were commercially provided by GenePharma . These oligonucleotides or vectors were transfected into A549 and ATII cells using Lipofectamine 3000  for 24\u2009h at 37\u00b0C. The transfection sequences used were as follows: si-NC: 5\u2032-AACCATCACTTACAAGAAACC-3\u2032; si-CREB1: 5\u2032-GCTCGATAAATCTAACAGTTA-3\u2032; si-LTBR: 5\u2032-CCATCCATACTTCCCTGACTT-3\u2032; pcDNA3.1: 5\u2032-CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT-3\u2032; pcDNA 3.1-CREB1: 5\u2032- ATGACCATGGAATCTGGAGCCGAGAACCAGCAGAGTGGAGATGCAGCTGTAACAGAAGCTGAAAACCAACAAATGACAGTTCAAGCCCAGCCACAGATTGCCACATTAGCCCAGGTATCTATGCCAGCAGCTCATGCAACATCATCTGCTCCCACCGTAACTCTAGTACAGCTGCCCAATGGGCAGACAGTTCAAGTCCATGGAGTCATTCAGGCGGCCCAGCCATCAGTTATTCAGTCTCCACAAGTCCAAACAGTTCAGATTTCAACTATTGCAGAAAGTGAAGATTCACAGGAGTCAGTGGATAGTGTAACTGATTCCCAAAAGCGAAGGGAAATTCTTTCAAGGAGGCCTTCCTACAGGAAAATTTTGAATGACTTATCTTCTGATGCACCAGGAGTGCCAAGGATTGAAGAAGAGAAGTCTGAAGAGGAGACTTCAGCACCTGCCATCACCACTGTAACGGTGCCAACTCCAATTTACCAAACTAGCAGTGGACAGTATATTGCCATTACCCAGGGAGGAGCAATACAGCTGGCTAACAATGGTACCGATGGGGTACAGGGCCTGCAAACATTAACCATGACCAATGCAGCAGCCACTCAGCCGGGTACTACCATTCTACAGTATGCACAGACCACTGATGGACAGCAGATCTTAGTGCCCAGCAACCAAGTTGTTGTTCAAGCTGCCTCTGGAGACGTACAAACATACCAGATTCGCACAGCACCCACTAGCACTATTGCCCCTGGAGTTGTTATGGCATCCTCCCCAGCACTTCCTACACAGCCTGCTGAAGAAGCAGCACGAAAGAGAGAGGTCCGTCTAATGAAGAACAGGGAAGCAGCTCGAGAGTGTCGTAGAAAGAAGAAAGAATATGTGAAATGTTTAGAAAACAGAGTGGCAGTGCTTGAAAATCAAAACAAGACATTGATTGAGGAGCTAAAAGCACTTAAGGACCTTTACTGCCACAAATCAGAT-3\u2032 and pcDNA 3.1-LTBR: 5\u2032-ATGGAAGCGACAGGAATCTCATTAGCATCTCAATTAAAGGTGCCTCCATATGCGTCGGAGAACCAGACCTGCAGGGACCAGGAAAAGGAATACTATGAGCCCCAGCACCGCATCTGCTGCTCCCGCTGCCCGCCAGGCACCTATGTCTCAGCTAAATGTAGCCGCATCCGGGACACAGTTTGTGCCACATGTGCCGAGAATTCCTACAACGAGCACTGGAACTACCTGACCATCTGCCAGCTGTGCCGCCCCTGTGACCCAGTGATGGGCCTCGAGGAGATTGCCCCCTGCACAAGCAAACGGAAGACCCAGTGCCGCTGCCAGCCGGGAATGTTCTGTGCTGCCTGGGCCCTCGAGTGTACACACTGCGAGCTACTTTCTGACTGCCCGCCTGGCACTGAAGCCGAGCTCAAAGATGAAGTTGGGAAGGGTAACAACCACTGCGTCCCCTGCAAGGCCGGGCACTTCCAGAATACCTCCTCCCCCAGCGCCCGCTGCCAGCCCCACACCAGGTGTGAGAACCAAGGTCTGGTGGAGGCAGCTCCAGGCACTGCCCAGTCCGACACAACCTGCAAAAATCCATTAGAGCCACTGCCCCCAGAGATGTCAGGAACCATGCTGATGCTGGCCGTTCTGCTGCCACTGGCCTTCTTTCTGCTCCTTGCCACCGTCTTCTCCTGCATCTGGAAGAGCCACCCTTCTCTCTGCAGGAAACTGGGATCGCTGCTCAAGAGGCGTCCGCAGGGAGAGGGACCCAATCCTGTAGCTGGAAGCTGGGAGCCTCCGAAGGCCCATCCATACTTCCCTGACTTGGTACAGCCACTGCTACCCATTTCTGGAGATGTTTCCCCAGTATCCACTGGGCTCCCCGCAGCCCCAGTTTTGGAGGCAGGGGTGCCGCAACAGCAGAGTCCTCTGGACCTGACCAGGGAGCCGCAGTTGGAACCCGGGGAGCAGAGCCAGGTGGCCCACGGTACCAATGGCATTCATGTCACCGGCGGGTCTATGACTATCACTGGCAACATCTACATCTACAATGGACCAGTACTGGGGGGACCACCGGGTCCTGGAGACCTCCCAGCTACCCCCGAACCTCCATACCCCATTCCCGAAGAGGGGGACCCTGGCCCTCCCGGGCTCTCTACACCCCACCAGGAAGATGGCAAGGCTTGGCACCTAGCGGAGACAGAGCACTGTGGTGCCACACCCTCTAACAGGGGCCCAAGGAACCAATTTATCACCCATGACTGA-3\u2032.Lung epithelial A549 cell line was commercially provided by ATCC  and was cultured in F-12\u2009K Medium  supplemented with 10% FBS. Human ATII cells and HEK-293\u2009T cells were purchased from Procell  and cultured in human ATII cell complete culture medium and DMEM, respectively. Cells were maintained in a 37\u00b0C incubator in the atmosphere of 95% and 5% CO2. Next, cells were added with 10\u2009\u03bcL of silencing or overexpressing plasmids. Ten \u03bcL of CCK-8 solution was added, and then cells were cultured in the incubator for 2\u2009h. OD value at 450\u2009nm was read using a SpectraMax iD5 microplate reader .A CCK-8 kit  was applied to detect A549 and ATII cell viability. In brief, transfected cells were plated in 96-well plate and precultured in an incubator at 37\u00b0C and 5% CO\u03bcg/lane) was separated by 11% SDS-PAGE at 110\u2009V for 2\u2009h and transferred to 0.22\u2009\u03bcm PVDF membranes . The membranes were incubated with LTBR antibody , CREB1 antibody , IKK\u03b1 (phospho T23) antibody , IKK\u03b1 antibody , p52 antibody , RELB antibody , or GAPDH antibody  at 4\u00b0C overnight. After washing with PBS, membranes were incubated with the secondary antibody IgG  for 2\u2009h at room temperature. Proteins were detected by enhanced chemiluminescence  based on the manufacturer's instructions. Protein band grey intensity was quantified using the Quantity One software .Total protein was extracted from lung tissues and A549 and ATII cells using RIPA lysis buffer . Protein concentration was measured with a BCA protein assay kit . Total protein  for 15\u2009min at room temperature in dark. Finally, the cells were resuspended in 400\u2009\u03bcL 1\u2009\u00d7\u2009Binding Buffer, mixed on ice, and analyzed by FACS using flow cytometry  within one hour. Cells were gated into four groups: Annexin V\u2212/PI\u2212, Annexin V+/PI+, Annexin V+/PI\u2212, and Annexin V\u2212/PI+. In this study, cell apoptosis rate was defined as the percentage of Annexin V+/PI+ cells (in late apoptosis stage).A549 and ATII cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde overnight at 4\u00b0C. Next, cells were washed again, resuspended in 100\u2009\u03bcg, Abcam) or anti-IgG  for one night. After washing and elution, samples were treated with 5\u2009M NaCl for heating, and then incubated with proteinase K for 1\u2009h. The bound DNA fragments were purified via DNA Extraction Kit  and analyzed by real-time PCR.This assay was performed with a ChIP assay kit  following the manufacturer's instructions. Briefly, A549 and ATII cells were cross-linked with 1% formaldehyde and sonicated to shear DNA to lengths between 200~1000 base pairs. Cell lysates were incubated at 4\u00b0C with protein A/G beads coated with the anti-CREB1 antibody .Wild type (WT) LTBR gene promoter at different sites and the mutated (wt) sites were subcloned into the pGL3 luciferase reporter vector (GeneCopoeia). Mutation was performed using a QuickMutation\u2122 Site-Directed Mutagenesis Kit . HEK-293\u2009T tool cells at the concentration of 5 \u00d7 10t-test and one-way ANOVA with Dunnett's or Tukey's post hoc test. A p < 0.05 was deemed as statistically significant. For in vitro studies, three\u2009biological\u2009replicates \u00d7 three\u2009technical\u2009replicates were applied.Data are presented as mean \u00b1 SD. For expression and luciferase activity values, data are represented as fold changes to mean control. For cell viability values, data are represented as percentage (%) of mean control. GraphPad Prism 8  was used to perform statistical tests including unpaired two-tailed Student's 2 in A549 cells  expression data of LTBR in clinical lung tissues of BPD are lacked; (2) there is a close association of LTBR and WNT/ignaling , while t\u03baB pathway and further modulated lung epithelial cell viability and apoptosis. This study may aid in understating the pathogenesis and treatment of BPD.In conclusion, this work innovatively demonstrated the upregulation of LTBR and CREB1 in BPD. LTBR and CREB1 are positive regulators of cell apoptosis and negative regulators of cell viability of A549 and ATII cells under hyperoxia. CREB1 induced LTBR expression at the transcriptional level to regulate NF-"}
+{"text": "Synema Simon, 1864 is a relatively large genus of family Thomisidae Sundevall, 1833 and currently includes 124 species distributed worldwide, except for the Polar Regions. However, Synema can be regarded as being poorly represented in China, with only seven species, three of which are endemic.Synema from Guiyang City in China, is described under the name of S.guiyang J. Zhang, Q. Lu & H. Yu, sp. nov. Detailed descriptions and photographs of the new species are provided. DNA barcodes  of the species were obtained to confirm matching of the sexes and for future use in molecular studies.A new spider species of the genus  Thomisidae is one of the largest spider families, with 171 genera and 2159 valid species distributed worldwide, 51 genera and 306 species of which are recorded from China , Tmarus Simon, 1875 (226 species) and Thomisus Walckenaer, 1805 (143 species) . Currently, only seven Synema species are known from China, amongst them, three are endemic . Despite being common, the genus remains inadequately studied because: more than half of the species are known from a single sex or juveniles  ; original descriptions are rather brief and lack illustrations or illustrations are inadequate  . CurrentSynema specimens of crab spiders in the same location, which are with similar habitus, markings, leg spination and other characters .A DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for 21 specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters were taken at the widest point. The total body length does not include the length of the chelicerae or spinnerets. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited at the Museum of Guizhou Education University, Guiyang, Guizhou, China .J. Zhang, Q. Lu & H. Yusp. n.BB6AFCEC-3FC5-5F68-A0A0-CFB80E085396C659D2A5-4612-4DCA-9EA3-1A43DBB0A5DCType status:Holotype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO002; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435709; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: Hao Yu; individualID: YHTHO003; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: GenBank: ON435708; Taxon: order: Araneae; family: Thomisidae; genus: Synema; specificEpithet: guiyang; scientificNameAuthorship: J. Zhang, Q. Lu & H. Yu,; Location: continent: Asia; country: China; countryCode: CHN; stateProvince: Guizhou; county: Guiyang City; locality: Guiyang Forest Park; decimalLatitude: 26.55540319; decimalLongitude: 106.75898910; Identification: identifiedBy: Hao Yu; dateIdentified: 2021-11; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2021; month: 8; day: 10; Record Level: institutionID: MGEU; basisOfRecord: PreservedSpecimenMale (holotype)  Fig. A\u2013C. TotaCarapace Fig. A, C yellAbdomen Fig. A\u2013C elongLegs basically yellowish-brown Fig. E, all lePalp Fig. A\u2013D. TibiFemale .DNAbarcode: 5'TATTTGGAGCTTGATCTGCTATAGTAGGGACGGCTATAAGAGTGTTAATTCGTATGGAATTAGGA AGATCTGGAAGATTATTAGGAAATGATCATCTTTATAATGTAATTGTTACCGCTCATGCTTTTGTCATGATTTTTTTTATA GTAATACCTATTTTAATTGGGGGTTTTGGAAATTGATTAGTACCTTTAATGTTAGGGGCTCCTGATATATCTTTCCCTCG GATAAATAATTTATCTTTTTGATTATTACCCCCTTCATTATTTTTACTATTTATATCTTCTATAGTAGAGGTAGGTGTGGGG GCAGGATGAACTGTTTATCCTCCTCTAGCTTCTAGAGTTGGGCATATAGGAGGATCTATAGATTTTGCTATTTTTTCTTT ACATTTAGCTGGGGCTTCTTCTATTATAGGGGCGGTTAATTTTATTTCTACTATTATTAATATACGAACTAGAGGTATAAG AATAGAAAAGGTTCCTTTGTTTGTATGATCTGTATTAATTACAGCTATTTTACTTCTTTTGTCTTTACCTGTATTAGCAGG TGCTATTACTATATTATTAACTGATCGTAATTTTAACACTTCTTTTTTTGATCCTGCAGGGGGAGGGGATCCAATTTTATT TCAACATTTGTTTTGATTTTT3' .S.albomaculatum and S.chikunii in having the similar habitus  ; anterior metatarsi and tibiae with dense hairs (vs. few hairs in Spilosynema); PER slightly recurved (vs. distinctly recurved in Spilosynema). Strictly based on this diagnosis, it seems that the new species should be assigned to the genus Spilosynema . Therefore, it is very strange (or interesting) that S.guiyang sp. nov. exhibits typical somatic features of Spilosynema and genitalic features of Synema. In view of the fact that somatic characters are either poorly marked or variable, they are thus not sufficient for distinguishing the Spilosynema and Synema. Consequently, S.guiyang sp. nov. is assigned tentatively to the genus Synema in the present paper for the lack of a better solution.However, some genitalic characteristics are not mentioned in the diagnosis of ned Fig. B, D. In"}
+{"text": "Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from  Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create novel materials with tailored functions.\u00a0In this work, the authors engineered bacteria to grow novel macroscopic materials that can be reshaped, functionalized, and used to filter contaminated water while also showing that the stiffness of these materials can be tuned through genetic changes. Engineered living materials (ELMs)4 are inspired by naturally occurring living materials, but use synthetic biology to introduce tailored, non-natural functions. By incorporating engineered cells into a biopolymer matrix, these materials can function as living sensors5, therapeutics6, biomanufacturing platforms7, electronics8, energy converters9, and structural materials10. While cells confer functionality to ELMs, the matrix assembles the material and controls the bulk material composition, structure, and function11.Naturally occurring living biomaterials, such as bones or wood, grow bottom-up from a small number of progenitor cells into macroscale structures11 because secreting recombinant biopolymers at concentrations that gelate is challenging12 and because the assembly of micrometer-sized cells into centimeter-scale materials requires self-organization across length scales spanning four orders of magnitude. Engineering principles to achieve this assembly are unknown12. Therefore, most macroscopic ELMs have been produced by adopting a top-down approach (such as 3D printing) to incorporate living cells into an exogenous matrix14 or by processing microscopic ELMs that grow a synthetic biomolecular matrix into macroscopic materials19. The few autonomously produced, macroscopic ELMs have been created by genetically modifying existing nanocellulose matrices20 or genetically manipulating the mineralization of silica matrices. However, these two approaches to autonomously produced, macroscopic ELMs have afforded little genetic control over the mechanical properties, e.g., ~1.2\u20131.4-fold change in the storage modulus21. This tunability is much more limited than the tunability of naturally occurring materials, chemically synthesized materials, or macroscopic ELMs produced by processing23.Since the matrix plays such a key role in generating material properties, one primary goal of the field is to create ELMs that both have a synthetic biomolecular matrix\u2014that can control these properties\u2014and grow autonomously into macroscopic structures. However, such bottom-up, de novo ELMs are considered well beyond the current state-of- artEscherichia coli has been engineered to display interacting proteins, such as leucine zippers24 or antigen-nanobody pairs25, via outer membrane proteins. Engineered strains that display interacting pairs will self-assemble into cell\u2013cell aggregates that flocculate25; however, these aggregates are microscopic and must be processed to form larger materials18. In contrast, micron-sized colloidal particles  that display DNA have been programmed to self-assemble into both microscopic21 and macroscopic crystalline solids26. Over two decades of work on these systems has established central principles that underlie their self-assembly27. One of these central principles is that the interactions between particles must be mediated by high-density surface modifications, e.g., 1 DNA molecule per 27\u2009nm26. Since the outer membrane proteins used for bacterial adhesins are displayed at ~5% of this density, i.e., 1 nanobody per 640\u2009nm28, we hypothesized that a matrix composed of self-interacting proteins displayed on bacteria at high density could lead to the formation of macroscopic solid materials.We posit that new strategies for developing synthetic biomolecular matrices to self-assemble bacteria into macroscopic ELMs can be informed by prior work on surface-engineered bacteria and surface-modified colloidal particles. The surface of Caulobacter crescentus for high-density peptide display29 and biopolymer secretion23. The S-layer forms a 2D crystalline layer on the extracellular surface of C. crescentus, opening the possibility of displaying proteins at a density of up to 1 protein per 70\u2009nm30. Leveraging this prior work, here we describe the autonomous formation of macroscopic living material from C. crescentus engineered to display a synthetic, self-interacting, protein matrix based on the S-layer scaffold. We demonstrate that the mechanical properties\u00a0of this material can be genetically controlled over a factor of ~25x. We also describe unexpected findings indicating that the protein matrix plays a multifaceted role in the material formation and that material assembly occurs through a multi-step process mediated by the air\u2013water interface.We have previously engineered the surface layer (S-layer) of the oligotrophic bacterium C. crescentus31, we sought to create bottom-up ELMs composed of cells that interact at high density through a surface-bound, de novo matrix. To minimize native cell\u2013cell interactions, we started with a C. crescentus background that lacks the adhesive holdfast and therefore cannot form a biofilm33; we refer to this as the wild-type strain. Next, we designed a displayed bottom-up de novo (BUD) protein by replacing the native copy of the surface layer (S-layer) RsaA30  based on human tropoelastin that contains 60 repeats of the Val-Pro-Gly-X-Gly motif 37 (ELP60). ELPs form elastic materials, are flexible, and self-associate in a concentration-dependent fashion. Thus, this region is designed to influence material properties, promote solution accessibility, and add non-covalent self-interactions. SpyTag38 was used as a functionalization tag, as it covalently binds to fusion proteins containing SpyCatcher. We also introduced a FLAG tag as an epitope marker. Lastly, the C-terminal domain of the BUD protein, consisting of the last 336 residues of RsaA, was chosen to mediate protein secretion29 and to self-associate39. We refer to this BUD protein-expressing strain of C. crescentus as the BUD-ELM strain. In this way, we designed a C. crescentus strain to display a high-density, surface-bound, elastin-based matrix across its entire surface  on all sides , and the BUD protein shows this same apparent difference in molecular weight (observed 102 vs. expected 86\u2009kDa). Interestingly, we found secreted BUD protein in the medium under both static and shaking conditions  limit\u201390\u2009\u00b1\u20095% of cadmium was removed  to obtain the holo forms of the enzyme. After confirming the activity of holo GDH in both cases , which couples oxidation of glucose to the reduction of a soluble electron carrierier Fig.\u00a0. This deIn summary, we developed macroscopic living materials that autonomously grow from engineered bacteria and that can be genetically encoded to have a wide range of mechanical properties. Specifically, we show that the expression of a self-interacting protein\u2014the BUD protein\u2014enables macroscopic material formation Fig.\u00a0. When diE. coli with self-interacting proteins displayed at ~10% the density of our engineered C. crescentus strains lead to small cell\u2013cell aggregates25. However, additional studies that systematically vary the surface density are needed to test this hypothesis. Another design rule that will be critical to understand and explore in future work is the nature and strength of the self-interactions in the BUD protein. We selected the RsaA690-1026 and ELP60 domains because prior reports demonstrate they can self-aggregate44. However, additional studies are needed to identify the nature of self-interactions and their strengths in the existing BUD protein and the range of self-interactions that permit the assembly of macroscopic materials.Our work identifies design rules that lead to the autonomous formation of BUD-ELMs and suggests other design rules to be tested. We identify a secreted matrix as a design constraint for this class of centimeter-scale, autonomously forming BUD-ELMs. Our work also indicates that a surface-anchored protein matrix is necessary for these materials to be cell-rich. Our data also suggests that this surface-anchored protein matrix may need to be present at high-density for cell-rich materials. This suggestion is supported by previous literature that shows that 18, we suggest that the use of the air\u2013water interface to locally concentrate and order hydrophobic biomolecules into a matrix may represent a general assembly principle for macroscopic ELMs. The genetic tools and C. crescentus platform developed here will permit systematic exploration of design and assembly rules for programming the growth of centimeter-scale structures using living cells as building blocks.This work also identified assembly principles for the autonomous formation of macroscopic materials. We have demonstrated that nucleation of a pellicle at the liquid-air interface and hydrodynamically-driven coalescence and collapse of the pellicle are required to form macroscopic ELMs. Since pellicle formation is also a key step in nanocellulose-based living materialsC. crescentus BUD-ELM platform developed herein is the highly reproducible, autonomous formation of engineered living materials. Growing BUD-ELMs from an engineered strain of C. crescentus requires only control of the temperature, media composition, flask and culture volume, shaking speed, and shaking orbit. We envision this simplicity will enable the ready adoption of this platform by other researchers. The second advantage of this platform is that the modularity of the BUD protein and the ease of engineering protein biopolymers offer much greater opportunities for introducing desirable properties into the matrix11. The BUD-ELM variants described herein have storage moduli that ranges between 13\u2009kPa, comparable to nanocellulose-based materials, and 0.5\u2009kPa, comparable to printed curli fiber-based materials. Introducing sites for chemical crosslinking into the ELP domain could allow the BUD-ELMs to be developed into elastomers45. More broadly, this work enables leveraging known polypeptides and proteins that exhibit desirable optical, electrical, mechanical, thermal, transport, and catalytic properties46. We envision specific matrix properties that can be combined synergistically with existing cellular functions such as sensing, biomolecule production, and information processing. Thus, this work multiplies the opportunities to program ELMs tailored for applications in human health, energy, and the environment.By creating BUD-ELMs with a de novo, modular protein matrix, this work greatly expands the ability to tailor macroscopic ELMs for specific applications. One of the key advantages of the \u2206ELP60\u2014RCC004, and \u2206SpyTag\u2014RCC005) from the wild-type47 (MFm126), we cloned integration plasmids designed to facilitate the incorporation of synthetic DNA sequences into the rsaA locus using homologous recombination. The integration plasmid used to generate the original BUD-ELM strain (pSMCAF008) was cloned by inserting a target sequence into the multicloning site of the backbone plasmid pNPTS138 (GenBank: MK533795.1) using restriction enzymes (ApaI upstream and NheI downstream). The target sequence  encoded the ELP60-SpyTag flanked by 800\u2009bp of homology regions up- and downstream of the native rsaA central domain (rsaA750\u20132073). The integration plasmids used to generate the \u2206ELP60 and \u2206SpyTag BUD-ELM strains  were cloned from the plasmid pSMCAF008 using Golden Gate assembly (PCR primers listed below).All strains and plasmids used in this work are listed in Supplementary Table\u00a029: plasmid pSMCAF008, pSMCAF017 or pSMCAF018 was electroporated into E. coli WM3064 cells and subsequently conjugated overnight into C. crescentus NA1000 \u0394sapA::Pxyl-mkate2 (MFm126) on a PYE agar plate containing 300n\u2009\u03bcM DAP. The culture was then plated on PYE with 25\u2009\u03bcg/ml kanamycin to select for integration of the plasmid and removal of E. coli cells. Successful integrants were incubated in liquid PYE media overnight and plated on PYE supplemented with 3% w/v sucrose to select for excision of the plasmid and sacB gene, leaving the target sequence in the genome. Integration of the sequences was confirmed by colony PCR (with primers SMCAF070 and SMCAF065) using a touchdown thermocycling protocol with an annealing temperature ranging from 72\u201362\u2009\u00b0C, decreasing 1\u2009\u00b0C per cycle. The PCR amplicons have been fully sequenced.The BUD-ELM strains were generated using the two-step recombination techniqueSMCAF142: TGGGTCTCAAGGGCGGTTCGGGAGGAGGCSMCAF143: TGGGTCTCAGCAATCCAAACGAGAGTCTAATAGAATGAGGTCSMCAF144: TGGGTCTCCTTGCAACTGGTCTATTTTCCTCTTTTGSMCAF145: TAGGTCTCCCCCTTGTCATCGTCGTCCTTG.SMCAF168: CAGGTCTCTTGTCGCACCTGATTGCCCGASMCAF169: CAGGTCTCTGCTGGGACACCACCGCCAGGSMCAF170: CTGGTCTCCCAGCTGACCCGGCCTTCGGCSMCAF171:\u2009CTGGTCTCTGACAATCTATCGATTGTATGGGAAGCCCG.SMCAF070: TATGACGTTTGCTCTATAGCCATCSMCAF065: GAGGATCAGACGTTCGCTTAG.CCAATGATCGTAATACGACTCACTAGTGGGGCCCGCGCCACTCGGTCGCAGGGGGTGTGGGATTTTTTTTGGGAGACAATCCTCATGGCCTATACGACGGCCCAGTTGGTGACTGCGTACACCAACGCCAACCTCGGCAAGGCGCCTGACGCCGCCACCACGCTGACGCTCGACGCGTACGCGACTCAAACCCAGACGGGCGGCCTCTCGGACGCCGCTGCGCTGACCAACACCCTGAAGCTGGTCAACAGCACGACGGCTGTTGCCATCCAGACCTACCAGTTCTTCACCGGCGTTGCCCCGTCGGCCGCTGGTCTGGACTTCCTGGTCGACTCGACCACCAACACCAACGACCTGAACGACGCGTACTACTCGAAGTTCGCTCAGGAAAACCGCTTCATCAACTTCTCGATCAACCTGGCCACGGGCGCCGGCGCCGGCGCGACGGCTTTCGCCGCCGCCTACACGGGCGTTTCGTACGCCCAGACGGTCGCCACCGCCTATGACAAGATCATCGGCAACGCCGTCGCGACCGCCGCTGGCGTCGACGTCGCGGCCGCCGTGGCTTTCCTGAGCCGCCAGGCCAACATCGACTACCTGACCGCCTTCGTGCGCGCCAACACGCCGTTCACGGCCGCTGCCGACATCGATCTGGCCGTCAAGGCCGCCCTGATCGGCACCATCCTGAACGCCGCCACGGTGTCGGGCATCGGTGGTTACGCGACCGCCACGGCCGCGATGATCAACGACCTGTCGGACGGCGCCCTGTCGACCGACAACGCGGCTGGCGTGAACCTGTTCACCGCCTATCCGTCGTCGGGCGTGTCGGGTTCGGGCGGTTCGGGAGGAGGCTCGGGTGACTACAAGGACGACGATGACAAGGGAGTTGGCGTCCCAGGAGTTGGAGTCCCAGGAGGGGGCGTTCCGGGCGCAGGAGTTCCTGGAGTAGGAGTTCCAGGAGTGGGCGTGCCAGGGGTGGGCGTCCCAGGTGGGGGAGTTCCCGGAGCAGGTGTGCCTGGGGGCGGCGTGCCTGGAGTCGGAGTTCCGGGGGTGGGTGTACCGGGTGGAGGCGTACCAGGCGCGGGAGTGCCGGGCGTGGGCGTGCCAGGCGTCGGTGTACCCGGCGTTGGTGTTCCGGGCGGAGGTGTCCCCGGAGCTGGGGTTCCCGGTGGGGGTGTACCGGGCGTCGGGGTTCCCGGTGTGGGTGTCCCAGGTGGCGGCGTTCCCGGGGCGGGCGTACCTGGAGTGGGTGTGCCAGGAGTCGGCGTCCCAGGAGTCGGCGTACCAGGAGGTGGTGTTCCCGGGGCCGGAGTTCCCGGCGGAGGAGTTCCCGGCGTCGGCGTCCCTGGGGTCGGCGTCCCGGGAGGTGGAGTACCCGGAGCAGGAGTGCCGGGAGTCGGTGTACCTGGTGTCGGTGTCCCTGGTGTAGGTGTCCCGGGTGGTGGGGTGCCAGGTGCTGGCGTACCTGGGGGGGGGGTTCCTGGCGTAGGCGTTCCGGGGGTGGGCGTTCCGGGCGGCGGGGTGCCGGGAGCAGGTGTCCCCGGCGTTGGTGTACCGGGGGTTGGTGTCCCAGGCGTAGGTGTGCCCGGTGGAGGGGTGCCGGGAGCTGGAGTGCCTGGAGGGGGTGTACCAGGGGTCGGTGTTCCCGGTGTAGGAGTACCGGGGGGCGGAGTCCCAGGAGCCGGCGTGCCGGGTGTTGGAGTCCCGGGAGTCGGAGTCCCTGGGGTAGGCGTTCCAGGGGGAGGGGTCCCCGGTGCAGGGGTTCCTGGCGGTGGTGTCCCAGGCGGTTCGGGAGGAGGCTCGGGTGCGCATATCGTAATGGTCGATGCATACAAGCCCACGAAAGGAGGTTCAGGCGGCGGAAGCGGTGGTGGAAGCGGAGGTGGGTCAGGCGGAGGCTCAGGGGGAGGTTCGGGTGGCGGTTCGGGAGGAGGCTCGGGTGCTGACCCGGCCTTCGGCGGCTTCGAAACCCTCCGCGTCGCTGGCGCGGCGGCTCAAGGCTCGCACAACGCCAACGGCTTCACGGCTCTGCAACTGGGCGCGACGGCGGGTGCGACGACCTTCACCAACGTTGCGGTGAATGTCGGCCTGACCGTTCTGGCGGCTCCGACCGGTACGACGACCGTGACCCTGGCCAACGCCACGGGCACCTCGGACGTGTTCAACCTGACCCTGTCGTCCTCGGCCGCTCTGGCCGCTGGTACGGTTGCGCTGGCTGGCGTCGAGACGGTGAACATCGCCGCCACCGACACCAACACGACCGCTCACGTCGACACGCTGACGCTGCAAGCCACCTCGGCCAAGTCGATCGTGGTGACGGGCAACGCCGGTCTGAACCTGACCAACACCGGCAACACGGCTGTCACCAGCTTCGACGCCAGCGCCGTCACCGGCACGGGCTCGGCTGTGACCTTCGTGTCGGCCAACACCACGGTGGGTGAAGTCGTCACGATCCGCGGCGGCGCTGGCGCCGACTCGCTGACCGGTTCGGCCACCGCCAATGACACCATCATCGGTGGCGCTGGCGCTGACACCCTGGTCTACACCGGCGGTACGGACACCTTCACGGGTGGCACGGGCGCGGATATCTTCGATATCAACGCTATCGGCACCTCGACCGCTTTCGTGACGATCACCGACGCCGCTGTCGGCGACAAGCTCGACCTCGTCGGCATCTCGACGAACGGCGCTATCGCTGACGGCGCCTTCGGCGCTGCGGTCACCCTGGGCGCTGCTGCGACGCTAGCTGACTGGGAAAACCCTGGCGTTAATCGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC.C. crescentus strains into 80\u2009mL of PYE in a 250\u2009mL glass flask. All cultures were grown in an Innova 44 incubator shaker with a 2-inch orbit. These cultures were grown at 30\u2009\u00b0C at 250 rpm, and BUD-ELMs typically formed within ~24\u201330\u2009h. To explore the effect of growth parameters on BUD-ELM size, the flask volume, shaking speed, and culture volume were varied from 125 to 500\u2009mL, 0 to 250 rpm, and 25 to 160\u2009mL, respectively. The complete list of conditions tested can be found in Supplementary Tables\u00a0Unless indicated otherwise, BUD-ELMs were grown by inoculating a single colony of 2 piece was broken off from the desiccated material and inoculated into 80\u2009mL of fresh PYE and grown under standard conditions. We detected BUD-ELM formation in 48\u2009h for material dried over 7 or 14 days, and in 72\u2009h for material dried over 21 days.To test the ability of BUD-ELMs to re-seed their own growth, BUD-ELMs grown under standard conditions were collected, transferred in a petri dish for 7, 14, or 21 days and left on the bench at room temperature. The material dried out in 24\u201348\u2009h. To re-seed material growth, a ~0.3 to 0.5\u2009cmE. coli were assembled from existing constructs  by substituting the mRFP sequence with the GFP sequence (below). Similarly, plasmids pSMCAF032 and pSMCAF029 used for the expression of GDH and SpyCatcher-GSH from E. coli were assembled by introducing the GDH sequence in the same position. These plasmids were transformed into chemically competent BL21(DE3) cells (New England Biolabs \u2013 C2527H); single transformants were selected using ampicillin resistance.Plasmids pSMCAF015 and pSMCAF016 used for the expression of GFP and SpyCatcher-GFP from ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTTCCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAA.GACGTTCCGCTGACCCCGAGCCAGTTTGCGAAAGCGAAAAGCGAGAACTTCGACAAAAAAGTCATCCTGAGCAACCTGAATAAACCGCACGCTCTGCTGTGGGGTCCGGATAATCAGATTTGGCTGACCGAACGCGCAACCGGTAAAATTCTGCGCGTTAACCCGGAAAGCGGCAGCGTTAAAACCGTCTTTCAGGTTCCGGAAATCGTTAACGACGCAGACGGTCAAAACGGTCTGCTGGGTTTTGCGTTTCATCCGGACTTCAAAAACAACCCGTACATCTACATCAGCGGCACCTTCAAAAACCCGAAAAGTACCGACAAAGAGCTGCCGAATCAGACCATCATCCGTCGCTATACCTACAACAAAAGCACCGACACCCTGGAAAAACCGGTTGATCTGCTGGCAGGTCTGCCGAGTAGTAAAGATCATCAGAGCGGTCGTCTGGTAATTGGTCCGGACCAGAAAATCTACTATACCATTGGCGATCAGGGCCGTAACCAACTGGCATACCTGTTTCTGCCGAACCAAGCACAACATACCCCGACCCAACAAGAACTGAACGGCAAAGACTACCACACCTACATGGGCAAAGTTCTGCGTCTGAATCTGGACGGTAGCATTCCGAAAGACAACCCGAGCTTCAACGGCGTTGTTAGCCATATCTATACCCTGGGTCACCGTAATCCGCAAGGTCTGGCATTTACCCCGAACGGTAAACTGCTGCAGTCTGAACAGGGTCCGAATTCTGACGACGAAATCAACCTGATCGTTAAAGGCGGCAATTACGGTTGGCCGAACGTTGCAGGCTATAAAGACGATAGCGGCTATGCATACGCGAATTATAGCGCAGCGGCAAACAAAAGCATCAAAGACCTGGCCCAGAACGGTGTTAAAGTTGCAGCAGGCGTTCCGGTTACCAAAGAAAGCGAGTGGACCGGCAAAAACTTTGTTCCGCCGCTGAAAACCCTGTATACCGTCCAGGACACCTACAACTATAACGATCCGACCTGCGGCGAAATGACCTATATTTGCTGGCCGACCGTTGCACCGAGTTCTGCATACGTTTACAAAGGCGGCAAAAAAGCGATCACCGGTTGGGAAAATACCCTGCTGGTTCCGAGTCTGAAACGCGGCGTTATCTTCCGCATCAAACTGGATCCGACCTATAGTACCACCTACGACGATGCCGTTCCGATGTTCAAAAGCAACAACCGTTATCGCGACGTTATTGCAAGTCCGGACGGTAACGTTCTGTACGTTCTGACCGATACCGCAGGTAACGTTCAGAAAGACGACGGTAGCGTTACCAATACCCTGGAAAATCCGGGTAGCCTGATCAAATTCACCTACAAAGCGAAATGA.E. coli BL21(DE3) harboring plasmids pSMCAF015 and pSMCAF016, for expression of GFP and SpyCatcher-GFP, respectively, were inoculated in 25\u2009mL of RM minimal media with 0.2% w/v glucose and 100\u2009\u00b5g/mL ampicillin. After ~16\u2009h of growth at 37\u2009\u00b0C and 250 rpm, cells were used to inoculate 0.5\u2009L of RM minimal media with 0.2% v/v glycerol, 100\u2009\u00b5g/mL ampicillin and 0.0004% antifoam (Antifoam 204) to a final OD600 ~0.05. The cultures were allowed to grow at 37\u2009\u00b0C until mid-log phase. Protein production was induced with 0.2% w/v l-arabinose with incubation at 30\u2009\u00b0C for ~17\u2009h.Single colonies of g for 30\u2009min, resuspended in lysis buffer  and lysed using Avestin Emulsiflex C3 Homogenizer. The lysate was centrifuged at 12,000 \u00d7 g for 1\u2009h and the supernatant was collected for protein purification. The proteins were purified using Immobilized Metal Affinity Chromatography (IMAC) with a HisTrap FF column and buffers containing 50\u2009mM Tris pH 8.0, 300\u2009mM NaCl, 5% v/v glycerol, and 10\u2013250\u2009mM Imidazole. After protein purity was confirmed by SDS-PAGE, the protein was dialyzed into TEV-cleavage buffer  and the 6 x His-tag was cleaved using TEV protease by agitation at 4\u2009\u00b0C for 4\u2009h. The cleaved protein was stored at \u221280\u2009\u00b0C in 50\u2009mM NaPO4 pH 8.0, 300\u2009mM NaCl, and 5% v/v glycerol.Cells were harvested by centrifugation at 8000 \u00d7 2O and lyophilized for 5\u2009h in a Labconco Freezone 4.5 freeze dryer. Tubes were then weighted.Eight BUD-ELMs were grown under standard conditions. Samples were harvested from liquid cultures, placed in Eppendorf tubes, washed once in ddHl-lysine coated silicon substrates were immersed in Falcon\u2122 round-bottom polypropylene culturing tubes containing 3\u2009mL fresh C. crescentus cell culture at an OD600 of 0.3\u20130.5. Culture tubes were then centrifuged at 3000 \u00d7 g for 10\u2009min to immobilize the cells onto the silicon substrate. The silicon substrate was washed with 2\u2009mL of sterile PYE to remove loosely-bound cells before being mounted to a metal puck and transferred to the AFM sample stage. In situ AFM was performed on an Asylum Cypher AFM using soft tapping mode. A fluid cell and two syringe pumps were assembled to control liquid flow and PYE medium was supplied to maintain cell viability during imaging. The AFM probe consisted of a sharp silicon tip on a silicon nitride cantilever  with a spring constant of 0.09\u2009N/m. Cells were imaged in native state without fixation. A 100\u2013200\u2009mV amplitude setpoint was used to apply minimum forces (~0.2\u2009nN) to cells during imaging.Poly-2 precleaned silicon substrate was dipped into the pellicle forming cell culture with an entry angle of ~60\u00b0 perpendicular to the water surface. The silicon substrate was then retrieved, and the pellicle structure was dried under an N2 atmosphere for 2\u2009h. The dried pellicle structure on the substrate was mounted to a metal puck and transferred to the AFM sample stage. AFM imaging was performed on an Asylum Cypher AFM using soft tapping mode in air. A Tap-150 tip (BudgetSensors) with a 5\u2009N/m force constant was used to image the pellicle structure.To bind the BUD-ELM pellicle to a silicon substrate, a 2\u2009cmSmall pieces of BUD-ELMs grown under standard conditions were placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30\u2009mm (MatTek Corporation) and imaged with an optical inverted microscope. Optical microscopy data were acquired using the software NIS-Elements AR (version 4.51.01). All microscopy pictures presented were generated using ImageJ software (version 2.0.0-rc-69/1.52p).d-xylose\u2014to induce the expression of mKate2, in a 125\u2009mL flask and grown for 24\u2009h at 30\u2009\u00b0C at a shaking speed of 250\u2009rpm. BUD-ELMs of similar dimensions were collected and washed twice with 1\u2009mL of 0.01\u2009M Phosphate-buffered saline (PBS), in a centrifuge tube. They were then incubated in 1\u2009mL of 0.01\u2009M PBS, at 30\u2009\u00b0C, with the following staining agent: 80\u2009\u00b5g of SpyCatcher-GFP or GFP for 1\u2009h, 1% Congo Red (Thermo Fisher Scientific\u2014D275) or 100\u2009\u00b5g DiO (DiOC18(3) \u2212 3,3\u2032-Dioctadecyloxacarbocyanine Perchlorate) for 20\u2009min. Samples were washed three times with 1\u2009mL 0.01\u2009M PBS and then a small amount was placed between a slab of PYE agarose (1.5% w/v) and a glass coverslip-bottomed 50-mm Petri dish with a glass diameter of 30\u2009mm (MatTek Corporation). To acquire the low-magnification images  to distinguish the matrix from the cells.Single colonies of BUD-ELM strain RCC002) were inoculated in 30\u2009mL PYE with 0.15%  were inoC. crescentus BUD-ELM strain (RCC002) were cultured in standard (shaking) or static (not shaking) conditions until they reached stationary phase . The supernatant of each culture was extracted and loaded onto a TGX Stain-Free\u2122 gel (Biorad). After running, the gel was transferred to a 0.2\u2009\u03bcm nitrocellulose membrane and blocked for 1\u2009h at room temperature with SuperBlock\u2122 blocking buffer (Thermo Scientific). Membranes were then washed four times in TBST buffer before incubation in a 1:5000 dilution of Monoclonal ANTI-FLAG\u00ae antibody  antibody from Sigma Aldrich \u2013 A8592-.2MG) produced in mouse, clone M2, purified immunoglobulin, buffered aqueous glycerol solution) solution for 1\u2009h at room temperature. Membranes were washed an additional four times in TBST buffer before Clarity Max Western ECL Substrate (Biorad) was applied and the membrane was imaged for chemiluminescence. Each sample lane represents an independent sample, grown from separate individual colonies.For immunoblot analysis of culture supernatant, cultures of \u0394ELP60 BUD-ELM strains were grown under standard conditions. Planktonic cells from each colony were collected and washed 3 times in fresh PYE media to remove any free BUD protein not attached to the cell surface. After washes, all samples were normalized to an OD600 value of 0.225 in 0.5 x PYE, 1 x Laemmli buffer. Each sample was then boiled for 5\u2009min, and 10\u2009uL was loaded onto a TGX Stain-Free\u2122 gel (Biorad). All subsequent steps follow the protocol for immunoblotting described above. Protein molecular weight has been determined by image analysis with ImageJ.For whole-cell immunoblot analysis, the original and L) values.To determine the apparent BUD-ELM size, the bottom of glass flasks where BUD-ELM grew was imaged using a Canon EOS 77D camera. Flasks were positioned within a reflective photobox on a clear plastic surface such that the bottom of the flask stood approximately 11.5\u2009cm above the camera lens. These images were separated into RGB channels using MATLAB R2020b. A subset of the blue channel images was then input into the image classification software ilastik (version 1.3.3), as a training set for the autocontext workflow. The first stage of training separated images into three different classifications: background, scattered material, and bundled material. Scattered material was defined as overlapping regions of small aggregates not associated with each other, whereas bundled material referred to larger, connected pieces of material. The second stage of training distinguished bundled material from the rest of the image. Both stages of training utilized all 37 features provided within the ilastik workflow . From the results of the training set, the second stage segmentation masks for all blue channel images were calculated, and loaded into MATLAB R202b. From these masks, the flat area of each piece of material was calculated, and the top five percentile of size from each image was averaged to yield a representative size measurement. For each image, a conversion rate between pixels and squared centimeters was determined using the standard flask diameter as a reference point. Size measurements were averaged between samples and plotted with respect to their calculated (d5)(kLa); Ne\u2019 \u2013 modified Newton number (dimensionless); P \u2013 power input (W); Re \u2013 Reynold\u2019s number (dimensionless); VL \u2013 culture volume (m3); \u03b7app \u2013 dynamic apparent viscosity (Pa\u2219s); \u03c1 \u2013 liquid density (kg/m3); d0 \u2013 orbital shaking diameter (m); D \u2013 diffusion coefficient (m2/s); v \u2013 kinematic viscosity (m2/s); g \u2013 acceleration of gravity (m/s2).ELP60, and \u2206rsaA1\u2013250 strains, respectively) were evaluated on a strain-controlled rheometer (ARIES G2) equipped with an 0.1\u2009rad 8-mm diameter cone plate. BUD-ELMs were grown in standard conditions. An approximate volume of 100\u2013200\u2009\u00b5L of BUD-ELMs were collected into a 1.5\u2009mL centrifuge tube and spun for 10\u2009s at 3200 rcf with a mini centrifuge (VWR\u00ae \u2013 C0803). This allowed for the material to collect at the bottom as a homogeneous paste. The supernatant was removed and 150uL of fresh PYE were added on top of BUD-ELMs to prevent desiccation. Strain sweep experiments from 0.1 to 100% strain amplitudes were performed at a fixed frequency of 3.14\u2009rad/s. Frequency sweep experiments from 100 to 0.1\u2009rad/s were performed at a 0.35% strain amplitude. Data were acquired using TRIOS software (version 4.2.1.36612).The rheological properties of BUD-ELMs produced from strains RCC002, RCC004 and MFm152  and incubated with 7\u2009mL of 6 ppm CdCl2 (Sigma Aldrich \u2013 202908) in ddH2O for 90\u2009min on an orbital shaker. After incubation, the Cd2+ concentration of the supernatant was measured by ICP-MS. Specifically, 5\u2009\u00b5L of the supernatant was diluted in 4.995\u2009mL 1% HNO3 with 5\u2009\u00b5g/mL Indium (In), as standard for data analysis (Perkin Elmer N9303741). This diluted solution was run on a Perkin Elmer Nexion 300 ICP-MS with two isotopic measurements (Cd2+ 111 and Cd2+ 112) and In 115 as the internal standard. Data was acquired using Syngistix software.To measure the ability of BUD-ELMs to bind CdE. coli BL21(DE3) harboring pSMCAF032 and pSMCAF029, for expression of GDH and SpyCatcher-GDH, respectively, were inoculated in 25\u2009mL of Terrific broth (TB) with 100\u2009\u00b5g/mL ampicillin. After ~16\u2009h of growth at 37\u2009\u00b0C and 250\u2009rpm, cells were used to inoculate 0.5\u2009L of TB with 0.02% antifoam (Antifoam 204) and 100\u2009\u00b5g/mL ampicillin to a final OD600 ~0.05. The cultures were allowed to grow at 37\u2009\u00b0C until the mid-log phase. Protein production was induced with 0.2% w/v L-arabinose with incubation at 30\u2009\u00b0C for ~17\u2009h.Single colonies of g for 5\u2009min, resuspended in lysis buffer (0.01\u2009M MOPS) and lysed using sonication. After centrifugation at 12,000 \u00d7 g for 1\u2009h, the GDH (or SpyCatcher-GDH) in the supernatant was reconstituted by adding a final concentration of 3\u2009mM Ca2+ and 0.06\u2009mM PQQ and incubated for 15\u2009min at 4\u2009\u00b0C. BUD-ELMs, washed once with 0.01\u2009M PBS, were incubated with reconstituted and non-reconstituted cell lysates for 2\u2009h at 4\u2009\u00b0C and then washed three times with 0.01\u2009M PBS. A small piece of functionalized BUD-ELMs , 1\u2009mL DCPIP (20\u2009mg dissolved in 5\u2009mL of DI water), and 1\u2009mL phenazine methosulfate (PMS) (45\u2009mg dissolved in 5\u2009mL of DI water) were prepared. Analytical samples (~3\u2009uL) were mixed with the reagent to 190\u2009\u00b5L. The reaction was initialized by adding 10\u2009\u00b5L glucose (2\u2009M). Glucose consumption was correlated to the consumption of DCPIP (2:1 ratio), which was quantified colorimetrically by absorption at 600\u2009nm. A representative curve is shown in Supplementary Fig.\u00a0The activity of GDH functionalized material was quantified with a modified colorimetric 2,6-dichlorophenol (DCPIP) assayFigure\u00a0Figures\u00a0Figures\u00a0The blots shown in Fig.\u00a0Further information on research design is available in the\u00a0Supplementary InformationReporting SummaryPeer Review File"}
+{"text": "Liquid\u2013liquid phase separation (LLPS) governs a variety of mesoscale cellular processes. However, less is known about how cells utilize LLPS to drive cellular function. Here, we examined the destruction complex (DC), an organelle which controls Wnt signaling and whose components phase separate. Through a combination of advanced microscopy, CRISPR, computational modeling, and optogenetics, we find that the DC is nucleated by the centrosome and that this nucleation drives efficient signal transduction. Our work not only uncovers a biological function for LLPS but also highlights nucleation as a general method for controlling the function of intracellular condensates. Finally, our findings suggest a thermodynamic coupling between Wnt signal transduction and the cell cycle which could lead to insights into Wnt-driven cancers. Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate \u03b2-catenin stability. Overexpressed DC scaffolds undergo liquid\u2013liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in \u03b2-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of \u03b2-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3\u03b2 partitioning on the centrosome controls \u03b2-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle. The canonical Wnt signaling pathway is a conserved , morphog2Extracellular Wnt ligands inhibit DC function through a mechanism that is still unclear, but likely involves selective recruitment of DC components to the signalosome, a biomolecular condensate on the plasma membrane that includes Wnt/Frizzled/LRP5/6 clusters  and, whe8The \u201cmolecular crucible\u201d model posits that DC LLPS promotes \u03b2-cat degradation in Wnt OFF conditions  via concA biophysical mechanism for regulating condensates is control over their nucleation. This principle was recently explored with a synthetic optogenetic system , but theSlimb  and localization of \u03b2-cat at the cell membrane, consistent with previous work in fixed specimens  (Movies S2 and S3). \u03b2-cat puncta became more difficult to distinguish at higher cytoplasmic concentrations produced by activated Opto-LRP6, but dissolution nearly always preceded appreciable dilute-phase \u03b2-cat accumulation, indicating that they were not simply obscured by higher background levels. Further, of light-stimulated cells, those that were resistant to optogenetic activation maintained their \u03b2-cat puncta . Together, these results indicate that activation of the Wnt pathway causes perinuclear puncta to dissolve, and the presence of these puncta is inversely related to Wnt pathway activation at the population and single-cell levels.To establish whether directly activating the Wnt receptor controls the existence of the puncta, we transduced to-LRP6) K and L. to-LRP6) F. We fout puncta I and J. CSNK1A1, GSK3B, and AXIN1, genes encoding the kinases CK1\u03b1 and GSK3\u03b2 that sequentially phosphorylate \u03b2-cat in the DC, and the primary DC scaffold.We next sought to determine 1) what, if any, cellular structure was organizing these puncta, 2) whether all DC components were colocalized with puncta, and 3) whether these were solid or liquid-like condensates. Because of the sensitivity of LLPS systems to protein concentration , we deciSI Appendix, Fig. S2A): We observed single condensates in G1, two condensates in G2/S, and a \u201cfinger-like\u201d pattern\u2014suggesting association with the mitotic spindle\u2014during late mitosis. These observations, combined with previous reports of perinuclear enrichment of CK1\u03b1, GSK3\u03b2, and Axin1 in fixed cells  confirmed that tdmRuby3-CK1\u03b1, tdmRuby3-GSK3\u03b2, and tdmRuby3-\u03b2-cat puncta were indeed colocalized to the centrosome.We found that all tagged proteins were localized into one or two perinuclear puncta A. Timelaed cells \u201331, led ed cells B and GM1Left) that colocalized with centrosomal markers  and replicated following cell cycle progression . As protein concentration increased, Axin1, but not APC, caused formation of extracentrosomal puncta throughout the cytoplasm . Interestingly, extracentrosomal Axin1 condensates did not reliably induce formation of extracentrosomal GSK3\u03b2 condensates in these experiments, but often resulted in deenrichment of centrosomal puncta . We reason that this was due to extracentrosomal Axin1 condensates competing for relatively scarce GSK3\u03b2, thereby diluting across all condensates in the cytoplasm.When overexpressed, Axin and APC cross the phase boundary and form liquid condensates in the cytoplasm that are hypothesized to concentrate DC kinases and \u03b2-cat . The facytoplasm C\u2013E. To dSI Appendix, Fig. S2F). These findings establish that all DC components necessary for phosphorylating \u03b2-cat, prior to its ubiquitination, are localized at the centrosome throughout the cell cycle and suggest that DC centrosomal nucleation is generalizable to multiple cell types.The 293Ts are commonly used in experiments probing DC mesoscale structure in\u00a0vivo , 10, butNext, we sought to determine the material state of the centrosomal DC using FRAP on CRISPR-tagged CK1\u03b1, GSK3\u03b2, and \u03b2-cat, as well as of Axin1 and APC at low levels of induction. All centrosomal DC components exhibited mean half-maximal recovery times (\u03c41/2) between 10 s and 60 s E\u2014like inSI Appendix, Fig. S3 A\u2013D). Indeed, synthetic DC scaffolds with these simple attributes have been shown to rescue aberrant Wnt signaling . We found that the nucleated system processed \u03b2-cat and its intermediates more quickly . See SI Appendix, Fig. S3F). This efficiency gain was maintained over a large range of reaction rates . As expected, in systems with high reaction rates, the effect of nucleated phase separation is no longer observed.To test the effects of nucleation on \u03b2-cat processing, we compared simulations in the presence and absence of a nucleation region C. We fou quickly D over a SI Appendix, Fig. S3 H and I, Movie S6). Together, these results demonstrate that nucleation of DC components has the potential to increase \u03b2-cat processing and that a tunable control parameter of this process is the free energy of mixing.Given our findings that nucleation drives efficient processing of \u03b2-cat, we hypothesized that In silico analysis of the DC indicates that processing efficiency in the presence of a nucleator is dependent on client condensation. Imaging of GSK3\u03b2 showed relatively weak enrichment in centrosomal puncta compared to CK1\u03b1, suggesting that increasing nucleation of GSK3\u03b2 would increase the degradation rate of \u03b2-cat in\u00a0vivo. Changing concentration alters both propensity to undergo LLPS and reaction rate  and therMovie S7). Notably, activation of Opto-GSK3 strictly resulted in the formation of one or two perinuclear puncta and did not form extracentrosomal condensates, contrasting with results from studies using Cry2 alone  and eGFP to human GSK3\u03b2 (\u201cOpto-GSK3\u201d hereafter) and stably transduced it into 293Ts A. Upon ly2 alone . Thus, wSI Appendix, Fig. S4 A and B). We observed a similar effect when analyzing total \u03b2-cat by Western blotting and immunofluorescence staining . Given the modest accumulation of \u03b2-cat in response to Wnt-3a in 293Ts, we tested to see whether Opto-GSK3 clustering was sufficient to blunt \u03b2-cat accumulation induced by either CHIR or a Dox-inducible \u03b2-cat overexpression construct. Indeed, activation of Opto-GSK3 also inhibited both methods for driving \u03b2-cat accumulation in a light-dependent manner . These results demonstrate that increasing DC client nucleation at the centrosome dictates Wnt signal transmission across a wide range of activation regimes.To determine whether increased centrosomal condensation of GSK3\u03b2 controlled Wnt signal transmission, we activated Opto-GSK3 in cell lines with three distinct methods for increasing the cellular concentration of \u03b2-cat: ligand-induced, kinase inhibition, and dox-induced gene up-regulation. We found that Opto-GSK activation abolished both Wnt-3a-induced \u03b2-cat accumulation and transcriptional activation as measured by TOPFlash fluorescence D and E. Changes in \u03b2-cat concentration differentiate a variety of stem cell populations, including human embryonic stem cells (hESCs) , 40. HavBuilding on recent discoveries suggesting that LLPS plays a role in DC structure, we sought to understand how the biophysics of DC proteins regulate DC function in live cells. Through a combination of superresolution microscopy, in silico modeling, and optogenetic methods to isolate and probe the phase diagram, we discovered that the mesoscale structure of the DC is a liquid condensate nucleated by the centrosome. The complementarity of these methods allowed us to identify a function for nucleation: acceleration of the catalytic action of DC proteins, thereby promoting efficient processing of \u03b2-cat.The presence of many cytoplasmic Axin1 and APC droplets in mildly overexpressed cellular conditions  has beenOur results support a \u201cmolecular crucible\u201d model of \u03b2-cat degradation, in which multivalent DC scaffolds concentrate DC clients in nucleated droplets to increase \u03b2-cat phosphorylation rate. Assembly line models for \u03b2-cat degradation have been proposed , 42, andWe found that centrosomal DCs cease to concentrate \u03b2-cat under Wnt ON and GSK3\u03b2 chemical inhibition, but the mechanism for this change remains unclear. Multiple DC components that bind \u03b2-cat\u2014including Axin1, GSK3\u03b2, and CK1\u03b1\u2014are also binding partners of the Frizzled-LRP6 signalosome , 45, a kDrosophila embryos leads to only minor tissue-level defects in canonical Wnt signaling and overall morphology  coupling the DC to the centrosome? Axin1 is known to associate with \u03b3-tubulin  and is arphology , 48, indFinally, centrosomal nucleation of the DC suggests a potential function in coordinating cell cycle progression with Wnt signaling. We found two DC droplets in cells with duplicated centrosomes, suggesting that the DC is split along with centrosomes during mitosis. Nonnucleated droplets would be randomly partitioned into daughter cells, leading to potentially detrimental asymmetry in Wnt signaling capacity of the growing tissue. Cell cycle synchronization could be a method of reducing heterogeneity in Wnt-induced stem cell differentiations.Overall, our studies suggest an integral role for LLPS nucleation in regulating the activity of membraneless organelles in\u00a0vivo. The power of observing proteins in their endogenous contexts, coupled with the ability to precisely tune interaction strength without altering protein function or concentration, enables the functional dissection of membraneless organelles.2 Dulbecco\u2019s Modified Eagle Medium, high glucose GlutaMAX  medium supplemented with 10% fetal bovine serum , and 1% penicillin-streptomycin. The hiPSC WTC was gifted by the B.P. laboratory (purchased from Coriell). The hiPSCs were propagated on Matrigel-coated tissue culture plates using serum-free essential 8 (Gibco) culture conditions in standard environments consisting of 5% carbon dioxide at 37\u2009\u00b0C. Experiments in hESC lines were performed using the H9 hESC cell line purchased from the William K. Bowes Center for Stem Cell Biology and Engineering at University of California, Santa Barbara (UCSB). Cells were grown in mTeSR Plus medium (Stem Cell Technologies) on Matrigel (Corning)-coated tissue culture dishes and tested for mycoplasma in 2-mo intervals.Human 293T cells were cultured at 37\u2009\u00b0C and 5% COPiggyBac (CMV) Backbone fwd: tgacgcccgccccac rev: ggtaagctttttgcaaaagcctaggcc. H2B + 18AA linker fwd: cctaggcttttgcaaaaagcttaccatgccagagccagcgaagtc rev: GCATATTTTCCTTGATGAGTTCACTCATccCagTatGtcCgcCggAg. mTagBFP2 fwd: ATGAGTGAACTCATCAAGGAAAATATGCACATG rev: CGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCATTGAGCTTATGGCCGAGTTTGCTG.-3\u2032X-Flag-NLS-Cas9-HA-Avidin fwd: GGAAGTTTGTTGACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAgactataaggaccacgacggagactac rev: gctgcgggtcgtggggcgggcgtcaggatccagacgccgcagThe pPig_H2B-mTagBFP2::t2A::Cas9-Avidin was constructed via subcloning human H2B, mTagBFP2, and Cas9-Avidin provided by M.Z.W. into an expression vector bearing a cytomegalovirus CMV promoter and flanking PiggyBac transposase-compatible inverted terminal repeats using Gibson Assembly  according to supplier instructions. Each of the PCR fragments used was amplified using the following primers:rd gen tet ON-responsive promoter, and EF1\u03b1-driven Blasticidin selection cassette. The following primers were used: XLone Backbone fwd: taaactagtagaccacctcccctgcg, rev: ggtacctttacgagggtaggaagtgg, human Axin1 fwd: cacttcctaccctcgtaaaggtaccatgaatatccaagagcagggtttcccc, rev: CCATgctTCCgCCgCCACTACCgCCgtccaccttctccactttgccgatgatc, 7AA link- tdmRuby3 fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: gcaggggaggtggtctactagtttaCTTGTACAGCTCGTCCATGCCGXLone-Axin-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: Flag-Axin1 purchased from Addgene (#109370), tdmRuby3 from M.Z.W. into XLone-GFP purchased from Addgene (#96930) containing-3\u2032XLone-bCat-tdmRuby3 was constructed via PCR and Gibson Assembly, subcloning from the following constructs: XLone-Axin-tdmRuby3 (above) and Human Beta-catenin GFP purchased from Addgene (#71367). The following primers were used: XLone Backbone fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: ggtacctttacgagggtaggaagtgg, human bcat fwd: cacttcctaccctcgtaaaggtaccatggctactcaagctgatttgatggagttg, rev: CCATgctTCCgCCgCCACTACCgCCcaggtcagtatcaaaccaggccagcpPig-Hygro Backbone fwd: GGACGTGGAAGAAAACCCGGGTCCAatgggtaaaaagcctgaactcaccgc, rev: cattccacagggtcgacagtacaagc,Cuo + CMV fwd: cttgtactgtcgaccctgtggaatgcgttacataacttacggtaaatggcccgc,rev: actgatcatatgaagctgcagccatgaattcggtaccggatccagtcgactag,APC fwd: atggctgcagcttcatatgatcagttgttaaagcaag,rev: CCATgctTCCgCCgCCACTACCgCCaacagatgtcacaaggtaagacccagaatg,7AAlinker-tdmirfp670nano fwd: GGcGGTAGTGGcGGc,rev: ggcgccaaaacccggcgcggaggccttaGGACTGCTGTATTGCAATGCCAACTAC,UbC-CymR-V5-T2A fwd: ggcctccgcgccggg,rev: TGGACCCGGGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCcgtagaatcgagaccgaggagaggThe pPig_CuO-APC-tdmIRFP670::CymR was constructed via PCR and Gibson Assembly from the following constructs: pCuo CA Rac1 CMV + cumate operon purchased from Addgene (#84643), human APC open reading frame (ORF) purchased from Addgene (#16507), tdmirfp670nano from M.Z.W., and human ubiquitin C-driven CymR Cuo repressor purchased from Addgene (#119907) into pPig-Hygro transposase backbone from M.Z.W. PCR fragments were amplified using the following primers:Arabadopsis thaliana, tdmIRFP from M.Z.W. and human GSK3\u03b2 purchased from Addgene (#16260) ORFs were supplied to VectorBuilder for cloning and EF1\u03b1-driven expression into third-generation lentiviral backbone. Vectorbuilder provided the desired final, sequenced plasmid.The pLV_Cry2-tdeGFP-GSK3b was obtained via synthesis and cloning services provided by Vector Builder Inc. Full details are available upon request, but, briefly: primary plasmids containing ATGGCTGAAGGCAGCGTGGCCCGACAGCCAGACCTTTTGACTTGTGACGATGAACCAATCCACATACCGGGGGCAATACAACCTCATGGTCTCCTTCTGGCGCTTGCTGCCGACATGACTATAGTGGCCGGCTCTGACAACTTGCCGGAATTGACCGGACTTGCTATTGGGGCGTTGATTGGGCGCTCTGCCGCTGATGTATTTGATTCCGAGACACATAATAGGCTTACTATAGCCCTCGCCGAACCAGGGGCTGCCGTCGGCGCTCCTATAACAGTTGGGTTCACGATGCGAAAAGATGCTGGGTTCATTGGTAGCTGGCATCGCCACGATCAACTTATCTTCCTTGAGCTTGAACCCCCTCAACGGGACGTTGCGGAACCCCAAGCTTTCTTTAGAAGGACCAATTCAGCCATAAGGCGCCTTCAGGCCGCAGAGACATTGGAGTCCGCGTGTGCGGCAGCAGCGCAGGAAGTACGAAAGATCACGGGATTTGACCGGGTTATGATTTACAGATTCGCATCTGATTTCTCCGGGGAAGTCATCGCGGAGGATCGGTGTGCAGAAGTGGAAAGCAAGCTTGGTTTGCATTACCCCGCATCTACGGTTCCGGCCCAAGCGAGGAGACTGTATACGATAAACCCAGTGAGGATCATACCTGACATAAATTATAGACCGGTTCCCGTTACGCCAGACCTGAACCCCGTCACAGGCAGGCCAATAGACTTGTCTTTTGCAATCCTGCGGTCAGTCTCACCTGTTCACCTCGAGTTTATGAGGAACATAGGGATGCATGGGACGATGAGCATCTCAATCCTGAGAGGTGAACGGCTCTGGGGACTTATTGTTTGTCATCATCGCACACCGTATTACGTTGACCTTGATGGTCGCCAGGCCTGCGAACTCGTAGCTCAAGTATTGGCCTGGCAGATCGGTGTTATGGAGGAAAGCGGTCATGGGACTGGGAGTACAGGTAGCGGCAGCTCTAGTGGCACCTCCandTAGCGGCAGCTCTAGTGGCACCTCCATGGCAGAAGGGTCCGTAGCAAGGCAACCTGACTTGTTGACCTGTGATGATGAACCGATTCACATTCCTGGAGCAATTCAACCGCATGGGCTGCTCCTTGCTTTGGCAGCGGACATGACGATCGTCGCCGGCTCCGATAACCTGCCCGAGTTGACGGGCTTGGCGATAGGAGCCCTGATAGGCCGCTCAGCCGCTGACGTATTCGATAGCGAAACGCATAACCGGCTTACAATCGCCTTGGCTGAACCGGGCGCGGCCGTGGGAGCACCGATTACTGTAGGCTTTACAATGAGAAAAGACGCCGGCTTTATCGGGTCATGGCACCGACATGACCAGCTGATTTTCCTGGAATTGGAGCCCCCGCAGCGGGATGTAGCCGAACCACAGGCCTTCTTCCGGCGCACTAACTCCGCAATTAGGAGACTGCAGGCAGCTGAGACTTTGGAATCAGCATGCGCGGCAGCTGCACAAGAAGTCCGGAAAATCACGGGTTTTGACCGAGTCATGATCTATAGATTCGCGAGCGATTTCTCAGGAGAAGTTATTGCGGAAGACCGATGCGCGGAGGTAGAATCTAAGCTTGGGTTGCACTACCCCGCCTCCACCGTTCCGGCGCAAGCCAGACGGCTCTATACCATTAATCCGGTGCGGATCATTCCAGATATAAATTACCGGCCTGTACCTGTGACACCGGATTTGAACCCTGTCACGGGCCGACCGATAGACCTCAGCTTCGCTATATTGCGATCTGTGTCACCGGTCCACCTCGAGTTTATGAGGAATATAGGCATGCATGGTACAATGTCCATTTCCATTCTCCGGGGTGAACGGCTTTGGGGCCTCATCGTTTGTCACCATCGAACACCGTATTACGTCGATCTCGACGGCAGACAGGCATGTGAGTTGGTCGCTCAGGTACTCGCTTGGCAGATAGGGGTAATGGAGGAGThe pPig_8XTOP_tdIRFP_Puro was constructed via PCR and Gibson Assembly from the following constructs: pPig_H2B-mTagBFP2::t2A::Cas9-Avidin (above), M50 Super 8\u00d7 TOPFlash purchased from Addgene (#12456), and codon-optimized tandem (td) IRFP ordered from Twist Biosciences as overlapping gene fragments with the sequences:PiggyBacPuro backbone fwd: ACCTGCGGGGACGTGGAAGAAAACCCGGGTCCAatgaccgagtacaagcccacggtg,rev: cattccacagggtcgacagtacaagcaaaaag.8\u00d7 TOPFlash fwd: cttgtactgtcgaccctgtggaatgaagtgcaggtgccagaacatttctc,rev: GTCGGGCCACGCTGCCTTCAGCCATggtggctttaccaacagtaccgg.tdIRFP1 fwd: ATGGCTGAAGGCAGCGTGGC,rev: GGAGGTGCCACTAGAGCTGC. tdIRFP2fwd: TAGCGGCAGCTCTAGTGGCAC,rev: GGTTTTCTTCCACGTCCCCGCAGGTCAACAAACTTCCGCGACCTTCTCCGCTCCCCTCCTCCATTACCCCTATCTGCCAAGCGPCR fragments were amplified using the following primers:E. coli Transformation Kit and Buffer Set (Zymo Research #T3002), cultured on lysogeny broth agar plates to select for antibiotic resistance using standard workflows for molecular cloning and DNA production  and pMD 2.G at a 1:0.88:0.11 mass ratio using standard PEI-based transfection procedures (50). Cells were incubated for 24 h before replacing with fresh media and allowing for lentiviral production for an additional 48 h. Supernatant was harvested, filtered through a 0.22-\u03bcm filter and added to plated cells for transduction. Note that all steps for lentiviral production, transduction, and subsequent maintenance of cell lines were carried out in the presence of far-red light or the complete absence of light in an attempt to eliminate the possibility of Cry-2 opto-GSK3 clustering interference with cell growth or virus production.Genomic edits in 293Ts were carried out in cells constitutively expressing Cas9 to maximize editing efficiency.fwd: acgcgccctgtagcgrev: cttaatgcgccgctacagggcgcgtggtacctctagagccatttgtctgc,assembled, cloned, purified, and verified as described in the previous section. The baseline pCab_minimal construct was then subsequently used for production of gRNAs targeting exon1 of the human genomic loci of CTNNB1, CSNK1a1, and GSK3B. Primers creating one to three (depending on protospacer adjacent motif site availability/predicted on/off-target editing scores) unique protospacers targeting the 50-bp window surrounding the first codon of each gene were annealed and cloned into the pCAB_minimal via BbsI digestion and ligation  using standard protocols (CTNNB1_1 fwd: caccgTGAGTAGCCATTGTCCACGC rev: aaacGCGTGGACAATGGCTACTCACTNNB1_2 fwd: caccgTGAAAATCCAGCGTGGACAA rev: aaacTTGTCCACGCTGGATTTTCAcCTNNB1_3 fwd: caccGCGTGGACAATGGCTACTCA rev: aaacTGAGTAGCCATTGTCCACGCCSNK1a1_1 fwd: caccGGCCAAGCCCCGACACCTCT rev: aaacAGAGGTGTCGGGGCTTGGCCCSNK1a1_2 fwd: caccgAGGCTGAATTCATTGTCGGA rev: aaacTCCGACAATGAATTCAGCCTGSK3B fwd: caccCGAAGAGAGTGATCATGTCA rev: aaacTGACATGATCACTCTCTTCGA vector expressing guide RNA (gRNA) and Cas9 obtained from M.Z.W. was subcloned to remove the unnecessary Cas9 ORF via PCR using the following primers:rotocols . The folAXIN1 gRNAs were ordered complete from IDT. Four different protospacer sequences were used (in separate reactions) with the same homology-directed repair (HDR) template to maximize chance of target locus cutting. Cells from each reaction were then pooled 7 d after transfection and subsequently enriched together.AXIN1_1: GGCCGTCCTGCCCGTCTTTGAXIN1_2: GTCTTTGAGGAGAAGATCATAXIN1_3: gTCTTTGAGGAGAAGATCATCAXIN1_4: GGAGAAGATCATCGGCAAAG.Protospacer sequences:tdmRuby3: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGCTTATAAAGGAAAATATGAGAATGAAAGTTGTCATGGAAGGTTCAGTGAATGGCCATCAGTTTAAATGTACAGGTGAAGGCGAGGGACGCCCTTATGAAGGAGTCCAAACTATGAGGATCAAAGTCATAGAGGGAGGTCCTCTCCCCTTCGCCTTCGATATCCTCGCCACCTCTTTCATGTATGGTTCAAGAACATTTATCAAGTATCCTGCCGATATACCAGACTTCTTTAAGCAGTCATTTCCAGAAGGTTTCACTTGGGAACGAGTCACTAGGTATGAGGACGGCGGGGTTGTGACAGTAACTCAAGACACCTCTTTGGAAGATGGTGAGTTGGTCTACAACGTGAAGGTACGCGGGGTTAATTTCCCTTCTAACGGGCCTGTTATGCAAAAGAAGACAAAGGGTTGGGAGCCAAATACCGAGATGATGTATCCTGCAGATGGTGGCCTGCGGGGCTATACCGACATCGCTCTGAAGGTAGACGGCGGGGGCCACCTCCATTGTAATTTTGTAACCACTTACAGGTCTAAGAAGACCGTGGGTAACATTAAGATGCCAGGGGTTCATGCTGTCGACCATAGATTGGAGCGGATAGAAGAAAGCGACAACGAGACCTACGTCGTGCAACGCGAAGTCGCAGTAGCCAAGTATTCCAATCTCGGGGGAGGTATGGATGAACTCTATAAAGGCGGATCCGGTGGTGTGTCCAAGGGAGAAGAACTGATCAAAGAGAACATGAGGATGAAGGTCGTGATGGAGGGCAGCGTCAACGGACACCAATTCAAGTGCACCGGAGAGGGAGAAGGCAGACCATACGAGGGCGTGCAGACAATGAGAATTAAGGTGATCGAAGGCGGACCACTGCCTTTTGCTTTCGACATTCTGGCTACAAGCTTCATGTACGGCAGCAGGACCTTCATTAAATACCCCGCTGACATCCCTGATTTTTTCAAACAAAGCTTCCCTGAGGGCTTTACCTGGGAGAGAGTGACAAGATACGAAGACGGAGGCGTCGTCACCGTCACACAGGATACAAGCCTGGAGGACGGAGAACTGGTGTATAACGTCAAAGTCAGAGGAGTGAACTTTCCCAGCAATGGCCCCGTGATGCAGAAAAAGACCAAAGGCTGGGAACCTAACACAGAAATGATGTACCCAGCCGACGGAGGACTGAGAGGATACACAGACATTGCCCTCAAAGTGGATGGAGGAGGACATCTGCACTGCAACTTCGTCACAACCTACAGATCCAAGAAAACAGTCGGAAATATCAAGATGCCTGGCGTGCACGCCGTGGATCACAGGCTGGAAAGGATTGAGGAGTCCGATAATGAAACATATGTGGTCCAGAGGGAGGTGGCCGTCGCTAAATACAGCAACCTGGGCGGCGGCATGGACGAGCTGTACAAGGGGGGATCAGGAGGaGGctctCTNNB1 fwd: ATAAAAAGACATTTTTGGTAAGGAGGAGTTTTCACTGAAGTTCAGCAGTGATGGAGCTGTGGTTGAGGTGTCTGGAGGAGACCATGAGGTCTGCGTTTCA CTAACCTGGTAAAAGAGGATATGGGTTTTTTTTGTGGGTGTAATAGTGACATTTAACAGGTATCCCAGTGACTTAGGAGTATTAATCAAGCTAAATTTAAATCCTAATGACTTTTGATTAACTTTTTTTAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAGGcGGTAGTGGcGGcGGAagcrev: TAGGGAACCACCTAACAGTTACTCACTGAATCAGTGGAAGAATGGTACTGCATCCAGGCTCCAGAAGCAGTCATCCAGACTAGATTCCTGCTGGTGGCTT GTTTGCTATTTCACCAAGCCATTAGGAGGAGTGAGCAGAAAATGGAGCAAAAGGTAGCCTGACAAGTAAGCAGGGAGAGAGGAAAGCAGGGGGATCTCAGCCAGACTGGCTTAATGGCAACGAAGCAGAGCCCCAATTCAGTAACTAAAGATTTAATGACACAAACCTTGAGTAGCCATagagCCtCCTCCTGATCCCCCBlunt-end PCR products were used in conjunction with gRNAs to template genomic edits containing desired knock-ins. Blunt-end, double-stranded HDR templates were created from templates obtained via DNeasy Blood and Tissue genomic prep  of the 293T cell line to be edited (see next section). PCR was conducted using primers targeting amplicons of a 500- to 1,000-bp window centered on the intended cut site. The following primers were used to amplify genomic loci homology regions:CSNK1a1fwd: CCAGCCCGCGACGTC rev: CTTGACCCTTTTAGGGAGACAGCGGSK3B fwd: GATTTGCCCTCTCTTTTCTCTCCTCC rev: CCAAATAAATATCATATTATCTCAATTCAAGGTTAATGAGACCGNote that CTNNB1 homology arms were synthesized (requiring no genomic amplification step) and provided as a generous gift from Integrated DNA Technologies.Generic tdmRuby3 insert fwd: GGcGGTAGTGGcGGcGGAagcATGGTTAGCAAAGGGGAGGAGC, rev: agagCCtCCTCCTGATCCCCCCTTGTACAGCTCGTCCATGCCCSNK1a1 upstream homology arm rev: gctTCCgCCgCCACTACCgCCCCTGAGAGACGAAGATGGAGGCCSNK1a1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGGCGAGTAGCAGCGGCGSK3B upstream homology arm rev: gctTCCgCCgCCACTACCgCCGATCACTCTCTTCGCGAATCACCGSK3B downstream homology arm fwd: GGGGGATCAGGAGGaGGctctATGTCAGGGCGGCCCAXIN1 upstream homology arm fwd: CTTCACCCACATGTGGTCATTGCACAXIN1 upstream homology arm rev: CGGCAAAGTGGAGAAGGTGGACGGcGGTAGTGGcGGcGGAagcAXIN1 downstream homology arm fwd: GGGGGATCAGGAGGaGGctctTGATAGGCTGGTGGGCTGGCCAXIN1 downstream homology arm rev: CACCTGAAGCTGGCAGCAGGThe above amplicons were then used in a second round of PCR to obtain separate upstream and downstream homology arms that flanked desired knock-ins, and overlap extension was used to construct the final desired amplicons bearing tdmRuby3 and 7AA GS linker. The following primers were used:Note that original upstream fwd and downstream rev primers listed above for isolating genomic loci were reused in the present step and thus not repeated here.Bare 293T cells were first cotransfected using polyethyleniminePEI  with theCRISPR chassis cells were then cotransfected with one of the constructed gRNA plasmids and respective HDR templates at a 2:1 HDR template:gRNA plasmid molar ratio and allowed 72 h to reach steady-state expression. Similar to the process described above, cells were subject to two rounds of FACS  to obtain a clonal population. Knock-in validation was accomplished via a combination of fluorescence microscopy, genomic PCR, and sequencing . In the case of all intended knock-ins, spatiotemporal fluorescence expression of cell populations was binary (either fluorescent or not) and uniform , suggesting that selected clones were broadly representative of overall edited populations.The 293Ts were cotransfected as described in the previous section with PiggyBac and compatible XLone-Axin-tdmRuby3 and pPig_CuO-APC-tdmIRFP670::CymR expression cassettes. Seventy-two hours after transfection, cells were selected in 1 \u03bcM Blasticidin  and 100 \u03bcg/mL Hygromycin B Gold . Blast+/Hygro+ cells were then clonally sorted via FACS as described in the previous section to obtain a uniform population for experiments. For iPSCs, Both Piggyback and Donor plasmids were chemically transfected when cells reached 30% confluency using Lipofectamine Stem Transfection Reagent (manufacturer\u2019s protocol). Following transfection, Blasticidin selection (1 \u03bcM) was initiated 5 d later. At the end of Blasticidin selection, 12 clones were manually picked under a dissection microscope and continuously cultured in Blasticidin (1 \u03bcM) for an additional week. Upon fluorescence signal confirming successful integration, Blasticidin (1 \u03bcM) treatment ceased, and one clone was chosen for the remaining experiments.CHIR 99021 (STEMCELL Technologies #72052) was resuspended in DMSO according to supplied manufacturer recommendations and diluted to 5\u00d7 concentrated stocks in culture medium immediately prior to use on cells. In all cases, CHIR was used at 10 \u03bcM. Doxycycline hyclate (Sigma Aldrich #D9891-1G) was resuspended in phosphate-buffered saline (PBS) and diluted to 5\u00d7 desired concentration in culture medium prior to use. Stock cumate solution (System Biosciences #QM100A-1) was diluted to 5\u00d7 in culture medium prior to use.SI Appendix, Fig. S4 I and J was 100 ng/mL.The \u201cLow\u201d dose of Dox referred to in Recombinant Human Wnt-3a (R&D Systems 5036-WN-010) was resuspended in in PBS containing 0.1% bovine serum albumin according to supplied manufacturer recommendations and diluted to 5\u00d7 concentration in culture medium immediately prior to use. In all cases, Wnt-3a was used at a final concentration of 1 \u03bcg/mL.Primary antibodies used for immunofluorescent markers of the centrosome were \u03b1-GM130  and \u03b1-\u03b3-tubulin . The secondary used for both stains was \u03b1-Ms-Alexa-488 . Tissue fixation and staining was carried out using standard protocols using cold methanol . Immunof2. Glass-bottom culture plates (Cellvis #P96-1.5H-N) were pretreated with bovine fibronectin (Sigma #F1141) in the case of 293Ts or Matrigel in the case of H9 and iPSCs, and cells were allowed to adhere to the plate before subsequent treatment or imaging. FRAP was performed via custom Nikon NIS Elements JOBs function and 488-nm FRAP laser .All live and fixed cell imaging experiments were carried out using a Nikon W2 SoRa spinning-disk confocal microscope equipped with incubation chamber maintaining cells at 37\u2009\u00b0C and 5% COSpatial patterning of light during timelapse fluorescent imaging sessions was accomplished via purpose-built microscope-mounted LED-coupled digital micromirror devices (DMDs) triggered via Nikon NIS Elements software. Stimulation parameters  were optimized to minimize phototoxicity while maintaining continuous activation of Cry-2. For DMD-based stimulation on the microscope, the final settings for \u201cLight ON\u201d were 25% LED power (\u03bb = 455 nm), 2-s duration pulses every 30 s. For experiments that did not require frequent confocal imaging, cells were stimulated via a benchtop LED array purpose built for light delivery to cells in standard tissue culture plates (\u201cOptoPlate\u201d) adapted from previously established designs . The samAll quantification of raw microscopy images was carried out using the same general workflow: background subtraction > classification > measurement > normalization > statistical comparison. Subcellular segmentation of nuclear fluorescence was performed via custom Matlab scripts using H2B-mTagBFP2 brightness, size, and circularity to mask objects. When experimental conditions did not permit segmentation via H2B-mTagBFP2 nuclear fluorescence (such as with live-cell optogenetic stimulation), cells were selected at random using custom ImageJ macro that generates random regions of interest (ROIs) (available upon request). Unless otherwise noted, mean fluorescent intensity of ROIs were measured and subsequently processed. Raw measurements were compiled, processed, and plotted via custom Matlab scripts, available upon request.Statistical parameters are provided in https://fenicsproject.org/) to solve the modified Cahn\u2013Hilliard partial differential equations using the finite element method. In our simulation, we represent the volume fraction of each DC protein, SI Appendix, Fig. S3B), and Ri is the added reaction term such thatWe used the Python-based FEniCS computing environment . We generate a grid mesh with closed boundary conditions to mimic the closed system within a cell. A layer is generated for each simulated component, and \u00b15% noise of the initial value is added to induce inhomogeneities. The FEniCS package partial differential solver is called to generate the chemical potential with respect to each component. The final step is to define the output file path and then use the built-in Newton solver to generate the simulation. The simulations are then rendered using Paraview software. A detailed Python notebook of the simulations is available on https://github.com/MZWLab/Lach2022.First, all parameters are defined . This allowed us to test the sensitivity of a single metric to alterations in our model\u2019s parameters. In Movie S6) and also increased the nucleation efficiency.We defined the nucleation efficiency of \u03b2-cat processing for a given simulation by comparing the ratio of the integrated P4-\u03b2-cat to \u03b2-cat between identical simulations with and without a nucleator (Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File"}
+{"text": "Ctenidae Keyserling, 1877 has a worldwide distribution with 584 species belonging to 49 genera. Amongst these, 141 species are from Asia, including 130 species assigned to Cteninae Keyserling, 1877.The spider family Cteninae are reported from Asia: Amauropelmakrabi sp. n. , Am.phangnga sp. n. , Am.saraburi sp. n. ; Anahitamedog sp. n. ; Bowieninhbinh sp. n.  and B.vinhphuc sp. n.  from the robustus-species group; B.borneo sp. n.  from the chinagirl-species group; B.engkilili sp. n. ; B.sabah sp. n.  from the scarymonsters-species group. The male of An.popa J\u00e4ger & Minn, 2015 and the female of B.fascination J\u00e4ger, 2022 (robustus-species group) are described for the first time. B.fascination J\u00e4ger, 2022 is reported from China for the first time. In addition, the DNA barcodes of all the species in this study were obtained, except for B.vinhphuc sp. n.Nine new species belonging to three genera of  Ctenidae Keyserling, 1877 has a worldwide distribution, but mainly occurs in the tropical and subtropical regions   . Both Amrom Asia .Amauropelma, Anahita and Bowie are described from Asia. This brings the total number of Ctenidae to 150 species in Asia, of which 139 species belong to Cteninae . Photos were stacked with Helicon Focus\u00ae (Version 7.6.1) or Zerene Stacker\u00ae (Version 1.04) and processed in Adobe Photoshop CC2019\u00ae. All measurements are in millimetres (mm) and were obtained with an Olympus SZX16 stereomicroscope with a Zongyuan CCD industrial camera. Total length does not include the chelicerae. Palp and leg measurements are shown as: total length . Leg segments were measured on their dorsal side. The distribution map was generated with ArcGIS 10.2 (ESRI Incorporated Company). References to figures in the cited papers are listed in lowercase (fig. or figs); figures from this paper are noted with a capital letter . The type material is deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing, China.Size classes are used according to Terminology and taxonomic descriptions follow B.vinhphuc sp. n. A partial fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced, using the following primers: forward: LCO1490 (5\u2019-CWACAAAYCATARRGATATTGG-3\u2019) and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3')  and reverse: HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3')  , except ATCA-3') . COI p-dATCA-3') . For addS. Li & Yaosp. n.C5E9E60C-A668-5AE7-A9FF-89D05F8A802757EA8144-E44C-4F59-8B3A-0D16719CB5D5Type status:Holotype. Occurrence: recordedBy: Z. Chen; individualCount: 1; sex: female; lifeStage: adult; Taxon: order: Araneae; family: Ctenidae; genus: Amauropelma; Location: country: Thailand; stateProvince: Krabi; verbatimLocality: Ao Luk District, Klang Cave; verbatimElevation: 36 m a.s.l.; verbatimLatitude: 8\u00b020.268'N; verbatimLongitude: 98\u00b044.707'E; Event: year: 2015; month: 10; day: 12; Record Level: institutionCode: IZCAS-Ar 43530MaleUnknown.Female (IZCAS-Ar 43530): PL 3.3, PW 2.4, AW 1.6, OL 3.1, OW 1.6. Eye diameters and interdistances: AME 0.10, ALE 0.13, PME 0.11, PLE 0.11, AME\u2013AME 0.04, AME\u2013ALE 0.11, PME\u2013PME 0.06, PME\u2013PLE 0.24, AME\u2013PME 0.06, ALE\u2013PLE 0.08, clypeus AME 0.12, clypeus ALE 0.17. Palp and leg measurements: palp 3.8 , I 10.3 , II 9.2 , III 9.0 , IV 12.2 . Leg formula 4123. Spination of palp and legs: palp 130, 100, 1111, 1212; femora I p002, d111, r010, II p010, d111, r010, III p111, d111, r012, IV p002, d111, r102; patellae I\u2013IV 001; tibiae I\u2013II v22222, III p11, d111, r11, v222, IV p111, d11, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I\u2013II with 3, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 0.76, II 0.72, III 0.68.Copulatory organ Fig. a and b. Colour Fig. c and d. Ctenidae . The new species can be distinguished from all known congeners by the median plate roughly heart-shaped and with a mating plug :OP561682).TGTTTGGAGCTTGAGCTGCTATAGCAGGAACTGGAATAAGAGTGTTGATTCGAATAGAGTTAGGTCATCCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTTATTGTAACTGCTCATGCTTTTGTAATGATTTTTTTTATAGTAATACCAATTTTGATTGGTGGATTTGGAAATTGATTAGTTCCGTTGAGATTGGAGCACCTGATATATCATTTCCTCGAATAAATAATTTGTCGTTTTGATTACTACCTCCTTCTTTATTTTTATTAATAATATCATCAATAGTAGAAATAGGTGTTGGAGCGGGATGAACTGTTTATCCTCCTTTAGCATCTAGTATTGGGCATATAGGAAGATCTATAGATTTTGCTATTTTTTCTCTTCATTTGGCTGGAGCTTCTTCTATTATAGGAGCAGTAAATTTTATTTCTACTATTATTAATATACGGTTGTATGGAATGAGTATAGAAAAGGTTCCTTTGTTTGTGTGGTCTGTTTTTATTACTGCTATTTTGTTATTATTGTCGTTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCGGGAGGGGGAGATCCTATTTTGTTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.8, AW 1.2, OL 3.0, OW 1.9. Eye diameters and interdistances: AME 0.09, ALE 0.12, PME 0.10, PLE 0.10, AME\u2013AME 0.04, AME\u2013ALE 0.13, PME\u2013PME 0.08, PME\u2013PLE 0.21, AME\u2013PME 0.05, ALE\u2013PLE 0.08, clypeus AME 0.17, clypeus ALE 0.22. Palp and leg measurements: palp 3.8 , I 13.5 , II 11.6 , III 10.9 , IV 14.8 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1101; femora I p021, d211, r112, II\u2013III p012, d111, r012, IV p102, d111, r012; patellae I\u2013IV 001; tibiae I p010, v22222, II p100, r100, v22222, III p11, d111, r11, v222, IV p11, d11, r11, v222; metatarsi I v222, II p112, r010, v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Tarsi and metatarsi without scopula. Claw tufts arising separately, but intermingle with each other distally. Leg claws I with 7 and II with 6 secondary teeth. Position of tarsal organ: I 1.37, II 0.92, III 0.85.Palp Fig. a\u2013c. Pate9Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43532): PL 3.4, OL 2.4.Ctenidae . The new species can be distinguished from all known congeners by the embolus tip with an extension :OP718556).GGTGGGTTCGGAAATTGATTGGTTCCTTTGATGTTAGGAGCTCCTGATATATCATTTCCTCGTATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTGTTATTAATATCTTCTATGGTGGAAATAGGAGTGGGAGCAGGATGAACTGTCTATCCTCCTTTAGCTTCTAGAATAGGGCATGTGGGAAGATCAATAGATTTTGCGATTTTTTCTCTTCATTTAGCTGGAGTTTCTTCTATTATGGGAGCGGTTAATTTTATTTCTACTATTATTAATATGCGATTATATGGAATAACTATAGAAAAGGTTCCTTTATTCGTTTGATCAGTTTTTATTACTGCAGTTTTGTTGTTGTTATCATTACCTGTGTTAGCAGGTGCTATTACTATATTATTGACAGATCGAAATTTTAATACTTCTTTTTTTGATCCTGCAGGGGGTGGAGATCCAATTTTATTTCAACATTTATTCTGATTTTTTGGTCACCCTGGAAAGTTTAA : PL 4.5, PW 3.7, AW 1.6, OL 3.2, OW 2.1. Eye diameters and interdistances: AME 0.12, ALE 0.14, PME 0.14, PLE 0.14, AME\u2013AME 0.07, AME\u2013ALE 0.16, PME\u2013PME 0.08, PME\u2013PLE 0.26, AME\u2013PME 0.07, ALE\u2013PLE 0.13, clypeus AME 0.16, clypeus ALE 0.28. Palp and leg measurements: palp 5.2 , I - , II 15.2 , III 14.3 , IV 19.4 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 210; femora I p112, d111, r111, II p211, d111, r211, III p112, d111, r112, IV p112, d111, r002; patellae I 001, II\u2013IV 101; tibiae I p010, r110, v22222, II p100, r100, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I v222, II p110, r110, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Claw tufts arising separately, but intermingle with each other distally. Leg claws II with 1 and III\u2013IV with 2 secondary teeth. Position of tarsal organ: IV 1.58.Palp Fig. a\u2013c. Pate9Colour Fig. c and d. Female (IZCAS-Ar 43534): PL 5.6, PW 4.4, AW 2.8, OL 4.9, OW 3.1. Eye diameters and interdistances: AME 0.14, ALE 0.20, PME 0.15, PLE 0.16, AME\u2013AME 0.12, AME\u2013ALE 0.32, PME\u2013PME 0.16, PME\u2013PLE 0.53, AME\u2013PME 0.08, ALE\u2013PLE 0.20, clypeus AME 0.13, clypeus ALE 0.21. Palp and leg measurements: palp 6.0 , I 19.3 , II 18.3 , III 17.0 , IV 23.0 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 1111, 2112; femora I p021, d111, r111, II\u2013III p112, d111, r112, IV p112, d111, r002; patellae I\u2013II 000, III\u2013IV 101; tibiae I \u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III\u2013IV p112, d010, r112, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth, without denticles. Retromargin of chelicerae close to fang base without bristle. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Claw tufts arising separately, but intermingle with each other distally. Palpal claw with 3 secondary teeth, leg claws I with 3, II\u2013III with 2 secondary teeth.Copulatory organ Fig. a and b. Colour Fig. e and f. Variation: Second paratype female (IZCAS-Ar 43535): PL 4.6, OL 4.7.Ctenidae . The new species can be distinguished from all known congeners by the embolus slender .Male (IZCAS-Ar 43533):TGTTTGGAGCTAGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG (GenBank accession number OP572099).Female (IZCAS-Ar 43534):TGTTTGGAGCTTGATCTGCTATAGCGGGAACGGCAATAAGAGTTTTAATTCGTATGGAATTAGGAAATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTAATTGTGACAGCTCATGCTTTTATTATGATTTTTTTTATAGTAATACCGATTTTGATTGGTGGTTTTGGAAATTGATTAGTGCCTTTAATGTTAGGAGCTCCTGATATATCTTTTCCTCGGATGAATAATTTGTCTTTTTGATTACTTCCACCTTCTTTGTTTTTATTATTCATATCTTCTATGGTGGAAATGGGTGTAGGAGCTGGATGAACTGTTTATCCACCTTTGGCTTCTAGAATTGGTCATGCTGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCTGGGGCTTCTTCAATTATAGGAGCGGTGAATTTTATTTCTACTATTATTAATATACGATTATCTGGAATAAGAATGGAGAAGGTTCCATTATTTGTTTGATCTGTTCTTATTACTGCAATTTTATTATTATTATCTTTGCCGGTATTAGCTGGTGCTATTACTATATTGTTGACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGAGGGGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 2.7, PW 2.3, AW 0.9, OL 2.4, OW 1.4. Eye diameters and interdistances: AME 0.13, ALE 0.10, PME 0.22, PLE 0.19, AME\u2013AME 0.10, AME\u2013ALE 0.22, PME\u2013PME 0.16, PME\u2013PLE 0.23, AME\u2013PME 0.11, ALE\u2013PLE 0.14, clypeus AME 0.10, clypeus ALE 0.37. Palp and leg measurements: palp 4.1 , I missing, II 13.0 , III 10.9 , IV 16.0 . Leg formula 4123. Spination of palp and legs: palp 023, 000, 0211; femora II p112, d111, r012, III p112, d111, r112, IV p112, d111, r012; patellae II\u2013IV 101; tibiae II p110, d101, r100, v22222, III p11, d111, r11, v222, IV p11, d111, r11, v22; metatarsi II p111, r111, v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 6 tiny denticles along entire cheliceral furrow. Leg claws II with 9, III with 5 and IV with 7 secondary teeth. Position of tarsal organ: II 1.06, III 0.85, IV 1.14.Palp Fig. a\u2013c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43537): PL 2.9, PW 2.4, AW 1.2, OL 3.5, OW 2.0. Eye diameters and interdistances: AME 0.13, ALE 0.11, PME 0.19, PLE 0.18, AME\u2013AME 0.13, AME\u2013ALE 0.26, PME\u2013PME 0.21, PME\u2013PLE 0.24, AME\u2013PME 0.15, ALE\u2013PLE 0.17, clypeus AME 0.10, clypeus ALE 0.38. Palp and leg measurements: palp 3.2 , I 10.4 , II 9.3 , III 8.3 , IV 12.4 . Leg formula 4123. Spination of palp and legs: palp 020, 010, 010, 2012; femora I p011, d111, r021, II p011, d111, r011, III p012, d111, r112, IV p002, d111, r012; patellae I\u2013II 000, III\u2013IV 101; tibiae I v22212, II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Palpal claw with 5 secondary teeth, leg claws I\u2013II with 5, III with 4 and IV with 7 secondary teeth. Position of tarsal organ: I 0.79, II 0.72, III 0.70, IV 1.00.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The species resembles A.maolan Zhu, Chen & Song, 1999 (see 14A.maolan), by the median plate with a large n-shaped sclerite , by the lateral teeth pointing postero-medially  and by the spermathecae nearly cylindrical .Small 1999 see a\u2013c and f1999 see b, but caThe specific name refers to the type locality and is a noun in apposition.China :.OP572101).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG :OP572102).TGTTTGGAGCTTGAGCTGCTATAGCTGGAACAGCAATAAGAGTTTTAATTCGAATGGAATTAGGACATTCTGGTAGATTGTTAGGAGATGATCATTTATATAATGTAATTGTAACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTGATTGGGGGCTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCGGATATATCTTTTCCTCGAATAAATAATTTATCCTTTTGATTATTACCGCCTTCTTTATTTTTGTTGTTTATATCTTCTATAGTTGAGATAGGGGTTGGAGCAGGTTGAACGGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATATGGGAAGTTCAATGGATTTTGCTATTTTTTCTTTACATTTAGCAGGTGCTTCTTCTATTATAGGTGCTGTGAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATAACAATGGAGAAGATCCCTTTATTTGTATGATCGGTTTTTATTACTGCAATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTGACTGATCGAAATTTTAATACTTCTTTTTTTGACCCTGCTGGAGGTGGAGATCCTATTTTATTTCAACATTTATTTTGATTTTTTG : PL 3.3, PW 2.7, AW 0.9, OL 3.3, OW 1.8. Eye diameters and interdistances: AME 0.14, ALE 0.12, PME 0.22, PLE 0.17, AME\u2013AME 0.08, AME\u2013ALE 0.23, PME\u2013PME 0.16, PME\u2013PLE 0.21, AME\u2013PME 0.18, ALE\u2013PLE 0.13, clypeus AME 0.10, clypeus ALE 0.55. Palp and leg measurements: palp 3.9 , I 15.4 , II 12.7 , III 11.0 , IV 17.3 . Leg formula 4123. Spination of palp and legs: palp 151, 000, 122; femora I p021, d111, r112, II p112, d111, r112, III p111, d111, r111, IV p012, d111, r112; patellae I\u2013IV 101; tibiae I p010, d101, r110, v22222, II p10, d101, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p111, d001, r111, v222, II p111, d111, r111, v222, III p111, d012, r111, v222, IV p112, d011, r112, v222. Chelicerae with 3 promarginal, 4 + 2 retromarginal teeth and with elongated patch of 3 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 2 bristles. Sparse scopula restricted almost entirely to tarsi. Leg claws I with 6, II\u2013III with 5 and IV with 4secondary teeth. Position of tarsal organ: I 1.89, II 1.03, III 0.75, IV 1.56.Palp Fig. a\u2013c. Palp14Colour Fig. c and d. Female (IZCAS-Ar 43539): See Fig. Ctenidae . The species can be distinguished from all known congeners by the embolus arising at 12 o\u2019clock position, long and laminar, running around tegulum, its tip situated distally :OP572105).TATTTGGGGCTTGAGCTGCTATAGCGGGTACTGCAATAAGAGTTTTGATTCGAATGGAATTAGGACATCCTGGAAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATGATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATATTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGGTTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCTATAGTTGAAATAGGTGTAGGAGCAGGGTGAACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCAGGGAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGGGCTGTAAATTTTATTTCTACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCTTTGTTTGTTTGATCTGTTTTTATTACTGCAATTTTATTATTGTTATCTTTACCAGTGTTAGCTGGTGCTATTACAATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCTGCTGGAGGAGGAGATCCAGTTTTATTTCAGCATTTGTTTTGATTTTTTG :OP572104).TTTTTGGAGCTTGAGCCGCTATAGCGGGTACTGCAATAAGAGTTTTAATTCGAATAGAATTAGGGCATCCTGGGAGATTATTAGGTGATGATCATTTATATAATGTTATTGTAACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTAGTTCCTTTAATGTTAGGAGCTCCGGATATATCATTTCCTCGAATAAATAATTTATCTTTTTGATTATTACCTCCTTCTTTGTTTTTATTGTTTATATCTTCCATGGTTGAAATAGGTGTGGGAGCAGGATGGACAGTTTATCCTCCTTTAGCTTCTAGAATTGGGCATGCGGGAAGATCTATGGATTTTGCTATTTTTTCTTTACATTTAGCGGGTGCTTCTTCTATTATAGGAGCTGTAAATTTTATTTCGACTATTATTAATATACGATTAATAGGAATGACTATAGAGAAGGTTCCCTTATTTGTTTGATCTGTTTTTATTACTGCAATTTTATTGTTATTATCTTTACCAGTATTAGCTGGTGCTATTACGATGTTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCTGCTGGGGGAGGGGATCCGGTTTTATTTCAACATTTATTTTGATTTTTTG : See Fig. 28Female (IZCAS-Ar 43541): PL 9.9, PW 7.8, AW 4.1, OL 11.0, OW 7.3. Eye diameters and interdistances: AME 0.29, ALE 0.27, PME 0.32, PLE 0.39, AME\u2013AME 0.31, AME\u2013ALE 0.71, PME\u2013PME 0.43, PME\u2013PLE 1.32, AME\u2013PME 0.32, ALE\u2013PLE 0.38, clypeus AME 0.42, clypeus ALE 0.95. Palp and leg measurements: palp 10.0 , I 23.0 , II 21.4 , III 18.6 , IV 26.1 . Leg formula 4123. Spination of palp and legs: palp 131, 100, 131, 3020; femora I p021, d111, r111, II p112, d111, r111, III\u2013IV p111, d111, r112; patellae I\u2013II 000, III\u2013IV 101; tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p111, d012, r111, v222, IV p121, d012, r111, v2122. Chelicerae with 3 promarginal, 4 + 1 retromarginal teeth and with elongated patch of 27 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 11 bristles. Ventral tarsi and metatarsi I\u2013II with sparse scopula. Palpal claw with 5 secondary teeth, leg claws I\u2013II with 1, III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.41, II 1.34, III 1.25, IV 1.44.Copulatory organ Fig. a. EpigynColour Fig. e and f. Ctenidae . The species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth, RTA arising medially to distally from palpal tibia, female possesses a transversally median plate with lateral teeth situated mainly laterally and not reaching the epigastric furrow. It resembles B.candidate J\u00e4ger, 2022 (see B.candidate), by the spermathecae separated by less than their diameter and smaller chamber with distinct external rim  and by the fertilisation ducts pointing laterally . For the diagnosis of male, see Large-sized 2022 see a, but caChina :OP572108).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG :OP572107).TATTTGGATCTTGGGCTGCTATAGCTGGGACAGCTATAAGAGTATTAATTCGTATAGAGCTAGGTCATTCTGGTAGATTATTTGGTGATGATCATTTATATAATGTAATTGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATGGTTATGCCTATTTTAATTGGTGGTTTTGGAAACTGATTAGTTCCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGTATAAATAATTTATCTTTTTGATTACTCCCTCCTTCATTATTTTTGTTATTTATATCTTCTATGGTTGAGATAGGGGTGGGAGCTGGTTGGACAGTGTATCCTCCTTTAGCTTCTAGTATTGGCCATATAGGAAGATCAATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGAGCTTCTTCTATTATAGGGGCTGTTAATTTTATTTCTACAATTATTAATATACGTTTGTATGGAGTAAGAATAGAAAAGGTGCCTTTATTTGTATGATCTGTTCTAATTACTGCAGTATTATTGCTTTTATCTTTACCTGTATTAGCAGGTGCTATTACTATATTATTAACTGATCGTAATTTTAATACTTCTTTTTTTGACCCGGCTGGAGGAGGGGATCCAGTTTTATTTCAACATTTATTTTGATTTTTTG : PL 7.6, PW 5.9, AW 3.0, OL 5.8, OW 3.9. Eye diameters and interdistances: AME 0.26, ALE 0.19, PME 0.38, PLE 0.30, AME\u2013AME 0.19, AME\u2013ALE 0.29, PME\u2013PME 0.22, PME\u2013PLE 0.43, AME\u2013PME 0.18, ALE\u2013PLE 0.23, clypeus AME 0.11, clypeus ALE 0.55. Palp and leg measurements: palp 7.8 , I 21.3 , II 19.6 , III 15.9 , IV 23.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 101; femora I p021, d111, r112, II p112, d111, r112, III p212, d111, r112, IV p112, d111, r012; patellae I\u2013IV 101; tibiae I p110, d111, r210, v22222, II p110, d111, r110, v22222, III p11, d200, r11, v222, IV p11, d111, r11, v222; metatarsi I\u2013III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 19 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Ventral tarsi and metatarsi I\u2013II with sparse scopula. Right leg claws I\u2013III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.27, II 1.28, III 1.06, IV 1.36.Palp Fig. a\u2013c. RTA 28Colour Fig. a and b. FemaleUnknown.Variation: Paratype male (IZCAS-Ar 43543): PL 7.4, OL 5.7.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base, presence of retro-proximal cymbial outgrowth and RTA arising subdistally from palpal tibia. It resembles B.dodo J\u00e4ger, 2022 (see B.dodo) and by the RTA distally bifurcated .Medium-sized 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam :OP572110).TACTTGGATCTTGGGCTGCTATGGCAGGGACAGCTATAAGAGTATTAATTCGGATGGAATTAGGCCATTCTGGGAGATTGTTAGGTGATGATCATTTATACAATGTAATTGTTACTGCACATGCTTTTGTAATGATTTTTTTTATAGTAATGCCTATTTTAATTGGGGGTTTTGGAAATTGGTTAGTACCTTTGATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCGTTATTTTTATTATTTATATCTTCAATAGTTGAGATAGGAGTTGGAGCTGGATGAACGGTATATCCTCCTTTAGCTTCTAGTATTGGTCATATAGGGAGATCTATAGATTTTGCTATTTTTTCTTTACATTTAGCGGGGGCTTCTTCTATTATAGGAGCGGTAAATTTTATTTCTACGATTATTAATATGCGTTTGTATGGGATGACTATAGAGAAAGTACCTTTATTTGTGTGATCTGTTTTAATTACTGCGGTATTGTTATTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTGTTAACTGATCGAAATTTTAATACTTCTTTTTTTGATCCGGCTGGGGGTGGTGATCCTGTTTTGTTTCAACATTTATTTTGATTTTTTG : PL 6.4, PW 5.0, AW 2.4, OL 3.1, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.24, PME 0.28, PLE 0.26, AME\u2013AME 0.22, AME\u2013ALE 0.42, PME\u2013PME 0.25, PME\u2013PLE 0.41, AME\u2013PME 0.20, ALE\u2013PLE 0.21, clypeus AME 0.21, clypeus ALE 0.53. Palp and leg measurements: palp 7.4 , I 17.7 , II 15.9 , III 13.4 , IV 19.5 . Leg formula 4123. Spination of palp and legs: palp 141, 100, 1010; femora I p021, d111, r112, II p112, d111, r1111, III p1111, d111, r1111, IV p002, d111, r111; patellae I\u2013IV 101; tibiae I p110, d111, r21, v22222, II p20, d111, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p11, d002, r111, v222, II p111, r111, v222, III p111, d012, r211, v222, IV p122, d012, r111, v2122. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Leg claws I\u2013III with 3 and IV with 4 secondary teeth. Position of tarsal organ: I with 1.17, II 1.07, III 0.90, IV 1.09.Palp Fig. a\u2013c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43728): PL 6.7, PW 5.2, AW 3.1, OL 8.5, OW 7.0. Eye diameters and interdistances: AME 0.25, ALE 0.21, PME 0.28, PLE 0.26, AME\u2013AME 0.22, AME\u2013ALE 0.48, PME\u2013PME 0.29, PME\u2013PLE 0.57, AME\u2013PME 0.23, ALE\u2013PLE 0.23, clypeus AME 0.19, clypeus ALE 0.58. Palp and leg measurements: palp 6.9 , I 16.3 , II 15.0 , III 13.1 , IV 18.8 . Leg formula 4123. Spination of palp and legs: palp 131, 000, 1111, 2101; femora I p021, d111, r021, II p112, d111, r021, III p112, d111, r112, IV p111, d111, r001; patellae I\u2013II 000, III\u2013IV 101, tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III p112, d010, r112, v222, IV p112, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 10 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Palpal claw with 6 secondary teeth, leg claws I with 1, II\u2013III with 2 and IV with 3 secondary teeth. Position of tarsal organ: I 1.03, II 0.97, III 0.95, IV 1.42.Copulatory organ Fig. b. EpigynColour Fig. e and f. Variation: Paratype male (IZCAS-Ar 43545): PL 7.4, OL 6.5. Second paratype female (IZCAS-Ar 43729): PL 7.6, OL 7.8.Ctenidae . The new species is assigned to the robustus-species group with the characteristics of stout tegular apophysis, simple stout embolus with broad base and short apical part, presence of retro-proximal cymbial outgrowth, RTA arising subdistally from palpal tibia, female possesses a transversally oval median plate with lateral teeth situated laterally and not reaching the epigastric furrow. It resembles B.yassassin J\u00e4ger, 2022 (see 28B.yassassin), by the median plate with distinct humped areas best seen in posterior view , by the spermathecae separated by less than their diameter, larger chamber round and apically swollen  and by the fertilisation ducts pointing medially .Small to medium-sized 2022 see a\u2013c and l2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Vietnam : PL 3.8, PW 2.9, AW 1.2, OL 3.2, OW 2.2. Eye diameters and interdistances: AME 0.17, ALE 0.15, PME 0.22, PLE 0.21, AME\u2013AME 0.14, AME\u2013ALE 0.11, PME\u2013PME 0.24, PME\u2013PLE 0.27, AME\u2013PME 0.11, ALE\u2013PLE 0.13, clypeus AME 0.09, clypeus ALE 0.37. Palp and leg measurements: palp 5.1 , I 12.1 , II 10.9 , III 9.6 , IV 15.1 . Leg formula 4123. Spination of palp and legs: palp 151, 100, 1110; femora I p012, d111, r012, II\u2013III p112, d111, r112, IV p112, d111, r002; patellae I\u2013II 100, III\u2013IV 101; tibiae I p010, r010, v212222, II p100, d001, r110, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I p111, r011, v222, II p111, d002, r111, v222, III p111, d012, r111, v222, IV p111, d012, r111, v2212. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 8 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 4 bristles. Sparse scopula on all tarsi. Leg claws I\u2013II with 5 and III\u2013IV with 4 secondary teeth. Position of tarsal organ: I 1.07, II 0.89, III 0.72, IV 1.06.Palp Fig. a\u2013c. RTA 28Colour Fig. a and b. FemaleUnknown.Ctenidae . The new species is assigned to the chinagirl-species group with the characteristics of compact embolus, thick-walled, with rounded edges and with an entire margin, the tegular apophysis is longitudinally elongated, not covering the embolus. It resembles B.abdulmajid J\u00e4ger, 2022 (see B.abdulmajid), by the tibia having no distinct broad longitudinal ridge ventrally  and by the conductor nearly quadrilateral .Small 2022 see d, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572103).GGTTTGGTGCTTGGGCTTCTATAGCAGGTACGTCTATAAGAGTTTTGATTCGAATGGAATTAGGACATTCTGGAAGATTATTAGGGGATGATCATTTATATAATGTTGTTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTGATACCTATTTTAATTGGTGGTTTTGGAAATTGGTTGGTTCCTTTAATATTAGGAGCTCCTGATATATCTTTTCCTCGTATAAATAATTTGTCGTTTTGATTACTTCCTCCTTCTTTATTTTTATTGTTTATATCTTCTATGACTGAGATAGGGGTGGGAGCTGGTTGGACGGTTTATCCACCTTTGGCTTCTGGAATTGGTCATGCAGGAAGATCGATAGATTTTGCTATCTTTTCTCTCCATTTAGCAGGTGCTTCTTCTATTATAGGAGCTATTAATTTTATTTCTACGATTATTAATATACGATTATTAGGAATGAGAATGGAAAAGGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCAGTTTTATTATTATTATCTTTACCTGTTTTAGCGGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTCGATCCTGCTGGAGGAGGGGATCCAATTTTATTTCAACATTTATTTTGATTTTTTG : PL 4.5, PW 3.6, AW 2.3, OL 3.9, OW 2.3. Eye diameters and interdistances: AME 0.21, ALE 0.11, PME 0.26, PLE 0.23, AME\u2013AME 0.15, AME\u2013ALE 0.31, PME\u2013PME 0.27, PME\u2013PLE 0.35, AME\u2013PME 0.13, ALE\u2013PLE 0.17, clypeus AME 0.14, clypeus ALE 0.41. Palp and leg measurements: palp 4.7 , I missing, II 10.8 , III 10.2 , IV 14.4 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 2012; femora II p110, d111, r112, III p012, d111, r112, IV p002, d111, r112; patellae II 000, III\u2013IV 101; tibiae II v22222, III\u2013IV p11, d111, r11, v222; metatarsi II v222, III p112, d010, r112, v222, IV p112, d010, r112, v2222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 5 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 5 bristles. Sparse scopula restricted almost entirely to tarsi. Palpal claw with 7 secondary teeth, leg claws II\u2013IV with 3 secondary teeth. Position of tarsal organ: II 0.75, IV 0.85.Copulatory organ Fig. . EpigynaColour Fig. c and d. Ctenidae . The new species resembles B.withinyou J\u00e4ger, 2022 (see B.withinyou), by the median plate with two separate sclerotised patches laterally and a longer median bulge in anterior half  and by the spermathecae separated by more than twice their diameter .Small 2022 see b, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572106).TATTTGGGGCTTGAGCTTCTATAGCTGGTACATCTATAAGTGTTTTGATTCGTATGGAGTTGGGACATTCGGGGAGAATATTGGGAGATGATCATCTCTATAATGTTATTGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTATTTTAATTGGAGGTTTTGGTAATTGGTTGGTTCCTTTAATGTTAGGGGCTCCTGATATGTCGTTTCCTCGAATAAATAATTTGTCTTTTTGGTTACTTCCTCCTTCTTTATTTTTATTGTTTATGTCTTCTATAACTGAAATAGGGGTAGGAGCTGGTTGAACGGTGTATCCTCCTTTGGCTTCAAGAATGGGTCATGCTGGTAGATCTATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCGTCTTCTATTATAGGTGCTATTAATTTTATTTCTACTATTATTAATATGCGTTTATTAGGAATGAGAATAGAGAAAGTTCCTTTGTTTGTATGGTCTGTTTTTATTACTGCGGTATTGTTATTATTGTCTCTTCCTGTTTTGGCAGGAGCTATTACTATATTGTTAACTGATCGTAATTTTAATACTTCTTTTTTTGATCCGGCTGGAGGGGGAGATCCGATTTTATTTCAACATTTATTTTGATTTTTT : PL 6.4, PW 5.1, AW 2.3, OL 4.8, OW 3.6. Eye diameters and interdistances: AME 0.22, ALE 0.17, PME 0.27, PLE 0.23, AME\u2013AME 0.25, AME\u2013ALE 0.40, PME\u2013PME 0.34, PME\u2013PLE 0.49, AME\u2013PME 0.17, ALE\u2013PLE 0.29, clypeus AME 0.11, clypeus ALE 0.52. Palp and leg measurements: palp 8.2 , I 20.5 , II 17.4 , III 14.5 , IV 22.4 . Leg formula 4123. Spination of palp and legs: palp 161, 001, 111; femora I p021, d111, r1111, II p112, d111, r112, III p001, d222, r112, IV p012, d111, r112; patellae I\u2013IV 101; tibiae I p110, d111, r11, v22222, II p110, d11, r11, v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II p111, d012, r111, v222, III p111, d002, r111, v222, IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 23 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 6 bristles. Sparse scopula on all tarsi and metatarsi I\u2013III. Leg claws I with 5, II with 4, III with 5 and IV with 6 secondary teeth. Position of tarsal organ: I with 1.64, II 1.15, III 0.96, IV 1.28.Palp Fig. a\u2013c. RTA 28Colour Fig. c and d. Female (IZCAS-Ar 43733): PL 5.9, PW 4.4, AW 3.0, OL 5.2, OW 3.5. Eye diameters and interdistances: AME 0.23, ALE 0.19, PME 0.27, PLE 0.25, AME\u2013AME 0.20, AME\u2013ALE 0.44, PME\u2013PME 0.33, PME\u2013PLE 0.52, AME\u2013PME 0.17, ALE\u2013PLE 0.28, clypeus AME 0.13, clypeus ALE 0.51. Palp and leg measurements: palp 5.8 , I 13.9 , II 12.8 , III 11.3 , IV 16.7 . Leg formula 4123. Spination of palp and legs: palp 131, 001, 112, 203; femora I p021, d111, r011, II p012, d111, r111, III p112, d111, r112, IV p002, d111, r112; patellae I\u2013II 000, III\u2013IV 101; tibiae I\u2013II v22222, III\u2013IV p11, d111, r11, v222; metatarsi I\u2013II v222, III\u2013IV p111, d012, r111, v222. Chelicerae with 3 promarginal, 4 retromarginal teeth and with elongated patch of 12 tiny denticles along entire cheliceral furrow. Retromargin of chelicerae close to fang base with 7 bristles. Sparse scopula restricted almost entirely to tarsi, only metatarsi I\u2013II with sparse scopula hairs. Palpal claw with 5 secondary teeth, leg claws I with 2, II with 3, III with 4 and IV with 3 secondary teeth. Position of tarsal organ: I 0.97, II 0.84, III 0.75, IV 1.06.Copulatory organ Fig. a and b. Colour Fig. e and f. Ctenidae . The new species is assigned to the scarymonsters-species group with the characteristics of embolus with a basal, ventral bulge , tegulum bulging proximally at tegular apophysis base, the subdistally arising, apically pointed RTA in males, transversally oval median plate and lateral teeth situated at posterior margin of epigyne and spermathecae bottle gourd-shaped. It resembles B.neukoeln J\u00e4ger, 2022 (see 28B.neukoeln), by the tegular apophysis longitudinally orientated , by the conductor nearly quadrilateral  and by the constrictive anterior width/widest width: 1/2 .Medium-sized 2022 see a\u2013c and l2022 see a, but caThe specific name refers to the type locality and is a noun in apposition.Malaysia :OP572111).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC :OP572109).GGTTTGGAGCTTGAGCTTCTATAGTAGGAACATCTATAAGAGTATTAATTCGTATAGAATTAGGACATTCTGGAAGATTATTAGGAGATGATCATTTATATAATGTAGTTGTTACTGCTCATGCTTTTGTTATGATTTTTTTTATAGTAATGCCTATTTTAATTGGAGGTTTTGGAAATTGATTAGTTCCTCTAATATTAGGGGCTCCTGATATATCTTTTCCTCGAATAAATAATTTATCATTTTGATTACTTCCTCCTTCTTTATTCTTATTATTTATGTCTTCTATAACTGAGATAGGAGTAGGAGCTGGTTGAACAGTATATCCTCCCTTAGCTTCTAGAATAGGACATATGGGAAGATCAATGGATTTTGCTATTTTTTCTCTTCATTTAGCTGGGGCTTCCTCTATTATAGGAGCTATTAATTTTATTTCTACAATTATTAATATACGATTATTGGGAATAAGAATAGAGAAAGTTCCATTATTTGTGTGGTCTGTTTTTATTACTGCGGTATTGTTGTTATTGTCTTTACCTGTTTTAGCAGGTGCTATTACTATATTATTAACGGATCGTAATTTTAATACTTCTTTTTTTGACCCAGCTGGGGGGGGGGATCCCATTTTATTTCAACATTTATTTTGATTTTTGC , Am.krabi sp. n. , Am.matakecil Miller & Rahmadi, 2012 , Am.phangnga sp. n.  and Am.saraburi sp. n. , the PME and AME are almost the same size  also is a cave-dwelling species and its eyes are strongly reduced in size and pigments are absent  are almost the same size (The posterior median eyes of all Li, 2022 . For theame size . These re absent . It is wame size , so thes"}
+{"text": "Drosophila melanogaster, insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed inDrosophila. Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster. For example, nutrient sensing pathways, like IIS, mTOR, and the amino acid response (AAR) pathway, function within adultDrosophilaadipose tissue to control distinct stages of oogenesis . However, insects as a group that potentially includes 5.5 million species  have diverse life histories. For example, female reproductive strategies are influenced by differences in ovarian dynamics , egg carbon:nitrogen ratios , and ovariole numbers . Insects also vary in distribution of the adipose tissue within the body, which could potentially affect inter-organ communication.Drosophila is therefore an excellent model for one such suite of traits but is not representative of all insects. We must develop additional diverse insect models in order to understand the variety of mechanisms controlling life history responses to dietary stress across insects.Resource allocation is inextricably linked to tradeoffs between life-history traits, allowing organisms to adapt to a dynamic environment . Nutritional control of the tradeoff between survival and reproduction is one of the best-characterized examples of this phenomenon  and has been described for a variety of organisms, including insects. In several insect species, nutrients are reallocated from the energy intensive process of oogenesis to support somatic tissue maintenance during nutrient deprivation . This ensures that organisms survive beyond suboptimal nutritional conditions and do not waste resources on reproduction when the likelihood of offspring survival is low. While it is likely that multiple tissues sense and respond to nutrient input, we are just beginning to understand how inter-organ communication mediates whole organism responses in the heavily studied insect model,Drosophila melanogasterand various butterfly species within the Nymphalidae (Lepidoptera). These species differ in fat body distribution , ovarian dynamics , egg C:N ratio  and completeness of the adult diet .Given that nutrient sensing signaling pathways control the response of insect fat body to changes in dietary input, which in turn affects allocation to egg production and other life history functions , we are interested in comparing the operation of these pathways and their outcomes amongDrosophila.Upon insulin receptor activation, Akt, the main effector kinase, acts on several targets to regulate cellular metabolism, survival, proliferation, and growth as well as reproduction . When phosphorylated by Akt, the transcription factor FOXO is restricted from the nucleus, where it controls expression of genes that mediate the negative effects of reduced IIS . In addition, Akt indirectly activates the mTOR kinase by inhibiting the negative regulatory complex of mTOR, Tsc1/Tsc2 . The mTOR kinase can also be activated by the Ragulator complex which senses amino acid status . In both cases, mTOR activation happens at the lysosome.The response to dietary stress of the IIS pathway has been well characterized inDrosophila, the transcriptomes for our non-model lepidopteran species have very limited similarity to those ofDrosophila melanogaster. Therefore, the likelihood of successful use ofD. melanogasterantibodies in the lepidopteran species is low. In contrast, our identified FOXO and TOR transcriptomes for the three lepidopteran species had high percent similarity (> 95%), indicating likely success in developing an antibody for each protein that would work in all three butterfly species.To gain a better understanding of how IIS functions within lepidopteran fat tissue, we want to visualize changes in localization of FOXO and mTOR. One approach to tracking localization of these components is to use species-specific antibodies for immunocytochemistry. While the required antibodies for FOXO and mTOR are available forAgraulis vanillae andHeliconius charithonia, we first determined the protein sequences for those genes for each species. We downloaded amino acid sequences forBombyx mori(Lepidoptera: Bombycidae) andHeliconius melpomene melpomene for FOXO and TOR from uniprot.org. We then used GeneiousPrime to generate transcriptome sequences for these proteins. We blasted these sequences againstA. vanillae andH. charithoniatranscriptomes to locate the sequences for each gene for these species. Transcriptome data forH. charithoniacame from Catalan et al (2018), and forA. vanillaefrom Hanly et al (2019). Additionally, we used transcriptome and amino acid sequences forSpeyeria mormonia, because we are interested in experiments with that species in the future. These RNA sequences were converted to amino acid sequences for determination of epitopes joint among the species. Epitope identification and antibody production were done by Genscript. Rabbits were used for polyclonal antibody production. We also used GeneiousPrime to compare the similarity of FOXO and TOR RNA sequences among our three species, as well as withDrosophila melanogaster(Diptera: Drosophiladae).In this report, we describe the development of Lepidoptera-specific antibodies for FOXO and mTOR to track changes in localization of FOXO and mTOR. To generate antibodies for FOXO and TOR inDrosophilasince adipocytes in fruit flies from nutrient-rich conditions show low-nearly undetectable levels of FOXO nuclear localization . The majority of mTOR immunoreactivity shows diffuse cytoplasmic localization with slight enrichment at cell-cell junctions . Under nutrient replete conditions, mTOR localizes to lysosomes where it can be activated by amino acids . Thus, future studies will assess if the mTOR staining pattern in butterfly adipocytes colocalizes with lysosomal markers. For both FOXO and mTOR immunolabeling, we are confident that the observed staining patterns are specific since there is no fluorescence in samples incubated without primary antibody . Moreover, the nuclear localization of FOXO is quite different from the cytoplasmic localization of mTOR . There is stronger fluorescence intensity at the lowest antibody concentration for tissue treated with Tween 20 than that treated with Triton X-100 . At higher antibody concentrations, detergent type is not a major factor in immunostaining quality. Taken together, we achieve the best signal-to-noise ratio for both antibodies when using Tween 20 as the detergent and 2.5 \u03bcg/mL of anti-FOXO and 5 \u03bcg/mL of anti-mTOR .We used a standard whole-mount immunostaining protocol to test immunoreactivity of these antibodies in lepidopteran fat body. We varied antibody concentration  and permeabilization solution detergent (Triton X-100 versus Tween 20). We find that both antibodies show immunoreactivity in lepidopteran fat body at all three concentrations with varying robustness  in all three butterfly species tested. For both antibodies, higher concentrations produced stronger signal . FOXO immunoreactivity showed enrichment in the nucleus with diffuse labeling in the cytoplasm . Since FOXO translocation to the nucleus results from reduced IIS pathway activity, this data suggests that the butterflies from which the fat body originated may have been under nutrient stress. Alternatively, lepidopterans may have a higher baseline of FOXO-dependent gene expression thanDrosophilato other insects that differ in their fat body distribution, mode of ovarian function (e.g. stem cell-supported versus not), and adult diet macronutrient composition?In the future, we will further optimize the immunostaining protocol by modifying additional steps in the protocol, such as fixative type and fixation time. This work demonstrates the feasibility of using these newly developed lepidopteran antibodies to answer questions like how generalizable are nutrient sensing mechanisms inButterfly collectionAgraulis vanillae females were collected in Sunrise, Florida in January 2022.Heliconius charithonia females came from Davie, Florida and south Texas in January 2022 and fall 2021 respectively.AntigensFOXO: MSLPRGGSYQSPWSSQTALSELEGAMGEFEPVGELAEVGFEPQTRARSNTWPQPRPENYVDAEDPGSKKNSNQNLSGAPPLPTSATTKKNSSRRNAWGNLSYADLITQAITSSQEKRLTLSQIYEWMVQNVPYFKDKGDSNSSAGWKNSIRHNLSLHNRFMRVQNEGTGKSSWWMINPDAKPGKSVRRRALSMETSKTEKRRGRVKKKPDALRNGVTADATPSPSSSISESLDIFPDSPIHSSFQLSPDFRQRASSNASSCSRLSPIPSLIPTEPEWSTDYTPSGDFASTSDFSPADYTQDQDLAGSLADSMKLHGADPFLNTYVPTTSSSSSGGSYRFTYGACPRHPHGGCACAPLYPAHPAHPTHSHQHSLDHFVRPPPPADPADIMQTENSQTQMVATSDATLMNGGIMVQTGPMGPTTVMGQIMGALNTGLSEDLNIEALEHSFDCNVDEVIRHELSMDGTLDFNFPQQNTAMAAEAESQFRAPSAPVPTTLSGGGASQTPYTVAPSWVH.TOR: MYMFLLKGHEDLRQDERVMQLFGLVNTLLQADPDTFRRDLVIQRYAVIPLSTNSGLIGWVPHCDTLHSLIKDYREKRKILLNIEHRIMQRMASDLEKLMLMQKVEVFEHALEHTAGDDLAKLLWLKSPSSEVWFERRTNYTRSLAVMSMVGYILGLGDRHPSNIMLDRVTGKFLHIDFGDCFEVAVTRDKFPEKIPFRLTRMLINAMEVTGIEGTYRRTCESVMEVLHRHRDSVMAVLEAFVYDPLLNWRLIDAGRRAGAPDDDGDASPQRPDDAPAETNLNKRALAIVNRVRDKLTGRDFTHIEENSVSVQRQVDLLIQQATSNENLCQCYVGWCPFW.Whole-mount adipose tissue immunostaining\u03bfC until mounted on slides.Females were sacrificed by crushing the ventral nerve and their abdomens were immediately dissected in cold phosphate buffered saline (PBS) to obtain fat body. Fat bodies were placed in 1.5ml microfuge tubes that were previously coated with 3% bovine serum albumin (BSA). Fat bodies were fixed in 1ml of 4% paraformaldehyde diluted in cold PBS (PFA) for 30 minutes at room temperature, rocking on a nutator. The PFA was then removed, and samples were rinsed twice with 1ml of PBS containing either 0.1% Triton X-100 (PBTX) or Tween 20 (PBTW) as a detergent. After rinsing, samples were washed three times with 1ml of 0.1% PBTX or PBTW, rocking each time for 15min on a nutator at room temperature. After the last wash, samples were incubated in 1ml of a blocking solution (0.1% PBTX or PBTW in 5% BSA) overnight at room temperature on a nutator. Samples were incubated in the indicated primary antibody concentration, diluted in blocking solution, overnight at room temperature. After washing three times for 15 minutes with PBTX or PBTW, samples were incubated in goat anti-rabbit AlexaFluor 488 secondary antibody diluted 1:250 in blocking solution and protected from light by covering with foil. Samples were then washed three times for 15 minutes with 0.1% PBTX or PBTW, protected from light. Following removal of wash solution, two drops of VectaShield containing DAPI were added. Samples were stored at 41X PBS \u2013 made from 10X PBS 3% BSA \u2013 made from 30% BSA 4% PFA \u2013 made from 16% PFA Triton X-100 - VWR, 0694-1LTween 20 \u2013 VWR, 0777-1LBlocking solution \u2013 0.1% PBTX or PBTW and 5% BSA in 1XPBSRabbit anti-FOXO \u2013 GenScriptRabbit anti-TOR \u2013 GenScriptGoat anti-rabbit IgG AlexaFluor 488 \u2013 Invitrogen, A27034"}
+{"text": "Caenorhabditis elegans synthetic lethality screening. We identified the bromodomain protein, BET\u20101, as a key regulator of actin function and longevity. Overexpression of bet\u20101 preserves actin function at late age and promotes life span and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET\u20101. Together, our discovery assigns a key role for BET\u20101 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.The actin cytoskeleton is a three\u2010dimensional scaffold of proteins that is a regulatory, energyconsuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well\u2010understood. Here, we performed a multipronged, cross\u2010organismal screen combining a whole\u2010genome CRISPR\u2010Cas9 screen in human fibroblasts with in vivo  Caenorhabditis elegans (2), we identified BET\u20101/BRD4 as a novel regulator of the actin cytoskeleton (3). BET\u20101/BRD4 promotes actin filament stability in C.\u00a0elegans and senescent cells, which has direct impact on organismal health, survival, and life span.Using a cross\u2010species screening approach combining genome\u2010wide CRISPR\u2010Cas9 screening in human fibroblasts (1) and synthetic lethality screening in  ACSRactin cytoskeletal stress responseEVempty vectorGOgene ontologyRNAiribonucleic acid interference1Caenorhabditis elegans  , mediated by the heat\u2010shock transcription factor, HSF\u20101. HSF\u20101 is activated under thermal stress and promotes protein homeostasis through the upregulation of chaperones and other genes related to protein quality control , which promotes actin function, longevity, and healthspan in a multicellular eukaryote.To identify previously unidentified regulators of actin function, we adopted a multipronged, cross\u2010organismal screening approach. Since actin is one of the most highly conserved proteins, both in terms of sequence and functional homology, we held the rationale that an evolutionary\u2010conserved perspective could provide a powerful method to identify key regulators of actin function. We first utilized a CRISPR/Cas9\u2010driven growth\u2010based genetic screen in human cells to identify genes required for survival under actin stress. Actin stress was applied by exposure to cytochalasin D, a drug that inhibits actin polymerization by binding to F\u2010actin filaments and preventing the addition of actin monomers   and exhibits visible perturbations of actin function without affecting whole organismal physiology. Animals treated with 10% act\u20101 RNAi develop normally to adulthood but display notable perturbations of actin quality in muscle cells  Figure\u00a0. Importas Figure\u00a0. Therefobet\u20101, egrh\u20101 (Early Growth factor Response factor Homolog), F53B6.5, and ikb\u20101 (I Kappa B homolog) resulted in delayed development when combined with 10% act\u20101 RNAi but exhibit no physiological phenotypes when knocked down alone. RNAi knockdown of ZK512.4 did not exhibit a developmental defect but showed sterility (no eggs were visible on the plate at day 1 of adulthood) when combined with 10% act\u20101 RNAi, despite showing visible progeny formation when knocked down alone. ZK512.4 is a predicted ortholog of human SRP9 , a critical part of protein targeting to the endoplasmic reticulum , a major transcription factor that has been implicated in multiple diseases including cancer , which removes methionine residues from nascent polypeptide chains and are also implicated in cancer  for 24\u2009h to induce senescence , the heat\u2010shock response (HSR), or oxidative stress response. We did observe a mild activation of genes involved in the endoplasmic reticulum UPR (UPRER). These data were further verified by direct comparison with a dataset identifying targets of the UPRER transcription factor, XBP\u20101    Figure\u00a0. Consist6 Figure\u00a0, which e1 Figure\u00a0, suggest2.4bet\u20101B overexpressing worms was act\u20103 , which has been previously linked to aging , another gene whose reduction has been linked to longevity , we opted for a secondary screening platform in bet\u20101, the C. elegans ortholog of human BRD2 and BRD4. bet\u20101 encodes a double bromodomain protein that has been originally characterized for its role in cell fate decisions  dosage has been previously linked to longevity in C57B6/J mice, although the underlying molecular basis was not identified , but may be detrimental where actin dynamics are required .What was intriguing was that BET\u20101's impact on actin function is seemingly context dependent. We found that bet\u20101 overexpressing animals, with act\u20103 specifically being highly induced. We also observed changes in other cytoskeletal genes, including different regulatory factors. These genes showed opposing effects when compared to bet\u20101 loss of function animals and were largely dependent on the HAT, mys\u20101. Importantly, the transcriptional changes induced by BET\u20101B were largely distinct from the UPRMT, HSR, and the oxidative stress response.Since we see that all visible BET\u20101B protein is found within the nucleus and localizes to puncta which in no way resembles actin filaments, we believe that BET\u20101B does not impact actin through direct interactions. Moreover, the beneficial effects of BET\u20101B on actin and life span are dependent on MYS HATs, which acetylate histones at specific lysine residues, allowing BET\u20101B binding to alter gene expression , which can impact organismal health. An exciting area of future work is to investigate whether a BET\u20101B driven ACSR \u2013 possibly in coordination of other cytoskeletal regulators including DAF\u201016 and HSF\u20101 \u2013 can drive overall stress resilience and longevity.However, we did observe a mild overlap with UPRbet\u20101B overexpression was dependent on the insulin/IGF\u20101 transcription factor, DAF\u201016. While DAF\u201016 itself has not been directly tied to cytoskeletal function, FOXO transcription factors have direct impacts on actin function in mammalian systems  expressing hTERT and Cas9 were used for the CRISPR\u2010Cas9 based screen as previously described  and live/dead Aqua  staining was performed as per manufacturer's protocol. Briefly, cells were stained with both dyes for 20\u2009min at room temperature in Annexin V binding buffer . Dyes were washed once with Annexin V binding buffer, resuspended with Annexin V binding buffer, and flow cytometry  was used to quantify staining. For statistical analysis, two\u2010way ANOVA with Dunnett's test was used to compare the ratio of live cells between the 0\u00a0\u03bcM control against each drug\u2010treated group. 3 technical replicates of 2 biological replicates were performed.For Annexin V staining, BJ fibroblasts were treated with cytochalasin D at 0\u201310\u00a0\u03bcM concentrations for 24\u2009h and 3\u2010h treatment with 30\u2009mM H4.2Human primary IMR\u201090 fibroblasts were grown in DMEM, supplemented with 10% FBS, 100\u2009U/ml penicillin, 100\u2009\u03bcg/ml streptomycin, and 2\u2009mM glutamine at 37\u00b0C in a humidified atmosphere with 5% CO2 and 3.5% oxygen  per 100\u2009\u03bcl culture medium. The old medium from senescent IMR\u201090 cells was aspirated and 120\u2009\u03bcl MTS assay solution per well was added. After 2\u00a0h incubation, 100\u2009\u03bcl MTS assay solution aspirated from the plate in a fresh 96\u2010well clear plate and the absorbance at 495\u2009nm was taken immediately using Microplate Reader.To examine the adhesion capacity of senescent cells after drug treatments, VybrantTM Cell Adhesion Assay Kit  was used according to the manufacturer's protocol with modification. Briefly, 30\u201340% confluent IMR\u201090 cells were treated with etoposide and BET inhibitor/degraders drugs in 6\u2010wells plates as previously mentioned. After 8\u2009days, cells from each well were trypsinized and pelleted down after neutralizing the trypsin with complete media. The cells were washed once with PBS and resuspended in 1\u00a0ml serum free DMEM containing 5\u00a0\u03bcM Calcein AM solution for 30\u2009min. Then cells were washed twice with complete media to remove the excess Calcein AM. Finally, the Calcein AM labeled cells were suspended in 700\u2009\u03bcl complete media and 100\u2009\u03bcl of suspension was seeded per well in a PhenoPlate\u201096 (PerkinElmer). Cells from each treatment were seeded in total 6 wells of 96 wells. After 4\u00a0h incubation, 3 wells from each treatment were washed 3 times with PBS to assay the adhering cells and 3 wells were maintained without washing to assay the total cells. Fluorescence was measured using Clariostar Multi\u2010Mode Microplate Reader using FITC channel. The percentage of adhered cells were calculated from the fluorescence of adhered cells against total cells seeded after background subtraction.To detect the level of actin polymerization, we used Alexa Fluor 488 Phalloidin , because this bicyclic peptide specifically binds to polymerized F\u2010actin. Approximately 12,000 IMR90 cells were seeded in a PhenoPlate\u201096 (PerkinElmer) and treated with etoposide and drugs, as previously mentioned. After 8\u2009days of etoposide treatment, cells were fixed with 4% paraformaldehyde. After washing the fixed cells with PBS three times, cells were permeabilized with 0.2% Triton\u2010X and blocked with 1% BSA solution in PBS for 30\u2009min to reduce the background staining. After blocking, F\u2010Actin of the cells was stained with 165\u2009nM Alexa Fluor 488 Phalloidin for 1\u2009h at room temperature. Cells were washed with PBS three times and nuclei were stained with DAPI. The fluorescence of the wells was measured by using Clariostar Multi\u2010Mode Microplate Reader.4.3E. coli B strain and switched to growth at 20\u00b0C on HT115 E. coli K strain for all experimentation. HT115 bacteria carrying a pL4440 empty vector control or expressing double\u2010stranded RNA containing the sequence against a specific target gene were used for all experimentation. Experiments are performed on age\u2010matched animals synchronized using a standard bleaching protocol  and bleached in 1.8% sodium hypochlorite and 0.375\u2009M KOH diluted in M9 until all carcasses were digested. Intact eggs were then washed 4\u00d7 with M9 solution followed by L1 synchronization by floating eggs in M9 overnight in a 20\u00b0C incubator on a rotator for a maximum of 16\u2009h. Synchronized animals are always grown on standard RNAi plates .All strains used in this study are derivatives of the N2 wild\u2010type worm from the Caenorhabditis Genetics Center (CGC) and are listed below. Worms are maintained at 15\u00b0C on OP50 bet\u20101 overexpression, isoforms A, B, and C were defined as per  as a co\u2010injection marker, and 100\u2009ng/\u03bcl of pD64 vehicle as filler DNA. Worms positive for the fluorescent pharynx were selected to identify stable arrays. Integration was performed by gamma irradiation where L4 worms were irradiated with 4000\u20134400 rems of radiation and integration events were selected by finding animals that maintained 100% frequency of co\u2010injection marker in the F3 generation. Lines were then backcrossed into N2 a minimum of 8x to eliminate mutations. For overexpression of 3xHA::GFP::bet\u20101B, a GFP sequence containing introns and a 3xHA cassette was cloned upstream of the bet\u20101B coding sequence. Injection and integrations were performed by SUNY Biotech.For bet\u20101(uth41) mutant line, we used a Cas9\u2010RNA protocol as published on the IDT website via the Dernberg lab. Briefly, a mixture of 15\u2009\u03bcM trRNA, and 22\u2009\u03bcM crRNA (accatgggcaagtccgcgac) were incubated for 5\u00a0min at 95\u00b0C, cooled, then mixed with 24\u2009\u03bcM Cas9 protein and left at room temperature for 5\u00b0C. 2.5\u2009ng/\u03bcl of pEK2 (myo\u20102p::tdtomato) was added as a co\u2010injection marker and this mixture was injected into C. elegans gonads. All progeny positive for the co\u2010injection marker were selected, then sequenced for INDELs that incorporated a premature stop codon. The bet\u20101(uth41) mutant has a premature stop codon at amino acid 17.To synthesize the 4.4ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGC CATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGC GATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAATCCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAGCTCCAAAACCTGCACCAGTACCAGCACCAACATCATCAAGACCTCCGGCAGCACCGAGACCACCGTCAAAACCAAAGAAAACGGGTGGAGCGAGTATTCTTGATACTCTACTTCCAGATACATTTGGAGCATCACCTCCCCAGTTTTTCCAGTCGCAACCAACAACGTCGGCTACGATTAGATCACCAACGGAAAGCCAACCCGGGAATGGTGAAGACGAGCAGACCAGGATTCAGAGGATGCGGATGGAGGCAAAGCGAGCCCGCCAAAAAGAAGACGAAGGCAGTGTCTCGTTGTCAAACCAAATGGAAATGATGGCTGCATTTGAATTTGATAATACATATTAA4.5ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACC GGCAAAAAGTGACGGTGGATCAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTATGAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTT CAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGA AAGAAAAAGAAAAACTTCAATATGTCCGAATCCTCGGATGATGAGACGTCGAATAGTCGAAAACGTCGAAAGAGAGAGAGCAGTGAATCACAGAGCTCTTCGTCCAGTGATGATGATTCAGATGATGAGGATAGGCCGAGTATTCCCCGTAAATCAGGTCAACCACCATCAACATCACGTGAATGGAATCAATCATCAGCTCCTCCACCACGAATGGGAGGAATGGGAGGACAACCACCAATGTCACGAGTACCTGCATCATCATCCACATCTGTATCAGCAATCGGAAAGAACAACGCAGCCGCCTCGTCGAATTCATATCAAAAATTTTATAATTGTTTTCACAGTTATACTCCACCTTTAAAAGbet\u20101CTTGAAAAAAAAATCATCAAATTACTGGTAAATTTTTGTTAA ATGTCTGAGGGCAGCGGAGACCAATCACAACAACGACCATGGGCAAGTCCGCGACAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGTGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGC AGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCGCGAAGCCAGAACGAAAAGTGGCCGGAAAGAAGACGGGAAAACGAAAAGCCGAATCAGAAGATGACGAGAAGCCGGAACCTTTGA GAGCAAAACGAGAGGTGGCTGTTGTCAAAAAAGAAGTTCATCAGCCATTGCTCCCAAGTAT GAAGCCCTGTCTGAAGCTGCTCAATGATTTTTCTACAAAAAAATATCAGGAATTTGCTTGGCCATTCAACGAACCAGTAGACGCTGAACAACTGGGACTCCATGATTATCATAAAATTATCAAAGAACCAATGGATCTGAAATCAATGAAAGCAAAAATGGAAAGTGGAGCATACAAGGAACCTTCAGATTTCGAGCATGATGTTCGTTTAATGCTCAGGAATTGTTTTCTTTATAATCCAGTCGGTGATCCGGTTCACAGTTTTGGTCTTAGGTTTCAAGAAGTTTTTGATAGACGATGGGCTGAACTAGGTGATTCGAGTTCTCGTGCTTCATCAGTTGCACCTCAATCAGCTCCGATTGCTCCAACTCCGAAAGTAGCAAAATCAAGTGCTCCAAAAGAACCGAAAGAGTCTCGAAAAGAGCATAAAAAGGAGACGACTTTTGAAGCAAGCGGTGCAAAATCGGAGGATTTAATGCAGATAAACAACGCGTTGAGCATGATTCGAGAACGTGAGGAAAAGCTTAAAGCAGAGCTCGCCGCTGCACAAGCGATAAAGGATAAACTGACGAGTGTGAAGAATCGACGAGAAGATAATCCGAATGAGCCATTTCCGGAGAAGCTTATCAATGAGACAAGAGCCTTGTGCACGACGCAAGTTGGACAAAATGCTTCAAGTTCTTCAGCTTCTTCTGCTGCTTTGAGGAACGGACGAAGCAAAAAAGCAGCATCCGCACGTCTCTATGGTTACGAATTTGATTCGGATGATGAGGATAATAAGATGGCACTGACTTATGAGGAAAAACGAAACTTGAGCAATCTGATTAATAATTTACCCAACAATCAACTCAACACCATAATTTCGATTATTCAACGGAGAGAACGAAGCGCTCTGATGCAACAACAACTCGATGACAGTGAGGTTGAACTGGATTTCGAATCACTTGGAGATATGTGCCTGAGAGAAATGGGTGCATTTATCAAAACAATTCCAACATTAAACGGAAATGGCGATGATGAGAAGCCGAAAACGTCTTCGAAT CCGACATCTTCTGGAGCAACAGGATCAAAGGGTTCGTCGTCGTTGGAGAGCAAAAATGGAAAGAAAATAA4.6CACAGGTCTCTAGTGTATCCACTTCGAATGCGATGCCCGAAACCTCTTCATCCATCCGTCTCCTTCTCGCTCTCTCTCTCTCTCTCTCTTCTCCATCTCTCTCCACATTTTGCCTGCTATCTCGTGATTGTCGTCCCGTCCGTGTTCCGCCGCACACACTGCCTGTCTTCTCTTAACCGTGTGTCGATCAACTCCCAAACCGCTACGCTATTTCTCTCTCCCTCTCTCTCTCTCTTCGGCGGTGACATTTCTGACTAGATGGTCATACAAAACGCGTGCTGCGCGCGCGCTCCGCAAAAATCGACGCGAATCGATTAATGTGCGTCTCGTTTCTCTATCTCTGACCGCCCCCGCTTCAACCTAACACTATTTTTGAATGCTTTTCAACTGTAACTTGCAGCTAATTAGAAGTTGAGAGATAACCTGTTGCGATTGGCTCCGGGCAAGGGTTGGGAGGTCGCACCAGAAATTTTAGAGCTCTAGGATTTCAAATTTTTGGGTTTCAAGACCGTAACATGATTTTCTTGGAAATTTATCACAAATCATGTAGAAAATCGATATCAGTAAGAGGGAGTGAGTGATCTATCATTTTTTATCTTTCGATCTGAAATTCCACAGCGAAGGTTTTCTGCCGAAATTTCGAAATTGGTATTTTGAACTATCCGATAATTCGTAGAACATCAAGATAAAGTGTCAACCTATAGAAAATCACATGATTCGTCAGAAAATAACATTAATTTCATATGAAATAGTTGAGAAAGTGCTCAAAAATGGCCTAAAATTATCCAATAATCGACATTTGACAACTTTCAGCACACTTTTGAACCGTTTATCAATTGTTTCTGCTGAAATAGACGTATTTTTCGGACGAATCGAGTGATTTCCTATAGTTTTACACTGATTTTTGACAAAAAAATATTGATAGAACATGGTGCATTAGGCAATTTTTTAGAATTGCCGTCTACACCTGATTTCGATGGGTCCTCGTGACAAGACCCAAAATTTTATTATTTTTATCGTTGAAAAAAATCAAATCAATAACACCGCAATCAC CATTTGCAAAGTTTAATTAAATACAATTTTTATTAAAATATTTCAGGAATAAAAATATTAGTCAGAATAATCCCATGTTCTTCTAGCGATTTCAACTAATTCTTTGAAAATAACATTTCTTGGAATT TAAGAATACGAAAATAGTCACTTCTTTGTATTCTAGAAACGCTAATTCCTGCAACCGACAAA TTAAAAGTACAAAAAATGATACGGCAAGCGCGCTCCAATTCAAATCGAGTCTCCCGCCTTCCTTGACGTCATTGCTAACAGCTGCTTCGGTTTTTTCCTCCAAATTTCGTGGTTCAAATTTTAT TTTTAATTGAATTTTAACAAAATAGGAAGCTAGTTGAGTAACATTTATTATTAATTTTGTAAAATATTCTGCAAATTCGGCGTTTTCTTTTAATTCAAATAAAAGTTTTCAATAAAAAAAATCGATATTTTCAG4.7CATCTCGCGCCCGTGCCTCTGACTTCTAAGTCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCACCCCCTATTTTTGTTATTATCAAAAAACTTCTCTTAATTTCTTTGTTTTTTAGCTTCTTTTAAGTCACCTCTAACAATGAAATTGTGTAGATTCAAAAATAGAATTAATTCGTAATAAAAAGTCGAAAAAAATTGTGCTCCCTCCCCCCATTAATAATAATTCTATCCCAAAATCTACACAATGTTCTGTGTACACTTCTTATGTTTTTTACTTCTGATAAATTTTTTTGAAACATCATAGAAAAAACCGCACACAAAATACCTTATCATATGTTACGTTTCAGTTTATGACCGCAATTTTTATTTCTTCGCACGTCTGGGCCTCTCATGACGTCAAATCATGCTCATCGTGAAAAAGTTTTGGAGTATTTTTGGAATTTTTCAATCAAGTGAAAGTTTATGAAATTAATTTTCCTGCTTTTGCTTTTTGGGGTTTCCCCTATTGTTTGTCAAGATTTCGAGGACGGCGTTTTTCTTGCTAAAATCACAAGTATTGATGAGCACGATGCAAGAAAGATCGGAAGAAGGTTTGGGTTTGAGGCTCA GTGGAAG4.8ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatac taactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTGTTATGGTGTTCAATGCTTCTCGAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTAT GTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcgg tactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGA ATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATA CAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA4.9TATCCATATGACGTGCCGGACTACGCGTACCCGTATGATGTTCCAGACTACGCCTATCCGTACGACGTACCAGATTATGCA4.10CAGCAACCAATCAAAGGAATCGTACAGCCACGAGTACTTCCACCATTCGGAAAGCCAACACGACACACAAACAAACTGGACTACATTATGACAACAGTACTCAAAGAGGCTGGAAAACATAAACATGTCTGGCCGTTTCAGAAGCCCGTCGATGCGGTTGCTTTATGTATTCCTCTATATCACGAGAGAGTCGCCCGACCAATGGACTTGAAAACAATCGAGAATAGACTGAAAAGTACTTATTACACATGCGCTCAAGAATGCATTGATGATATCGAAACAGTTTTCCAAAACTGCTACACATTCAATGGGAAAGAGGACGACGTGACAATTATGGCCCAAAATGTGCACGAAGTGATAAAAAAGTCACTGGAACAAGCACCTCGCGAAGAGCATGATATGGATGTTTATTGGGGAAAAAATAAGAAAAAACCGGCAAAAAGTGACGGTGGATCGAAATCTTCGTCGAGCAAGAAGAATGATGCTCGTGGACCATCTGAAGCACCGTCAGAGGCTGGAAGTGAAGTTTCGTCTGTAACAACAGCATCAGCAGCAGCCCCGACGGTTTCTGAGTCTGCGAGTGTTGCCG4.11AAAAAAGCAGGCTTGACCGAGCCGAAGAAGGAGATTATAGAGGACGAAAATCATGGAATAT CCAAGAAAATACCAACAGATCCCAGGCAATACGAGAAAGTTACAGAGGGATGCCGGTTATTGGTCATGATGGCTTCACAAGAAGAAGAAAGATGGGCCGAAGTTATTTCAAGATGCCGAGCTGCAAATGGTTCAATTAAATTCTATGTCCATTATATCGATTGCAACCGAAGACTTGACGAATGGGTTCAGTCTGATAGGCTCAATTTAGCGTCGTGTGAGCTACCAAAAAAAGGAGGAAAGAAAGGAGCACACTTGCGGGAAGAAAATCGAGATTCGAATGAAAATGAAGGAAAGAAAAGCGGCCGAAAACGAAAGATTCCACTACTTCCGATGGATGATCTCAAGGCGGAATCCGTAGATCCATTACAAGCAATTTCAACGATGACCAGCGGATCTACTCCAAGTCTTCGAGGTTCCATGTCGATGGTCGGCCATAGTGAAGATGCAATGACAAGGATCCGAAATGTCGAATGCATTGAACTAGGAAGATCACGAATTCAGCCATGGTACTTTGCACCTTATCCACAACAATTGACAAGTTTGGATTGTATTTATATTTGCGAATTTTGTCTGAAATATCTAAAGTCGAAAACTTGTCTGAAACGGCACNTGGAAAAATGTGCAATGTGTCACCCACCTGGCAATCAAATCTACAGTCACGATAAACTTTCATTTTTTGAAATCGACGGCCGCAAAAACAAAAGCTATGCTCAGAATCTATGCCTGCTTGCCA AACTT4.12C. elegans orthologs of human genes from the cytochalasin screen were identified using Ortholist 2 , images were acquired on either a Zeiss AxioObserver.Z1 microscope equipped with a lumencor sola light engine and Ziess axiocam 506 camera driven by Zeiss ZenBlue software using a 63x/1.4 PlanApochromat objective, standard dSRed filter (Zeiss filter set 43), and a DFC9000 camera; or a Leica Thunder Imager equipped with a 63x/1.4 Plan AproChromat objective, standard dsRed filter (11525309), Leica DFC9000 GT camera, a Leica LED5 light source, and run on LAS X software. For confocal microscopy (gly\u201019p::LifeAct), imaging was performed on a Stellaris 5 confocal microscope equipped with a white light laser source and spectral filters, HyD detectors, 63x/1.4 Plan ApoChromat objective, and run on LAS X software.For high magnification live\u2010cell imaging, animals are picked off of plates and mounted directly onto a microscope slide containing M9\u2009+\u00a00.1\u00a0M sodium azide. For standard wide\u2010field microscopy  floating in M9 in a rotator at 20\u00b0C, then washed 3x with M9 and plated on standard OP50 plates for 24\u2009h, then imaged.For imaging of fluorescent transcriptional reporters, animals were synchronized via bleaching and grown on standard RNAi plates and imaged at day 1 of adulthood. For imaging, animals were moved onto standard NGM plates without bacteria containing 5\u00a0\u03bcl of 100\u2009mM sodium azide to paralyze worms. Paralyzed worms were lined up and imaged immediately on a Leica M205FCA automated fluorescent stereoscope equipped with a standard GFP filter and Leica K5 camera and run on LAS X software. 3 biological replicates were performed per experiment with 4.14t\u2010test using Prism software.To measure gut bacteria invasion, animals were synchronized via bleaching and plated from hatch on RNAi of choice mixed with 20% HT115 bacteria expressing mCherry as previously described . The aqueous phase was mixed 1:1 with isopropanol then applied to a Qiagen RNeasy Mini Kit (74106) and RNA purification was performed as per manufacturer's directions.bet\u20101B overexpression, bet\u20101 RNAi, and bet\u20101(uth41) mutants were compared to N2 wild\u2010type control. In addition, bet\u20101B overexpressing worms were grown on mys\u20101 RNAi and compared to a mys\u20101 RNAi control.Library preparation was performed using a Kapa Biosystems mRNA Hyper Prep Kit sequencing was performed at the Vincent J Coates Genomic Sequencing Core at the University of California, Berkeley using an Illumina HS4000 mode SR100. Four biological replicates were measured per condition. Reads per gene were quantified using kallisto  using 1\u00a0\u03bcg of RNA. RT\u2010PCR was performed using NEB Q5 DNA polymerase as per manufacturer's guidelines using primers listed below. Four biological replicates were performed per condition. Image quantification was performed using ImageJ by drawing an ROI 974 of equal size around each band and quantifying for integrated density. Data was normalized to a 975 4.16Thrashing assays were performed on animals synchronized via bleaching and aged on plates containing FUDR from day 1. 100\u2009\u03bcl of 10\u00a0mg/ml FUDR were spotted on the bacterial lawn. At the desired age, plates containing adult animals were flooded with 100\u2009\u03bcl of M9 solution, and 30\u2009sec videos were acquired on an M205FCA stereomicroscope equipped with a Leica K5 microscope and run on LAS X software. Thrashing was measured by eye over a 10\u00a0s period. A single trash is defined as bending of >50% of the animal's body in the opposite direction. Representative data of three independent biological replicates are presented. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using nonparametric Mann\u2013Whitney testing.4.17A synchronized population of animals were collected via bleaching and 10 L4 animals were moved onto individual plates. Every 12\u2009h, animals were moved onto fresh plates and plates containing eggs were stored in a 15\u00b0C incubator for 2\u20133\u2009days. All live progeny on every egg\u2010lay plate were scored and summed to determine brood size. Dot plots were generated using Prism 7 software where every dot represents a single animal and lines represent median and interquartile range. All statistics were performed using non\u2010parametric Mann\u2013Whitney testing.4.18Caenorhabditis elegans life span assays were performed on standard RNAi plates and were all performed at 20\u00b0C as previously described (Bar\u2010Ziv et al.,\u00a0Caenorhabditis elegans experiments for manuscript revision. N.Dasgupta performed all senescent cell culture experiments. N.Dutta performed RTPCR analysis and assisted with life spans. H.Z., and W.F. performed all standard cell culture experiments. C.K.T., E.A.M., and O.S. performed all computational analysis for CRISPR\u2010Cas9 screening. D.M., A.A., S.H., and T.C.T. assisted with C. elegans experiments. P.D.A. supervised all senescent cell culture experiments. M.A.T. performed essential experiments that assisted in development of the manuscript. All authors edited the manuscript.G.G. and R.H.S. designed all experiments, performed or oversaw all experiments, and prepared the figures and manuscript. R.B.Z. performed computational analysis, figure construction, and writing for all transcriptomics data. M.A. performed all All authors of the manuscript declare that they have no competing interests.Figures S1\u2013S9Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file."}
+{"text": "Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle\u2019s passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain\u2019s property. The giant muscle protein titin is a tandem modular construction designed polyprotein containing more than two hundred individually folded domains, such as immunoglobulin-like (Ig) and fibronectin-type III domains . These dTo examine the linker effect, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the mechanical stability of human Ig domains. SMFS can manipulate a single molecule mechanically ,20,21,22Thus, we choose six consecutive Ig domains of titin, including I27, I28, I29, I30, I31, and I32, as a representative unit (I27\u2013I31), to study the interdomain linker effect b. The stA high-precision AFM measurement system has been used for accurate measurement and comparison of Ig domains in titin ,65,66,67n = 1148, Thus, we built polyprotein Coh-I(27\u201332)/9G-NGL with a 9G linker for measurement. The 9G linker is present between each Ig domain except for the two end I27 and I32 domains a. By appn = 1808, Then, we used polyprotein Coh-I(27\u201332)-NGL with natural sequence for AFM measurement and comparison. The same cantilever used previously for the polyprotein with the linker was used here again to minimize the error. As expected, a similar unfolding pattern was observed b,d,e. Hon = 860). Then, AFM measurement on Coh-I(28\u201330)-NGL with natural sequence showed a force of 325 \u00b1 35 pN (n = 965).To confirm this effect, we chose three Ig domains only and constructed two shorter polyproteins, Coh-I(28\u201330)/9G-NGL and Coh-I(30\u201332)/9G-NGL, for measurement. Indeed, stretching these polyproteins resulted in a shorter force-extension curve with only two 28 nm-peaks from I28, I29, or I31, I32, and one 11 nm-peak from I30, as expected . For Cohn = 1120). However, AFM measurement on the natural sequence showed a different result. Two peaks were observed in the histogram, with a force of 276 pN and 345 pN, respectively (n = 1804), which has not been observed before. For Coh-I(30\u201332)/9G-NGL, the histogram of unfolding force from I31 and I32 showed a single peak with an average force of 320 \u00b1 33 pN , and 186 \u00b1 55 pN (n = 225) without linker. For I(28\u201330), 9G linker is only present on the N terminus. The force was 197 pN (n = 570), and 172 \u00b1 24 pN (n = 910) without linker. Finally, for I(30\u201332), 9G linker is only present on the C terminus. The force was 159 \u00b1 23 pN (n = 477), and 175 \u00b1 24 pN (n = 404) without the linker /9G-NGL, Coh-I(27\u201332)-NGL, Coh-I(28\u201330)/9G-NGL, Coh-I(28\u201330)-NGL, Coh-I(30\u201332)/9G-NGL, Coh-I(30\u201332)-NGL were obtained after Gibson assembly-based method [Escherichia coli strain BL21 (DE3) and cells were cultured overnight in LB medium at 18 \u00b0C by the addition of 1mM IPTG. The cells were pelleted by centrifugation and the polyprotein purification by Ni-NTA affinity. After using wash buffer  to purify the target proteins, the polyproteins were eluted in elution buffer . Protein ligase AEP was obtained according to literature [d method . All platerature .Protein immobilization: The glass coverslips  and probes  were cleaned by plasma. Then, both probes and coverslips were immersed in 1% (v/v) APTES toluene solution for 1 h to add the NH2 group, followed by a reaction with Milli-Q water containing 2 mM ImSO2N3, 4 mM K2CO3, and 20 mM CuSO4 to add the N3 group. After flushing, they were further reacted with DBCO-PEG4-maleimide for at least 2 h to add the maleimide group. Peptide C-ELP20-NGL and GL-ELP20-C were respectively reacted onto the probe and coverslip. For AFM-SMFS measurement, 50 \u03bcL AFM buffer  containing 100 \u03bcM Ig proteins and 50 \u03bcM AEP were pipetted on the glass slides for 40 min. The cantilevers were incubated with 50 \u03bcL solution of 60 \u03bcM GL-CBM-XDoc and 50 \u039cm AEP in the AFM buffer.AFM-SMFS Experiment: Measurements using Coh-Doc interaction of high rupture force as a standard were carried out Nanowizard4 (JPK) atomic force microscope. Using the equipartition theorem, the spring constant of ~30 pN nm\u22121 was obtained by calibrating the MLCT-Bio-DC (Bruker) cantilever in the AFM buffer solution. The functionalized cantilevers and glass coverslip immobilize polyproteins in AFM buffer at pH 7.4. All AFM experiments were performed at a constant pulling speed of 1000 nm\u00b7s\u22121.9G-I28-9G-I29-9G-I30-9G-I31-9G-I32)Protein sequence (I27-GGGGGGGGGPLIFITPLSDVKVFEKDEAKFECEVSREPKTFRWLKGTQEITGDDRFELIKDGTKHSMVIKSAAFEDEAKYMFEAEDKHTSGKLIIEGIGGGGGGGGGRLKFLTPLKDVTAKEKESAVFTVELSHDNIRVKWFKNDQRLHTTRSVSMQDEGKTHSITFKDLSIDDTSQIRVEAMGMSSEAKLTVLEGGGGGGGGGGDPYFTGKLQDYTGVEKDEVILQCEISKADAPVKWFKDGKEIKPSKNAVIKTDGKKRMLILKKALKSDIGQYTCDCGTDKTSGKLDIEDRGGGGGGGGGEIKLVRPLHSVEVMETETARFETEISEDDIHANWKLKGEALLQTPDCEIKEEGKIHSLVLHNCRLDQTGGVDFQAANVKSSAHLRVKPRGGGGGGGGGVIGLLRPLKDVTVTAGETATFDCELSYEDIPVEWYLKGKKLEPSDKVVPRSEGKVHTLTLRDVKLEDAGEVQLTAKDFKTHANLFVKEPLIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLAASPDCEIIEDGKKHILILHNCQLGMTGEVSFQAANTKSAANLKVKEL"}
+{"text": "The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification. In SPAA (SPAAC) . As a re ethyl]-biotinamide, CAS: 717119-80-7, 5 mM, Amatek scientific, catalog: B-1328) under the catalysis of ADPRC  in 100 \u03bcl of ADPRC reaction buffer   was reacted with HEEB (4HCO3 in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 2 \u03bcl. The gradient was as follows: 0 min 0% B, 2 min 0% B, 4 min 20% B, 17 min 40% B, 19 min 100% B, 24 min 100% B, and 30 min 0% B. The retention time of NAD and ADPR-Biotin (product) was 9.393 min and 9.929 min, respectively. The corresponding compounds were purified from HPLC and then analyzed by LC\u2013MS by Agilent Technologies 6120 Quadrupole with a C18 column . The mobile phase was composed of (A) 0.1% formic acid in water and (B) acetonitrile at a flow rate of 0.4 ml/min. The injection volume was 5 \u03bcl. The gradient was as follows: 0 min 10% B, 4 min 100% B, and 7 min 100% B.Samples were analyzed by an Agilent Technologies 1260 Infinity with a C18 column  monitoring at 260 nm. The mobile phase was composed of (A) 100 mM NHTAATACGACTCACTATTACTGTGTCGTCGTCGTCTGCTGTCTCTCTCTCGCGGGC-3\u2032; boldface letters denote the sequence of T7 class II promotor (\u03d52.5)) and (anti-sense: 5\u2032-GCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032) ) and (anti-sense: 5\u2032-GGCAGCAAGCCGGAGGAGCAGAAGAACGACAGGGCCAGCGAGCCGCAGGCGGCAGCGCGCACGCCCACGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare spike-in ppp-RNA of 62 nt, oligonucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template ) and (anti-sense: 5\u2032-TGGCTGGCCGCATGCCCGCGGCAGAAGTGACATTATGGCTAAAGTTGTGGTAGCACTTCCCTAATAGTGAGTCGTATTA-3\u2032). To prepare spike-in NAD-RNA (500 nt) and m7Gppp-RNA (500 nt) with identical sequence, oligonucleotide without adenine was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032-AAGAAGAACACCAAGAAGCCGAACAACAGCAAGACGGCCAAGAAAAAGACGAAGAGGCAGAAGAAGAAGAACACCAGCAAGAGCCCCAGGAAGAAGCCGACCACCAAGAAGACGAAGCCCAACAGCACGAAGCGGAACACCAGGGAGACGCCCACGAACAACACCACGGCGCGGGACAAGAAGAAGCCGACGACCAAGAAGAAGAAGGAGCGCACCAGGACGAAGCCAACGGGCAAGGCGGACAAGAAGAAGACGAGCAGCAACAAGAGGACGGGGAAGCGGCAGAAGCACAGCACGCCGAAGGACAGGGAGGACACGAGGGAGGGCCAGGGCACGGGCAGCAAGCCGGAGGAGCAGAAGAACAACAGGGACAGCAAGCCGAAGGAGGCAACGCCCACGCCCACGCCGGACACGCAGAACAAGAGGCCGAAAACGACGCCGACCAGCACGACCAGGAAGGGCACCACCCCGGAGAACAGCACCACGCCCAAGCACACCATAATAGTGAGTCGTATTA-3\u2032). For in vitro transcription, 10 \u03bcM of double-stranded DNA (dsDNA) template in 100 \u03bcl transcription buffer , along with 1 mM of each of GTP, CTP and UTP, with 1mM ATP , 4 mM NAD (for NAD-RNA) or 4 mM m7GpppA  (for m7G-RNA), 10 \u03bcl of T7 RNA polymerase , 5% DMSO, 5 mM DTT and 2.5-unit RNase inhibitor were added and the transcription mixture was incubated at 37\u00b0C for 4 h. The reaction was incubated with 11-unit DNase I  at 37\u00b0C for 30 min to remove the DNA template. RNA was then extracted using acid phenol/chloroform and precipitated with isopropanol  at -80\u00b0C overnight. The RNA pellet was washed twice with 75% ethanol, air-dried, redissolved in DEPC-treated H2O, and stored at \u201380\u00b0C. For spike-in RNAs used as internal controls for gene-specific qRT-PCR assessment, NAD-RNA is 106 nt as described above. To prepare ppp-RNA, 88 nt oligo nucleotide was synthesized  and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 106 nt RNA ) and (anti-sense: 5\u2032-CAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGACCTCTCACACACAGTTCGCCTCTTTGATTAACGCCCAGCGTTTTCCCGGTATCCAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare 45 nt m7Gppp-RNA, oligo nucleotide was synthesized (Genewiz) and annealed to make double-stranded DNA template. Primer extension was used to increase the length of the template to encode 45 nt RNA ) and (anti-sense: 5\u2032-CGACAGGGCCCGCGAGAGAGAGACAGCAGACGACGACGACACAGTAATAGTGAGTCGTATTAGTGATC-3\u2032).To prepare short RNA of 38 nucleotides (nt), 10 \u03bcM template oligo DNA (template sequence: 5\u2032-GATCACGATC-3\u2032)  were com7G-RNA (500 nt) and ppp-RNA (500 nt) spike-ins with different sequences from that of NAD-RNA . The synthesis of m7G-RNA (500 nt) was as described in  and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032- TGGCCTTGTAGGTGGTCTTGACCTCAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGCTTGATCTCGCCCTTCAGGGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGTCTTCTTCTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGATGAACTCGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTCGAAGTTCATCACGCGCTCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTCGGGGATGTCGGCGGGGTGCTTCACGTAGGCCTTGGAGCCGTACATGAACTGAGGGGACAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTGGTCACCTTCAGCTTGGCGGTCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAACTCGTGGCCGTTCACGGAGCCCTCCATGTAATAGTGAGTCGTATTAGTGATC-3\u2032). To prepare ppp-RNA, oligonucleotide was synthesized (Genewiz) and were subjected to polyadenylation for poly(A) tails elongation ) and (anti-sense: 5\u2032-TCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATAATAGTGAGTCGTATTA-3\u2032).For boronate affinity experiment, we synthesized mribed in . To prep2) at 37\u00b0C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).NAD-RNA (1 \u03bcg) was reacted with 40 mM and 100 mM HEEB (1 M stock in DMSO) under the catalysis of ADPRC (25 \u03bcg/ml) with 0.4 U/\u03bcl of RNase Inhibitor  in 100 \u03bcl of ADPRC reaction buffer  were washed 3 times with immobilization buffer  , and the254nm (0.18 J/cm2) twice. Membrane was blocked in 5% BSA in PBST (0.1% Tween20 in PBS) for 1 h, followed by incubation with Alexa Fluor\u00ae 790 Streptavidin  at room temperature for 2 h in the dark. Membrane was washed with PBST and PBS for 3 times, respectively, and imaged on the Odyssey LiCor CLx scanner (Li-Cor Biosciences) with the software set to auto-detect the signal intensity at 800 nm channel. Finally, the membrane was stained with methylene blue solution (0.3% w/v methylene blue\u00a0+\u00a030% v/v ethanol\u00a0+\u00a070% v/v H2O)  which was then cross-linked by UVv/v H2O)  to visua7Gppp-RNA (45 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer  at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  and 0.4 U/\u03bcl of RNase Inhibitor  at 25\u00b0C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris\u2013HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H2O. The biotinylated RNA on the beads was extracted with Trizol LS  and chloroform. Input (see above) RNAs and the biotinylated RNA on the beads were analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of mreaction . RNAs we7Gppp-RNA (38 nt) was performed with 1 \u03bcl of NudC  in 25 \u03bcl of NudC reaction buffer  at 37\u00b0C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE\u2013urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) or m7Gppp-RNA (45 nt) were mixed together and incubated with 500 nM NudC  in 25 \u03bcl of NudC reaction buffer  at 37\u00b0C for 30 min. Product was purified with Trizol LS  and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m2O was then added and acid phenol/ether extraction was performed to stop the reaction . Total RNAs (100 \u03bcg) was incubated with 100 mM HEEB (1 M stock in DMSO), ADPRC  and 0.4 U/\u03bcl of RNase Inhibitor  in 100 \u03bcl of ADPRC reaction buffer at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated Hreaction . RNAs we2O. To ensure complete elution, biotin-conjugated RNAs were replaced from streptavidin beads by incubating with 1 mM biotin buffer  at 94\u00b0C for 8 min, followed by incubation with 500 nM NudC  in 25 \u03bcl of NudC reaction buffer  at 37\u00b0C for 30 min. After NudC treatment, biotinylated-RNAs that are resistant to NudC catalysis, potentially derived from contaminating m7G-RNAs, were retained on beads by incubation with high-capacity streptavidin particle  at 25\u00b0C for 30 min. Eluted RNAs in the supernatant were used for next step.After HEEB reaction, biotinylated RNAs were incubated with streptavidin bead particles  and 0.4 U/\u03bcl of RNase Inhibitor  at 25\u00b0C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris\u2013HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H7Gppp-RNA (38 nt) was performed with or without 1 \u03bcl of yDcpS  in 1X yDcpS reaction buffer  in 20 \u03bcl total volume at 37\u00b0C for 1 h. Product was purified with Zymo column  according to the instruction of manufacturer and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold  and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).De-capping of 200 ng NAD-RNA (38 nt) and m7Gppp-RNA (106 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with or without ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer  at 37\u00b0C for 1\u00a0h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates were used for qRT-PCR. Reverse transcription was performed with gene specific primers using SuperScript III SuperMix . qPCR was performed using SYBR Green master mix  to detect NAD-RNA, ppp-RNA or m7Gppp-RNA from three biological replicates using specific primers described in t test.Total RNAs (100 \u03bcg) and 100 ng of spike-in NAD-RNA (106 nt), ppp-RNA (106 nt) or mInput (see above) and NudC-eluted RNAs from four biological replicates of livers from 2- and 18-month old C57BL/6 mice were used for NGS library construction, in accordance with manufacturer's instructions . Library quality was assessed by Bioanalyzer 2100 , and quantification was performed by qRT-PCR with a reference to a standard library. Libraries were pooled together in equimolar amounts to a final 2 nM concentration and denatured with 0.1 M NaOH . Libraries were sequenced on the Illumina NovaSeq 6000 system .7Gppp-RNA, 1% NAD-RNA/99% m7Gppp-RNA, 5% NAD-RNA/95% m7Gppp-RNA or 10% NAD-RNA/90% m7Gppp-RNA, respectively. The mixture of total RNAs and spike-in RNA (500 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer  at 37\u00b0C for 1\u00a0h, followed by NudC-catalyzed NAD-RNA elution. Input (see above) and NudC-eluted RNAs from three biological replicates of livers from 18-month old C57BL/6 mice were subjected to polyA-selected RNA sequencing as mentioned above.Total RNAs (100 \u03bcg) were mixed with 1 ng of 3\u2032-end polyadenylated spike-in RNA (500 nt) that had 0% NAD-RNA/100% m7Gppp-RNA (38 nt) was ligated with 5 \u03bcM 3\u2032 adaptor oligo listed in 1 \u03bcg of NAD-RNA (38 nt) or mBoronic acid beads  were washed 3 times with binding buffer , and then incubated with RNA oligonucleotide in binding buffer in a thermomixer at 37\u00b0C for 2 h. Beads were then washed 3 times with wash buffer ; RNA oligonucleotide bound by the beads was extracted with Trizol LS ). Purified RNA product was analyzed by an 8% polyacrylamide TBE\u2013urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).7Gppp-RNA, and 1 ng of ppp-RNA). RNAs were incubated with yDcpS  in 50 \u03bcl of 1X yDcpS reaction buffer at 37\u00b0C for 1 h, to remove m7G cap. After purification with Trizol LS , yDcpS-treated RNAs were combined with 100 \u03bcmol oligo d(T)30VN in 100 \u03bcl 0.5\u00d7 SSC buffer , and incubated at 80\u00b0C for 5 min followed by 50\u00b0C for 60 min. Then, 1 \u03bcl RNase H  and 5.6 \u03bcl 10\u00d7 Reaction buffer  were added. The mixture was incubated at 37\u00b0C for 20 min, and purified with Trizol LS . This step aims to promote 3\u2032-end adaptor ligation by removing polyA sequence tract from the endogenous transcripts. Prior to 3\u2032-end ligation, polyA-depleted RNAs were treated with 2 U CIAP  in the presence of 40 U RNaseOUT at 37\u00b0C for 1 h to remove 5\u2032-terminal phosphate. RNA was extracted with Trizol LS . 5 \u03bcg of CIAP-treated RNA products were ligated with 100 \u03bcM 3\u2032 adaptor oligo listed in 2O. Reverse transcription was performed with adaptor-based primer listed in 7G-RNA (500 nt) was used as a negative control. Primers were listed in t-test.Total RNAs were extracted from three biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, total RNAs (50 \u03bcg) were mixed with polyadenylated spike-in RNAs   with theDESeq2 (v1.30.0) . NAD-RNAs were defined as fold change of normalized transcript counts\u00a0\u22652 and FDR\u00a0<0.05 in NudC-treated samples compared to those in input samples. Gene annotation information, such as chromosome, gene-types, gene-lengths and intron coordinates were retrieved from Gencode (M23) annotations. The violin plot, bar plot, line chart and scatter plot were generated by R package ggplot2 (v3.3.2) (Genes that had zero count in more than 25% (3 out of 12) sequencing libraries were removed before performing differential analysis. Principle component analysis (PCA) was performed with R function \u2018prcomp\u2019 using a transformed counts matrix by function \u2018vst\u2019. To identify NAD-RNA from total RNA-seq data, we used R package v1.30.0)  to perfo(v3.3.2) .gprofiler2 (v0.2.1)  in Cytoscape (v3.8.2)  . Pathway(v0.2.1)  and the (v0.2.1)  without (v3.8.2) . Network(v3.8.2) .2) at 37\u00b0C for 1 h. 100 \u03bcl of DEPC-treated H2O was then added and acid phenol/ether extraction was performed to stop the reaction  following the manufacturer's protocol with primers listed in Total RNA was extracted from four biological replicates of livers from 2-month old C57BL/6 mice as described above. For each replicate, 100 \u03bcg total RNA, mixed with 1 ng of NAD-RNA (106 nt) and 1 ng of ppp-RNA (106 nt), was incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 \u03bcg/ml) in 100 \u03bcl of ADPRC reaction buffer  ethyl]-biotinamide) as a reactant, allowing only one reaction to biotinylate NAD-capped RNAs. To avoid contaminating signals, we designed a NudC-based post-treatment to elute biotin-conjugated RNAs specifically derived from NAD, but not m7G-capped transcripts from streptavidin beads. Therefore, our method circumvented laborious steps to purify mRNAs followed by clearance of m7G-capped RNAs. With significantly simplified procedures, our method enabled NAD-RNA profiling directly from total RNA. Based on the same platform, qRT-PCR analysis on specific NAD-RNAs could be readily applied.Compared to previous methods that require multiple reactions, we introduced HEEB  and NAD-RNA (38 nt) with 100 mM HEEB to the reaction. While NAD-RNA reacted with HEEB, as indicated by the presence of a biotinylated form, m7G-RNA had no such a product, reflecting specificity  but not m7Gppp-RNA (38 nt) as shown by a lower-sized band corresponding to the de-capped product  to the same reaction. The result clearly showed that NudC was able to selectively de-cap NAD-capped (38 nt) but not m7G-capped RNA (45 nt)  but not m7Gppp-capped forms (38 nt) from streptavidin beads , but not NAD-RNA (38 nt) was sensitive to yDcpS treatment, a decapping enzyme that hydrolyzes the triphosphate linkage of m7G-capped RNA  Figure . We tests Figure  and\u00a0F. Apped RNA .7G-capped RNA (m7Gppp-RNA), respectively). HEEB reacted with NAD-RNA (106 nt) and m7Gppp-RNA (106 nt), but not ppp-RNA (106 nt), resulting in a band retained by the streptavidin beads (7Gppp-RNA (106 nt) could be detected on streptavidin beads; treatment of NudC, however, found no evidence to elute m7Gppp-RNA (106 nt)  Figure .7G-cap, followed by polyA tails. Presumably, endogenous transcripts may contain both NAD and m7G-capped forms, though the percentage may differ for particular genes. The rationale of this design is to mimic endogenous genes that have either low (0% or 1%) or relatively high (5% or 10%) NAD modification. Total RNAs were mixed with spike-ins that had different ratios of NAD-RNA and m7G-RNA. The sample that contained 100% m7G-RNA spike-in represented a gene with no NAD capping, which allowed the assessment of the specificity of ONE-seq platform. Other samples that contained 1%, 5%, and 10% of NAD- relative to m7G-RNA spike-ins were used to determine the capture sensitivity. We subjected 100 \u03bcg total RNA from mouse livers mixed with 1 ng spike-in RNA to ONE-seq experiment, followed by polyA-selected RNA sequencing. In the sample mixed with 100% m7G-RNA spike-in, we found no enrichment  but had either NAD or mt Figure . As demot Figure , represee Figure , suggeste Figure , reflect7G-capped RNAs.Fourth, we determined the noise-cancelling effect of NudC by comparative analysis of RNA-seq datasets. Total RNAs, after HEEB reaction, were captured by streptavidin beads, followed by either NudC-catalyzed elution (NudC+) or mock treatment (NudC-) . polyA-s7G and NAD-capping.We tested the utility of ONE-seq. We profiled NAD-RNAs from mouse liver tissues of young (2-month) and aged (18-month) cohorts. After quality control, we obtained in average \u223c12.3 million high-quality and uniquely mapped sequencing read pairs from each library . Assessmcis diols, occurring naturally at the 3\u2032-end of RNA, as well as in the 7-methylguanosine of m7G-cap and the nicotinamide riboside of NAD-cap  at the terminal is ligated to the 3\u2032-end of RNA, such that the vicinal-diol moiety of the ribose at the 3\u2032-end of RNA no longer exists. At this step, affinity binding can only occur between the boronyl group from boronic acid and 1,2-cis diols from the nicotinamide riboside of NAD-cap. Importantly, we perform reverse transcription using an adaptor-specific primer to harvest RNAs with 3\u2032end blocker, thereby abolishing false signals from boronate binding of RNAs that are not properly ligated with adaptor. Consequently, only RNA transcripts that contain NAD-cap can be selectively enriched by boronate affinity.We validated newly identified NAD-RNAs by an ADPRC-independent method. To do this, we applied boronic acid beads. Our rationale lies at the fact that boronyl groups of boronic acid can form relatively stable complexes with 1,2- NAD-cap . Inspire NAD-cap ,38, we d NAD-cap . First, 7G, but not NAD, from RNA oligos (38 nt). In the presence of RNA ligase, 3\u2032adaptor was efficiently ligated to the spike-in RNAs (38 nt), yielding an upper band in the gel  that were designed with different sequences. This experiment clearly demonstrated that NAD-RNA (500 nt) spike-ins, but not ppp-RNA (500 nt) and m7Gppp-RNA (500 nt), were selectively and significantly enriched by boronic acid beads  and transcription (e.g. RNA polymerase II) were found to contain NAD-cap. Also, transcripts with 5\u2032-NAD modification were enriched in cell cycle (e.g. centromere protein and CDK) and DNA damage responses (e.g. ATM and TP53). Not just housekeeping genes, transcriptional factors (e.g. BMP5 and Interferon), epigenetic pathways , and histone-related genes were also subject to NAD modification Figure . In mitoIn line with an age-modulated decrease of NAD , both thAbove evidence supported the notion that ONE-seq platform permits epi-transcriptome-wide profiling directly from total RNA, prompting us to extend its application for gene-specific assessment by qRT-PCR. To do this, we included non-capped ppp-RNA (106 nt) as a baseline negative control, and NAD-RNA (106 nt) as a positive control. Total RNA and internal spike-in controls were subjected to ONE-seq experiment, followed by qRT-PCR. The relative abundance was calculated between NudC\u00a0+\u00a0and input samples Figure . Our datN-acetylglucosamine (UDP-GlcNAc), can be incorporated at the 5\u2032-end of RNA during transcription initiation in both prokaryotes and eukaryotes , flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc) and uridine diphosphate karyotes . CapZymeNCIN cap . As a re7G) has been found to contaminate the NAD-RNA profile. As such, SPAAC-NAD-seq , Mup13 (http://genome.ucsc.edu/s/lda97/Mup13_ONE-seq), Mup19 (http://genome.ucsc.edu/s/lda97/Mup19_ONE-seq) and Mettl7b (http://genome.ucsc.edu/s/lda97/Mettl7b_ONE-seq) can be accessed at UCSC Genome Browser online sessions. All source codes for data analysis are available at Zenodo https://doi.org/10.5281/zenodo.7293917.All high-throughput RNA sequencing data as well as transcript quantifications have been deposited at the Gene Expression Omnibus under accession number GSE194271. Genome browser views for Cyp2c70 (gkac1136_Supplemental_FilesClick here for additional data file."}
+{"text": "In this announcement, we present the set of putative terpene synthase (TS) gene fragments detected in a subseafloor sediment sample collected off Shimokita Peninsula, Japan. This data set contains sequences with 72 to 100% identity to TS from actinobacteria and cyanobacteria. Terpenoids (TRPs) are the largest class of specialized metabolites , and manSubseafloor sediments represent an environment with unique microbiological communities and metabolic activities . HoweverChikyu shakedown cruise of CK-06-06  and frozen at \u221280\u00b0C immediately after the sampling. DNA was extracted from 5 g of the frozen sediment as previously described (The sediment sample was collected during the D/V TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCGAGATGCGSCGCAAGG-3\u2032) and geosmin-rev (5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGASCGSAKGTGCCACTCGTG-3\u2032) primers . The 2-MIB TS primers were mib-for (5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACGACDNBTACTGCGAGGAC-3\u2032) and mib-rev (5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGVCGGAAGTTGTTGAACTG-3\u2032) (331\u2009bp). The PCR mix consisted of 1\u00d7 EmeraldAmp Max PCR master mix (TaKaRa), 0.4 \u03bcM each primer, and 0.05\u2009ng of sediment DNA. The two-phase touchdown PCR protocol for increased specificity and sensitivity was used (The geosmin TS fragment (432\u2009bp) was amplified using primers geosmin-for . OTUs were filtered for chimeras using VSEARCH\u2019s implementation of UCHIME de novo (The obtained sequences  were pro de novo  training de novo .This data set can be used in studies on TS gene diversity and distribution in subseafloor environments.PRJNA846928. The GenBank accession numbers for OTUs are ON723903 to ON723912 (2-MIB) and ON723913 to ON723935 (geosmin).Raw reads were deposited in a BioProject at DDBJ/ENA/GenBank under the accession number"}
+{"text": "In the published article, there were errors. In the original article, there was an error in the description of the Reverse primer sequence in Materials and methods.Materials and methods, \u201cDeep amplicon sequencing of CMV-UL40 genomic DNA\u201d.A correction has been made to This sentence previously stated:\u201cThe following forward and reverse primers were used: Forward, 5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3\u2032 and Reverse, 5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGACGCATA-3\u2019.\u201dThe corrected sentence appears below:\u201cThe following forward and reverse primers were used: Forward, 5\u2032-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3\u2032 and Reverse, 5\u2032-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAACACGAGCGGACATA-3\u2019.\u201dMaterials and methods.In the original article, there was another error in the description of the concentration of the anti-HLA-E antibody in Materials and methods, \u201cImmunohistochemistry analysis\u201d.A correction has been made to This sentence previously stated:\u201cAfter antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1\u00a0h at room temperature to prevent nonspecific binding and stained with 10 \u03bcL/mL anti-HLA-E antibody [MEM-E/02]  or IgG from mouse serum (Sigma-Aldrich) overnight at 4\u00b0C.\u201dThe corrected sentence appears below:\u201cAfter antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1\u00a0h at room temperature to prevent nonspecific binding and stained with 10 \u03bcg/mL anti-HLA-E antibody [MEM-E/02]  or IgG from mouse serum (Sigma-Aldrich) overnight at 4\u00b0C.\u201dThe authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "We report an approach of combining CRISPR-Cas9 gene editing with florescence cell sorting to tag and purify human endogenous protein complexes from HEK cells for structural studies by single-particle cryogenic electron microscopy. This procedure is demonstrated by applying the method to study human proteasomal complexes, enabling rapid determination of high-resolution structures of several proteasomal complexes. We envision this approach will enable structural, biochemical, and biophysical studies of many important endogenous human macromolecular complexes. The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28\u03b1\u03b2\u201320S. Our structures reveal that PA28 with a subunit stoichiometry of 3\u03b1/4\u03b2 engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources. Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein\u2013DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial . Selecting the \u03b24-StrepII cell population, which had the highest knock-in efficiency, we expanded this cell line and purified the human proteasome by StrepII pull-down.To extract the endogenous human proteasome for structural studies, we incorporated a mNG2[11]-StrepII tag onto the C terminus of the 20S \u03b21 (Psmb6), \u03b22 (Psmb7), and \u03b24 (Psmb2) B subunitSI Appendix, Fig. S2), followed by cryo-EM structure determination of singly capped proteasomal complexes of PA28\u201320S, PA200\u201320S, and 19S\u201320S at \u223c2.6 to \u223c3.2 \u00c5 resolution, with and without MG-132 inhibitor .Affinity purification of human endogenous 20S(\u03b24-StrepII) pulls down multiple proteasomal complexes, including 20S, singly capped with 19S, PA200, and PA28. The purified proteasomal complexes were first visualized by negative-stain EM . Furthermore, the resulting cryo-EM map of PA28\u201320S purified from 20S(\u03b24-StrepII) pull-down shows side-chain density that matches PA28\u03b2 in some regions while matching PA28\u03b3 in other regions , suggesting that the cryo-EM map is an average of both PA28\u03b1\u03b2 and PA28\u03b3 complexes. However, the high similarity between the two complexes prevented using computational classification to separate them (Endogenous PA28 subunits are known to form two different heptameric complexes: a heteroheptamer composed of PA28\u03b1 and PA28\u03b2 subunits and a homoheptamer composed of PA28\u03b3 subunits. Pull-down of 20S(\u03b24-StrepII) is expected to yield both PA28\u03b1\u03b2\u201320S and PA28\u03b3\u201320S complexes. Indeed, mass spectrometry analysis of the purified proteins reveal all three PA28 subunits to be present in the purified sample . Additionally, the tagging of PA28\u03b1 with sfCherry2[11]-ALFA tag was performed in the cell line that already contained mNG2[11]-StrepII\u2013tagged 20S \u03b24, demonstrating the ability to multiplex orthogonal tags onto different subunits of a larger protein assembly to increase target specificity. The cryo-EM map of PA28\u03b1\u03b2\u201320S purified by ALFA-PA28\u03b1 pull-down allowed robust modeling of the side-chain densities of PA28\u03b1\u03b2, allowing us to distinguish the subunit stoichiometry of the complex.To isolate a homogeneous PA28\u201320S complex for structural studies, we engineered an sfCherry2[11]-ALFA tag onto the N terminus of the PA28\u03b1 subunit using the method described above, except here we used a split sfCherry system  rather tSI Appendix, Fig. S6 and Table S2), different from the 4\u03b1/3\u03b2 assembly observed previously.Previous crystallographic structural analysis of the mouse PA28\u03b1\u03b2 heteroheptamer found a stoichiometry of four \u03b1 and three \u03b2 subunits . The proSI Appendix, Fig. S9). There is one low-occupancy binding site situated between the 20S \u03b11 and \u03b12 subunits where a phenylalanine, 20S \u03b11 F29, may be sterically hindering the binding of the C-terminal tail of PA28\u03b2 . We suspect that this additional density does not represent endogenous substrates but rather is from ALFA peptide used to elute purified complex. ALFA peptide is present at high concentrations in the elusion buffer and likely enters 20S as substrates through the \u03b1-ring gate opened by PA28. Consistent with this argument, cryo-EM analysis of proteasomal complexes purified via StrepII pull-down of 20S(\u03b24-StrepII) did not show similar density inside any map of 20S, PA200\u201320S, or 19S\u201320S.Proteasome activators serve to open the 20S gate and facilitate the loading of unfolded polypeptides into the 20S inner chambers for degradation. Passing through the open gate in the \u03b1-ring, unfolded polypeptides first enter the antechamber formed between an \u03b1- and \u03b2-ring and then move into the proteolytic chamber formed between two \u03b2-rings for peptide cleavage. Interestingly, the cryo-EM map of PA28\u03b1\u03b2\u201320S purified by ALFA-PA28\u03b1 pull-down shows additional density inside the 20S antechamber that is proximal to the bound PA28 , which may serve to bind unfolded polypeptides. The ALFA peptide accumulates in the 20S antechamber and is likely degraded once it enters the proteolytic chamber. To visualize what might happen if proteolysis was blocked, we purified proteasome complexes via 20S(\u03b24-StrepII) by StrepII pull-down in the presence of the peptide-like inhibitor MG-132 and carried out cryo-EM analysis , consistent with a previous study of the yeast proteasome , and signal amplification using our recent tag-assisted split enzyme complementation method  could fuOne exciting future direction stemming from the concepts described here is the study of endogenous proteins from animal models. This can allow the structural and biophysical characterization of proteins in their physiological states, which can provide important insights into how proteins function in\u00a0vivo . EfficieTAATACGACTCACTATAGAAAAAAAGCACCGACTCGGTGCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAACGATGTTAGGAGCCCTGTTTGGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGTTATTGCATACTAGAATCCCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGGCCACCCACTGATGCCATTCGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGCCCGGTCATGGCCATGCTCAGTTTAAGAGCTATGCTGGAATAATACGACTCACTATAGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTAACATCATGTCCTCCCTCCCACTTGCCAGGGAACTTTTTTTTGATGGGCTCCTTTATTGACAAAAATGGCATCCATGACCTGGATAACATTTCCTTCCCCAAACAGGGCTCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATACTGGGATTCTAGTATGCAATAAGAGATGCCCTGTACTGATGCAAAATTTACAAGTACTTTTGGGAGACCAGATACCCAAATTCGCCGTTGCCACTTTACCACCCGCCGGCAGCACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATATGATGGGGAGTGCGTGGTCCCACCCTCAATTCGAGAAGTGAATGGCATCAGTGGGTGGCTGGCCGCGGTTCTGGAAGGTGGTGAGCATTGAGGCATCACTCCTCTGGAGATTGAGGTGCTGGAAGAAACAGTCCAAACAATGGACACTTCCGGCAGCCCTTCTAGATTAGAAGAGGAATTGAGACGACGATTGACTGAACCAGGATCTTATACTATCGTTGAACAATACGAGAGAGCTGAGGCAAGACACTCTACAGGTTCTGGCGCCATGCTCAGGGTCCAGCCCGAGGCCCAAGCCAAGGTGAGCGCCGCCCACTCCACTCCTTGTGCGGCGCTAGGCCCCCCGTCCCGGTCATGSI Appendix, Fig. S1). The reactions were completed in a 100-\u03bcL reaction containing 50 \u03bcL 2\u00d7 Phusion master mix, 2 \u03bcL ML557\u2009+\u2009558 mix at 50 \u03bcM, 0.5 \u03bcL ML611 at 4 \u03bcM, 0.5 \u03bcL of each gene-specific oligo at 4 \u03bcM, and 47 \u03bcL diethyl pyrocarbonate (DEPC) H2O. PCR program: 95\u2009\u00b0C, 3 min; 20\u00d7 ; 72\u2009\u00b0C, 60 s. The PCR product was purified using a Zymo DNA Clean and Concentrator Kit (Zymo Research D4013) and DNA was eluted in 10 \u03bcL. The expected DNA concentration is around 100 ng/\u00b5L. DNA was stored at \u221220\u2009\u00b0C until used for gRNA synthesis.The IVT template for LMNA gRNA was made by PCR (In vitro transcription was carried out using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB E2050S) with the addition of RNAsin (Promega N2111). In 20 \u03bcL, 300 ng of DNA template, 10 \u03bcL NTP buffer mix, 2 \u03bcL T7 polymerase, 1 \u03bcL RNAsin, and water was added. Addition of RNAsin is important to prevent RNA degradation. The reaction was incubated overnight and the RNA was purified using the RNA Clean and Concentrator Kit (Zymo Research R1017). RNA was eluted in 10 \u03bcL with an expected concentration >6 \u03bcg/\u03bcL. Single-guide RNA (sgRNA) was stored at \u221280\u2009\u00b0C immediately after measuring concentration and diluting to 130 \u03bcM.2O, and 4 \u03bcL sgRNA were mixed and incubated at 70\u2009\u00b0C for 5 min to refold the gRNA. During this step, 10-\u03bcL aliquots of purified Cas9 at 40 \u03bcM were thawed on ice and once thawed were slowly added to the diluted sgRNA in Cas9 buffer and incubated at 37\u2009\u00b0C for 10 min for ribonucleoprotein complex formation. Six microliters of HDR template donor was added to the ribonucleoprotein mix, and all samples were kept on ice until ready for nucleofection. For efficient recovery post-knock-in (KI), a six-well plate with 2 mL of Expi293F expression media (Thermo Fisher) per well was incubated at 37\u2009\u00b0C. An appropriate amount of supplemented Lonza Amaxa solution corresponding to the number of KIs to be performed was prepared at room temperature in the cell culture hood. For each sample, 65.6 \u03bcL of SF solution (Lonza Group Ltd.) and 14.4 \u03bcL of supplement was added to an Eppendorf tube for a total of 80 \u03bcL per KI. Lonza nucleofector instruments/computers were then turned on and kept ready for nucleofection. The Amaxa solution is toxic to cells, so it is important to make sure everything is ready to go beforehand.In a sterile PCR or microcentrifuge tube, 8 \u03bcL Cas9 Buffer, 12 \u03bcL DEPC Hg for 3 min.Supernatant was removed and cells were resuspended in 1 mL of phosphate-buffered saline to wash. The cells were centrifuged again at 500 \u00d7 g for 3 min. PCR tubes containing ribonucleoproteins were brought into the tissue culture hood. Cells were resuspended in 80 \u03bcL of supplemented Amaxa solution and the cell resuspension was added to the 40 \u03bcL of preformed ribonucleoprotein + HDR template mix. The 120-\u03bcL mixture was pipetted into the bottom of the nucleofection plate, avoiding air bubbles. The nucleofection was carried out in 100-\u03bcL cuvettes on a Lonza Nucleofector X Unit (Lonza AAF-1002X) attached to Lonza 4D Nucleofector Core Unit (Lonza AAF-1002B). Cells were nucleofected using the FS-100 program and immediately recovered using 400 \u03bcL of media from the prewarmed six-well plate and transferred to the corresponding well. Because the Amaxa solution is toxic to the cells, it is important to perform these steps quickly. The cells were incubated at 37\u2009\u00b0C and 8% CO2.Cells  were harvested by centrifugation at 500 \u00d7 2. The next day, the cells were transferred to a 125-mL vented flat-bottom Erlenmeyer flask containing 20 mL Expi293F media and incubated at 37\u2009\u00b0C and 8% CO2 with shaking at 120 rpm until the cell density reached 1 million/mL.Cells were monitored over 5 to 7 d. Many cells will look unhealthy and die, and growth rate will slow dramatically. Cells will begin to recover after 5 d. Once cells expanded to the point where they began to detach from the well bottom, the plate was transferred to a shaker operating at 120 rpm and incubated at 37\u2009\u00b0C and 8% COTo prepare the transfection mixture, 20 \u03bcg of pSFFV-mNG2 (1-10) plasmid was added to 1 mL of Opti-MEM (Gibco) in one tube and 53 \u03bcL of ExpiFectamine 293 (Gibco) was added to 1 mL of Opti-MEM (Gibco) in a separate tube. The mixture was incubated at room temperature for 5 min. The Opti-MEM containing Expifectamine 293 was then added to the solution containing DNA, inverted multiple times to mix, and then incubated at room temperature for 25 min. The transfection mixture was then added to 20 million Expi293F cells in 20 mL of media in a 125-mL vented flat-bottom Erlenmeyer flask. The cells were incubated at 37\u2009\u00b0C for 2 d with shaking at 120 rpm. If the knock-in was successful, some cells will show green fluorescence the next day, which will get brighter on day 2.2 for 2 d with shaking at 120 rpm. Cells were transferred to 20 mL of Expi293F media in a 125-mL vented Erlenmeyer flask. When the cell density reached 2 \u00d7 106 per mL, cells were transferred to 100 mL of Expi293F media in a 500-mL vented Erlenmeyer flask. When cells reached a density of 2 \u00d7 106 per mL, cells were harvested and frozen in aliquots.FACS sorting and flow cytometry were performed on a BD FACSAria II in the Laboratory for Cell Analysis at the University of California, San Francisco (UCSF). mNG2 signal was measured with the 488-nm laser and 530/30 bandpass filter. One million cells per sample were sorted into Eppendorf tubes and then plated in a six-well plate. The six-well plate was incubated at 37\u2009\u00b0C and 8% COTotal RNA was extracted from 1 million cells using the Monarch Total RNA Miniprep Kit (NEB T2010S). cDNA was prepared from 1 \u00b5g of extracted RNA using LunaScript RT SuperMix Kit (NEB E3010). No Template and No Reverse Transcriptase controls (NTC and NRT) were performed in parallel to cDNA preparations. cDNA was analyzed in a 2% agarose gel. Sequencing confirmation of amplicons was completed by Elimbio.2, and shaking at 120 rpm. When cells reached a density of 3 million cells per mL, cells were harvested by centrifugation at 1,000 \u00d7 g for 5 min. Cells were resuspended in 20 mL Buffer A  supplemented with SigmaFast protease inhibitor (Sigma). Cells were lysed by sonication and insoluble debris were removed by ultracentrifugation at 100,000 \u00d7 g for 20 min. For StrepII-tagged proteins, the supernatant was passed through a column containing 0.2 mL Strep-Tactin XT Sepharose resin (Cytiva) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted with 1 mL Buffer A supplemented with 50 mM biotin . For ALFA-tagged proteins, the supernatant was passed through a column containing 0.2 mL ALFA Selector PE resin (NanoTag Biotechnologies) by gravity flow. The resin was washed with 2 mL Buffer A and the bound proteins were eluted at room temperature with 1 mL Buffer A supplemented with 200 \u03bcM ALFA peptide . To acquire 19S-bound proteasome complexes, 1 mM AMPPNP (Sigma) and 2 mM MgCl were included throughout the purification. To acquire MG-132 bound proteasomal complexes, cells were treated with 5 \u03bcM MG-132 for 15 min prior to harvesting and MG-132 was maintained throughout the purification, along with 1 mM ATP. The eluted proteins were concentrated in a 100 K centrifugal concentrating device.Expi293F cell lines were grown in 100 mL Expi293 expression medium (Thermo Fisher) in 500-mL flat-bottom Erlenmeyer flasks at 37\u2009\u00b0C, 8% CONegative-stain EM grids were prepared following established protocol . SpecificisTEM . For PSMB2-mNG2 (11)-strepII with MG132, the 19S-20S classes were grouped and subjected to another round of 3D classification using cryoSPARC multirefinement (20-\u00c5 resolution limit) with eight classes to separate the different conformations. The resulting classes were refined to high resolution using cryoSPARC homogeneous refinement . Maps were filtered by local resolution in cryoSPARC. Atomic models were built into the cryo-EM density maps using Coot -strepII and mCherry -strepII . Images cisTEM . Particling Coot , 39 and ing Coot , 41 withing Coot .Supplementary File"}
+{"text": "The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition. How resistance to different classes of AKT inhibitors can emerge is unclear. Here, the authors show that resistance to allosteric inhibitors is mainly due to mutation of AKT1 while the ATP competitive resistance is driven by activation of PIM kinases in prostate cancer models. The AKT/PKB serine/threonine kinase functions as a central node in this pathway and is being investigated as a therapeutic target in oncology2. Three isoforms of AKT  exist in humans that each contain a Plekstrin homology (PH) domain, a kinase domain, and a C-terminal hydrophobic regulatory region3. Activation of these isoforms is mediated by recruitment to PtdIns-3,4-P2  and PtdIns-3,4,5-P3 (PIP3) at the plasma membrane and subsequent phosphorylation of T308 and S473 by 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and mTOR complex 2 (mTORC2), respectively. Aberrant activation of AKT in cancer may occur via several mechanisms including mutational activation of the catalytic subunit of PI3K, which generates PIP3 and, indirectly, PI3,4P2; loss of the PIP3 phosphatase PTEN; and, albeit less frequently, activating mutations in AKT4. Upon activation, AKT mediates various cellular processes including cell survival, metabolism and proliferation by regulating the activity of downstream proteins including proline-rich AKT substrate of 40\u2009kDa (PRAS40), glycogen synthase kinase 3 (GSK-3), Forkhead box class O (FoxO) transcription factors, tuberous sclerosis complex 2 (TSC2), Bcl-2 associated death promoter (BAD), mTOR complex 1 (mTORC1), eukaryotic translation inhibition factor 4E-binding protein 1 (4EBP1), and the S6 ribosomal protein kinase4.Enhanced activity of the\u00a0phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling pathway is among the most frequently observed changes in cancer and is associated with tumor invasiveness, survival, and proliferation2. Importantly, these inhibitors differentially exploit the on-off activity cycle of AKT. In its inactive state, AKT adopts a closed conformation in which the PH domain interacts with the kinase domain, also referred to as the PH-in state6. Upon recruitment to the membrane and phosphorylation at T308 and S473, the interaction between the PH and kinase domains is released, resulting in an open PH-out conformation conducive to ATP-binding5. Allosteric inhibitors preferentially bind to\u00a0the inactive PH-in conformation at a cavity formed between the PH and kinase domains, preventing phosphorylation and activation of AKT6. In contrast, ATP-competitive inhibitors selectively target the PH-out conformation, protecting AKT from dephosphorylation at T308 and S473 while simultaneously blocking ATP binding and kinase activity7. As a result, decreased AKT phosphorylation at both T308 and S473 is typically observed in allosteric inhibitor-treated cells while increased or sustained pAKT at both sites is characteristic of the ATP-competitive inhibitors.Two main classes of AKT inhibitors (AKTis) have entered clinical investigation in oncology: allosteric inhibitors such as MK-2206 and adenosine 5\u2032-triphosphate (ATP)-competitive inhibitors such as ipatasertib/GDC-00688, the therapeutic potential of AKTis is likely to be the greatest in indications associated with PI3K/AKT pathway activating alterations. One such indication is prostate cancer. Activation of the PI3K/AKT pathway is thought to comprise roughly 50% of metastatic castration-resistant prostate cancer (mCRPC), frequently via PTEN loss11. In fact, a randomized phase II study evaluating combined inhibition of AKT via ipatasertib and androgen signaling via abiraterone in patients with mCRPC showed superior antitumor activity of the combination compared to abiraterone alone, especially in patients with PTEN-loss tumors12. Additionally, in metastatic triple-negative breast cancer (mTNBC), another indication associated with frequent PI3K/AKT pathway activating alterations, the combination of ipatasertib and paclitaxel also improved progression-free survival compared to paclitaxel alone in a randomized phase II trial, with a more pronounced effect observed in patients with PIK3CA/AKT1/PTEN-altered tumors13. Phase III clinical trials are currently underway to further evaluate ipatasertib as a therapeutic agent in these indications.Given that intrinsic sensitivity to AKTis such as ipatasertib correlates with AKT pathway activation14. Although some mechanisms of intrinsic resistance to PI3K/AKT/mTOR pathway inhibitors have been described, including SGK1 signaling in breast cancer15, Wnt-\u03b2-catenin signaling in colon cancer16, androgen receptor signaling in prostate cancer17 and RAS/RAF pathway signaling across multiple cancers8, few studies have explored mechanisms of acquired resistance to AKTis following prolonged treatment. Further, it remains unknown whether overlapping or distinct mechanisms of resistance will arise to long-term treatment with allosteric vs. ATP-competitive inhibitors. Here, we aim to identify mechanisms of acquired resistance to both allosteric and ATP-competitive AKTis using an unbiased approach. We performed systematic analysis of cell lines with acquired AKTi-resistance (AKTi-R) using methods including RNA sequencing (RNA-seq) and whole exome sequencing (exome-seq) and explored potential functional dependencies and combination strategies using a chemical genetics screen. Our findings indicate that distinct mechanisms do arise to the two different classes of AKTis and that combination approaches may be taken to reverse this resistance.While treatment of tumors with targeted therapies can initially result in impressive clinical outcomes, resistance is likely to emerge over extended treatment times50s  or W80C AKT1 , pPRAS40 (T246) and pS6 (S235/236) levels comparable to that observed following overexpression of WT AKT1   Fig.\u00a0. In cont50) Fig.\u00a0. We alsoAKT1S1)/PRAS40 were detected in multiple G-R clones \u2009Given that a high number of alterations in both gene expression and exome sequence were detected in G-R cells compared to parental LNCaP cells  kinases  models, respectivelylls Fig.\u00a0. Given tlls Fig.\u00a0. Consist36. Interestingly, while PIM3 transcript levels are not increased in G-R cells compared with parental cells by RNA-seq analysis , each potently targeted by the pan-PIM kinase inhibitors included in this study, LNCaP cells primarily express PIM3 protein , suggesting that the low level PIM3 expression in the parental cells can antagonize the effect of AKT inhibition to some extent, the impact of PIM3 knockdown on ipatasertib sensitivity was more pronounced in G-R cells  -inducible overexpression experiments. While siRNA-mediated depletion of PIM3 resulted in some increase in sensitivity of parental cells to ipatasertib (~2.9-fold decrease in ICed) Fig.\u00a0. Therefoed) Fig.\u00a0. Furthered) Fig.\u00a0. AltogetAmong the prostate cancer cell lines characterized in Supplementary Fig.\u00a039), or a combination of ipatasertib and GDC-0339. Tumor growth inhibition was evident following treatment with ipatasertib in Par X1.6 but not G-R3 X1.2 tumor-bearing mice, demonstrating that G-R3 X1.2 retained AKTi resistance in vivo  mediating resistance appear to be context-dependent. For example, in PC3-LN4 prostate cancer cells, intrinsic resistance to both allosteric and ATP-competitive AKTis has been demonstrated to involve AKTi-induced PIM1 upregulation followed by a PIM1-dependent increase in receptor tyrosine kinase expression via cap-independent translation51. In various breast cancer cell lines, intrinsic resistance to the PI3K\u03b1 or AKT inhibitors has been demonstrated to involve PIM1, with PIM3 likely playing a less prominent role in this setting52. PIM2 expression has been linked to the resistance of breast cancer and MM cell lines to PI3K inhibitors GDC-0941 or BKM12053. Interestingly, PIM3 upregulation has been reported as a feedback mechanism in response to mTORC1 inhibition by rapamycin through miR-33 mediated suppression encoded by the SREBP loci54. In the current study, we did not observe increased PIM3 transcripts in the LNCaP parental or G-R cells in the presence of ipatasertib , AKT1 , AKT2 , AKT3 , pAKT(T308) , pAKT(S473) , pPRAS40(T246) , PRAS40 , pS6(S235/236) , S6 , p4EBP1 (T37/46) , p4EBP1 (S65) (#9456 1:1000), 4EBP1 , PARP (#9532 1:1000), Cleaved PARP , PTEN , PIM2 , PIM3 , pGSK-3\u03b2 (S9) , GSK-3\u03b2 , pBAD (S112) , and BAD  were obtained from Cell Signaling Technology. The PIM1 antibody was obtained from Abnova . An additional antibody to total PRAS40 was obtained from Invitrogen/ThermoFisher . Protein loading was assessed using antibodies to \u03b2-actin , \u03b2-Tubulin  or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) .Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author, Kui Lin (lin.kui@gene.com).56. LNCaP cells harbor a frameshift mutation (K6fs*4) (COSMIC # COSM4929) and loss of heterozygosity (LOH) in PTEN58. The presence of both alterations was confirmed in the LNCaP line in-house via exome sequencing and SNP array. AKTi-resistant (AKTi-R) cell lines were established by treating cells with gradually escalating doses of ipatasertib or MK-2206 until reaching a maximum dose of 5\u2009\u03bcM of AKTi. ipatasertib-resistant (G-R) and MK-2206-resistant (M-R) cell pools were then subjected to single cell sorting using FACSAria instrumentation and software (BD Biosciences) and surviving clones were expanded in the presence of AKTi at the maximum doses indicated above. MK-2206 or ipatasertib-resistant cell pools are denoted as M-Rpool or G-Rpool, respectively. Individual AKTi-R clones were assigned numbers, which are indicated following the M-R or G-R prefix. LNCaP G-RB cell lines were established following long-term exposure of LNCaP cells to 5\u2009\u03bcM ipatasertib, single cell sorting, and expansion of surviving clones in the presence of the AKTi. The Par X1.6 and G-R3 X1.2 selected lines used for in vivo studies were derived from LNCaP and LNCaP G-R clone 3 (G-R3) tumors that displayed growth in untreated male SCID.bg C.B-17 mice (Charles River Labs). The R0068 X1.2 line was established from mice bearing LNCaP tumors that had been treated with 50\u2009mg/kg ipatasertib over the course of 106 days. Whole tumors from individual mice were excised, minced in complete media, and plated in tissue culture flasks. Cell lines were established following one to two passages. After establishment in culture, G-R3X1.2 and R0068 X1.2 cells were maintained in the presence of 5\u2009\u03bcM ipatasertib. All cell lines were maintained at 37\u2009\u00b0C/5% CO2 in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (Sigma), 2 mM L-Glutamine, and 0.01\u2009M HEPES, pH 7.2. Growth medium for AKTi-R cell lines was additionally supplemented with AKTi at the indicated concentration for cell line maintenance.Cell lines were originally obtained from the American Type Culture Collection (ATCC) and genotyped by Genentech\u2019s cell banking facility. LNCaP is an approximately tetraploid epithelial line derived from a prostate adenocarcinoma metastasis6 cells into male NOD scid gamma (NSG) mice . Testosterone pellets  were implanted into the dorsal shoulder 5 days prior to cell inoculation. Animals were distributed into treatment groups (n\u2009=\u20099/group) when the tumors reached a mean volume of approximately 230 to 350 mm3. GDC-0068 and GDC-0339 were formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT) and were administered once daily (QD) via oral formulation  at 25 and 100\u2009mg/kg, respectively for 21 days. Tumor volumes were determined using digital calipers  using the formula (L x W x W)/2. Tumor volumes and body weights were recorded twice weekly over the course of the study. Mice with tumor volumes\u2009>2000\u2009mm3 or recorded body weight loss of >20% from their start of treatment were euthanized per Institutional Animal Care and Use Committee guidelines.All in vivo efficacy studies were approved by Genentech\u2019s Institutional Animal Care and Use Committee and adhere to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Tumor xenografts derived from the LNCaP Par and LNCaP G-R3 X1.2 cell lines were established by subcutaneous injection of 10\u2009\u00d7\u20091059. In brief, as tumors generally exhibit exponential growth, tumor volumes were subjected to natural log transformation before analysis. A generalized additive mixed model (GAMM) was then applied to describe the change of transformed tumor volumes over time using regression splines with auto-generated spline bases as this approach addresses both repeated measurements from the same study subjects and moderate dropouts before study end. Estimates of group-level efficacy were obtained by calculating a growth contrast, the difference in AUC-based growth rates  between the treatment and reference group fits. To calculate AUC-based growth rates, group AUC values are corrected for starting tumor burden and then subjected to slope equivalence \u201cnormalization\u201d. Slope equivalence \u201cnormalization\u201d of AUC results in the actual slope of a fit on the natural log (LN) scale in cases of log-linear growth. In the cases of non-log-linear growth, such \u201cnormalization\u201d results in the constant log-linear growth rate that would have been needed to yield the baseline-corrected AUC that was actually observed for a fit on the natural log scale. Mathematically, this \u201cnormalization\u201d is attained by dividing each estimated baseline-corrected AUC value by half of the square of the common study period resulting in units of natural log units per day. The more negative the Growth Contrast value, the greater the anti-tumor effect. The 95% confidence intervals are based on the fitted model and variability measures of the data.Analysis and comparison of tumor growth was performed as detailed previously using a package of customized functions in R v3.6.2  which integrate software from open source packages including lme4, mgcv, gamm4, multcomp, settings, plyr, and several packages from the tidyverse such as magrittr, dplyr, tidyr, and ggplot2A generalized additive mixed model (GAMM) was also employed to describe the change in raw body weights  over time with regression splines. After data fitting, raw body weight data at each time point from all individual animals and all group fits were normalized to the starting weight and reported as a percentage to yield % body weight change.8. Briefly, cells were plated in black/clear bottom 384 well plates  and incubated at 37\u2009\u00b0C under 5% CO2. The following day, cells were treated with a 9-point dose titration of indicated inhibitors or with DMSO control. All conditions were tested in quadruplicate within each experiment. Treated cells were then incubated for 4 days and viability was assessed using the CellTiter-Glo\u00ae (Promega) luminescent assay according to the manufacturer\u2019s instructions. Total luminescence was measured on a Wallac Multilabel Reader (PerkinElmer) and was considered to represent cellular viability. Dose response curves  generated with Prism (GraphPad) depict mean % viability (% DMSO control), with error bars representing standard error of the mean (SEM), from quadruplicate samples (y-axis) versus concentration of inhibitor (x-axis) from a single representative experiment. The inhibitor concentration resulting in the half maximum inhibitor effect (IC50) was calculated from % viability values from quadruplicate wells using a 4-parameter curve analysis . Scatter plots depict absolute IC50 values from independent biological repeats, with bars denoting mean absolute IC50 values and standard error of the mean (SEM). Mean IC50 values are denoted below x-axis and sample size (n) is indicated for each cell line/condition above scatter plot data. Unless otherwise stated, at least 3 independent experiments were performed to assess reproducibility. Where applicable, statistical significance is indicated above scatter plot data used to make comparisons.Cellular viability was assessed 4 days after addition of inhibitors using the CellTiter-Glo\u00ae (Promega) luminescent assay as described previouslyCells were washed with cold 1X phosphate-buffered saline (PBS) and lysed in Cell Extraction Buffer (CEB) (Biosource/Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Roche). Protein concentrations were determined using the Lowry-based RC DC protein assay (Bio-Rad) and normalized for equal protein loading. Lysates were loaded onto Tris\u2013glycine gels (Invitrogen) and proteins were separated by electrophoresis. Proteins were then transferred onto nitrocellulose membranes using the iBlot\u00ae dry blotting system (Invitrogen), and membranes were blocked with blocking buffer for fluorescent Western blotting (Rockland or LI-COR). Primary antibodies (see below) were detected using IR Dye 800-conjugated (Rockland or LI-COR) or Alexa Fluor 680 (Invitrogen) or IR Dye 680 (LI-COR) species-selective secondary antibodies. Detection and quantification were conducted using an Odyssey infrared scanner (LI-COR) using the manufacturer\u2019s software. Protein loading was assessed using antibodies to \u03b2-actin, \u03b2-Tubulin, or GAPDH. Raw immunoblot images for cropped blots are provided in the Supplementary Information.Par or AKTi-R cells were plated in duplicate in complete RPMI medium and, the following day, treated with DMSO, 5\u2009\u03bcM ipatasertib, or MK-2206 for 14\u2009h. Cells were then subjected to total RNA extraction using the RNeasy kit (Qiagen) and RNA concentrations were read using a NanoDrop 8000 spectrophotometer (Thermo Scientific). Following confirmation of RNA integrity with the 2200 TapeStation system (Agilent Technologies), RNA-seq libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) from 1\u2009\u03bcg of total RNA. Library size was determined using 2200 TapeStation and High Sensitivity D1000 screen tape (Agilent Technologies) and concentration was assessed by qPCR-based methodology . The libraries were multiplexed and sequenced on Illumina HiSeq2500 (Illumina) to generate 50 million paired-end 75 base pair reads. RNA-seq reads containing 30% or more bases with a Phred quality score of 23 or lower were excluded. The remaining high quality reads were then mapped to NCBI GRCh37 (hg19) using GSNAP and default settings. Multimapping reads were discarded. Gene expression levels were summarized into count and RPKM . Differential expression analysis was performed using limma. Genes with adjusted p-value < 0.05 and absolute value of log2FC\u2009>\u2009=\u20091 were considered to be differentially expressed. For the hierarchical clustering and heatmap of RNA-seq transcriptome analysis, the RPKM values for the top 100 most variably expressed genes were z-scored and clustered using Euclidean distance.Par or AKTi-R cells were plated in complete RPMI medium and the following day, DNA extraction was performed using the DNeasy kit (Qiagen). Prior to processing by whole exome sequencing, the concentration and integrity of DNA samples was determined using NanoDrop 8000 (Thermo Fisher Scientific) and 2200 TapeStation (Agilent Technologies), respectively. Exome capture was performed using 0.5\u2009\u03bcg of genomic DNA and SureSelectXT Human All Exon v5 kit (50 megabases [Mb]) according to manufacturer\u2019s protocol . Fragment size distribution of post-capture amplified libraries was determined with 2200 TapeStation using high sensitivity D1000 screen tape . Concentration of the libraries was measured by Qubit (Thermo Fisher Scientific). Exome capture libraries were sequenced on HiSeq 2500  to generate 75 million paired-end 75 base pair reads. High quality exome-seq reads were mapped to NCBI GRCh38 using GSNAP. Somatic SNVs and INDELs were called by comparing the treatment resistant clones against the parental clones using LoFreq with its default setting. Highly-confident variants were annotated using Ensembl Variant Effect Predictor and filtered with dbSNP 138, ExAC 0.3.1 and RepeatMasker 4.0.5. The functional consequences of somatic variants were annotated using SIFT, PolyPhen and Condel.60.DNA from parental or AKTi-R cells was extracted and assessed for quality and quantity as described above. Illumina HumanOnmi2.5-8 arrays were then used to assay genotype, DNA copy number, and loss of heterozygosity as described previouslyThe Lipofectamine RNAi Max (Life Technologies) transfection reagent was used to transfect cells with siRNA oligonucleotides (see table below) according to manufacturer\u2019s instructions. Briefly, siRNA oligonucleotides and Lipofectamine RNAi Max were each mixed with Opti-MEM\u00ae (Invitrogen) in separate microcentrifuge tubes or wells in multiwell plates. These mixtures were combined and incubated for 15\u2009min prior to mixing with cells in suspension. Cells were transfected with a final concentration of 25\u2009nM siRNA duplexes. ON-TARGETplus SMART pool and individual siRNA oligonucleotides are reported to be highly specific, as verified by microarray analysis, as a result of unique dual-strand modification patterns used to synthesize the reagents.LNCaP cells were transfected with a pCMV6-AC-GFP vector containing the human AKT3 sequence (NM_005465) (Origene) or with EV using Fugene HD FuGENE\u00ae HD (Promega Corporation). 48\u2009h later, cells were subjected to selection with 0.4\u2009mg/l G418/Geneticin (Invitrogen/Gibco) and surviving cells were expanded. Cells were then sorted FACSAria instrumentation and associated software (BD Biosciences) for positive GFP expression and were maintained in the presence of 0.4\u2009mg/l G418/Geneticin.The piggyBac transposon-based system (System Biosciences) was used to introduce a cumate-inducible version of AKT1 WT or W80C to LNCaP Par or M-R7 cells. Using NheI and BstBI, the following sequences were cloned into the B-Cuo-MCS-IRES-GFP-EF1-CymR-Puro Inducible cDNA Cloning and Expression Vector (System Biosciences #PBQM812A-1):AKT1 WTGGCGCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGGACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTGAGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGAGATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAGCCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGTGGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTCACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCGAGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTGCCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTCCCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGAGGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAGCTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGGCCCAGATGATCACCATCACACCGCCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCCCCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGAAKT1 W80CGGCGCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGCCACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCCAACACCTTCATCATCCGCTGCCTGCAGTGTACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTGAGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGAGATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAGCCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGGTGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGTGGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTCACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCGAGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGATTGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTCATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTGCCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGGCCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTACAACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTCCCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGAGGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAGCTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGGCCCAGATGATCACCATCACACCGCCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCCCCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGAThese sequences include a silent mutation to confer resistance to the ON-TARGET plus siRNA oligonucleotide (Dharmacon) targeting AKT1 with the sequence CAUCACACCACCUGACCAA. The piggyBac expression vector includes a GFP expression cassette separated from the AKT1 sequence by an internal ribosome entry site (IRES), enabling independent expression of both genes from a single transcript. Using either the PureFection\u2122 (System Biosciences) or FuGENE\u00ae HD (Promega Corporation) transfection reagents, LNCaP Par or M-R7 cells were transfected with the piggyBac transposase (System Biosciences) combined with the empty piggyBac expression vector, AKT1 WT piggyBac vector, or AKT1 W80C piggyBac vector. Transfected cells were then incubated for 48\u2009h prior to selection with 1\u2009\u03bcg/ml puromycin and subsequently expanded. Leaky expression was minimized by using FACSAria instrumentation and associated software (BD Biosciences) to select for cells that do not express GFP in the absence of cumate. In an effort to obtain populations that express similar levels of AKT1 WT vs. AKT1 W80C, cells were treated with 10\u2009\u03bcg/ml cumate for 5 days  and were subjected to sorting for specific GFP expression levels using FACSAria instrumentation and associated software (BD Biosciences).Cell sorting was performed on multiple FACSAria\u2122 Fusions running DIVASoftware v8.0.1 equipped with 5 lasers  (BD Biosciences). The instruments were set up with a nozzle size of 100 micron at a frequency of 32\u2009kHz and pressure of 20\u2009psi. The \u201cFour-Way Purity\u201d sort mode was used for coincident discrimination.http://www.cbioportal.org/index.do?session_id=5b5e1288498eb8b3d5672636). All AKT1 mutation information reported in those selected studies was then retrieved. The frequency of each AKT1 mutation detected within the same indication was calculated from these studies . Data bases used include TCGA: The Cancer Genome Atlas, https://portal.gdc.cancer.gov; METABRIC: Molecular Taxonomy of Breast Cancer International Consortium (Nature 2012 & Nat Commun 2016), Pierra et al., 2016 https://www.ncbi.nlm.nih.gov/pubmed/27161491; MSK-IMPACT: Memorial Sloan Kettering Cancer Center\u2019s Integrated Mutation Profiling of Actionable Cancer Targets .Cancer genomics studies in which AKT1 W80 alterations were detected in patients were first identified using cBioPortal  was constructed using standard PCR techniques and mutants were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies). Mutant or WT AKT1 was cloned into the pRetro-internal ribosome entry site (IRES)-GFP vector (Clontech). MEK1 N3 was constructed as previously described61 and cloned into the pMXs-puro retroviral vector (Cell Biolabs). Retroviral constructs expressing the WT or mutant protein were transfected into the Phoenix amphoteric packaging cell line using Fugene6 (Roche). Viral supernatant was harvested 48\u2009h after transfection and filtered using a 0.45-\u03bcM syringe filter. Ba/F3 cells were then infected with virus by spinoculation , and cells infected with WT or mutant AKT1 were sorted by flow cytometry based on GFP fluorescence. Infected cells were selected with 2\u2009\u03bcg/mL puromycin for 7 days. Pools of these cells were used for subsequent studies. All Ba/F3-derived cell lines were maintained in culture in the presence of 2\u2009ng/mL recombinant murine IL-3 (R&D Systems). For analysis of the impact of WT vs. mutant AKT1 on the response to AKT inhibition, Ba/F3 cells which stably co-express MEK1 N3 and WT or mutant AKT1 were washed three times with 1X PBS and plated in the absence of IL-3 in complete RPMI in 384 well plates (1000 cells per well). The next day, cells were treated with a 9-point dose-titration of MK-2206 or ipatasertib using a maximum dose of 20\u2009\u03bcM. DMSO controls were included and all conditions were tested in 4 replicate wells. Cellular viability was assessed 4 days after addition of inhibitors using the CellTiter-Glo\u00ae (Promega) luminescent assay as described previously8. For analysis of protein levels, lysates were prepared from cells cultured in the absence of IL-3 for 1 day and subjected to Western blotting as described above.The impact of WT or mutant AKT1 on sensitivity to MK-2206 or ipatasertib was assessed using the IL-3 independent viability assay in the Ba/F3 model. Survival of the Ba/F3 murine pro-B cell line is constitutively growth-factor dependent but can be rendered IL-3 independent via co-expression of AKT1 and an activated form of the MAP2 kinase mitogen-activated protein kinase (MAPK)/extracellular-signal- regulated kinase (ERK) kinase (MEK1) , termed MEK1 N362. The data was processed using Genedata Screener, Version 14 , with a four\u2011parameter Hill equation using compound dose-response data normalized to the median of 42 vehicle\u2011treated wells on each plate. A \u201cRobust Fit\u201d strategy was also employed by Genedata Screener, which is based on Tukey\u2019s biweight and is resistant to outlier data. The reported absolute IC50 is the dose at which cross\u2011run estimated inhibition is 50% relative to DMSO control wells. In addition to absolute IC50, mean fitted viability across the nine tested doses  was also computed.A library consisting of 426 compounds including targeted agents, chemotherapeutics, and tool compounds was used to screen for inhibitors associated with enhanced sensitivity in AKTi-R cells compared with Par cell lines. Compounds were obtained from in-house synthesis or purchased from commercial vendors. Cells were plated in 384 well plates  using seeding densities previous determined to achieve approximately 70\u201380% confluence at the final time point of the assay. AKTi-R cells were plated in the presence of inhibitor at the dose used to maintain the resistance and Par cells were plated in DMSO control media. The following day, cells were treated with a 9-point dose titration of each inhibitor or DMSO control and 4 days later, cell viability was assessed as described above. Screening drug management, quality control, and the calculation of drug response statistics were performed as described previously63 as well as Highest single agent (HSA) analysis64, as described previously65. Briefly, a Bliss expectation for a combined response (C) of drugs A and B was calculated by the equation: C\u2009=\u2009(A\u2009+\u2009B)\u2009\u2212\u2009(A\u2009\u00d7\u2009B) where A and B are the fractional growth inhibitions of each respective drug at a given dose. The difference between the Bliss expectation and the observed growth inhibition of the combination of drugs A and B at the same dose is the \u201cDelta.Bliss.\u201d Delta.Bliss scores were summed across the dose matrix to generate a Bliss sum. Bliss sum\u2009=\u20090 indicates that the combination treatment is additive (as expected for independent pathway effects); Bliss sum > 0 indicates activity greater than additive (synergy); and Bliss sum <0 indicates the combination is less than additive (antagonism). Using the Highest single agent (HSA) model64, scores were calculated at each dose matrix point based on the excess loss of viability in the combination in comparison to the highest single drug response. At least three independent experiments were performed to assess reproducibility unless otherwise indicated. Heatmaps depict % viability inhibition, Bliss score, or HSA score associated with each dose combination point following treatment of cells with the two agents. Mean Bliss sum values from three independent biological replicates are depicted in scatter plots, with mean Bliss sum values denoted below the x-axis and sample size (n) indicated for each cell line/condition above scatter plot data.Par or G-R cells were plated in 384 well plates and 24\u2009h later were treated with a 9-point dose titration of GDC-0068/ipatasertib, a second inhibitor, or a combination of the two. DMSO controls were also included. After a further 4 days, cell viability was assessed using the CellTiter-Glo\u00ae assay as described above. The combination effect of ipatasertib and other inhibitors was assessed by Bliss independence analysis66 were cloned into the BH1.4 Dox-inducible piggyBac vector with an IRES-turboGFP-nuclear expression marker . LNCaP Par cells were transfected with the piggyBac transposase combined with the empty BH1.4 vector or the PIM expression constructs. Transfected cells were then subjected to selection with puromycin and subsequently expanded. Leaky expression was minimized by FACS sorting to select for cells that do not express GFP in the absence of Dox. In an effort to obtain populations that express similar levels of PIM proteins, cells were treated with 100\u2009ng/ml Dox for 3 days and subjected to sorting for specific GFP expression levels.Sequences encoding the full length forms of human PIM1 (NP_001230115.1), PIM2 (NP_006866.2), PIM3 (NP_001001852.2), or a kinase-deficient PIM3 K69M mutantFlash frozen tumors were pulverized to powder by mechanical force (Covaris cryoPREP). Tumor powder was lysed with RIPA lysis buffer (Sigma) supplemented with a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific) and 1\u2009mM PMSF (Sigma). Tumor lysates were homogenized by mechanical disruption (SPEX SamplePrep) and cleared by centrifugation at 14,000\u2009rpm for 10\u2009min at 4\u2009\u00b0C. Protein levels were quantified by the BCA Protein Assay Kit (Pierce Biotechnology) and normalized to equal concentrations. Equal amounts of protein lysates were resolved using NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using the iBlot\u00ae dry blotting system (Thermo Fisher Scientific). Membranes were blocked with blocking buffer for fluorescent Western blotting (LI-COR). Primary antibodies were detected using IR Dye 800-conjugated (LI-COR) or IR Dye 680-conjuated (LI-COR) species-selective secondary antibodies. Detection and quantification were conducted using an Odyssey infrared scanner (LI-COR) using the manufacturer\u2019s software. Protein loading was assessed using antibodies to \u03b2-actin or GAPDH.Immunohistochemisty (IHC) for xenograft tumors were carried out using 5-\u03bcm paraffin sections of formalin-fixed tissue on a VentanaBenchmark XT instrument (VMSI) by deparaffinization, treatment with antigen retrieval buffer (VMSI) and incubation with primary antibodies against PTEN (#9559), pAKT (S473) (#4060), pPRAS40 (T246) (#2997), pS6 (S235/236) (#2211), cleaved caspase 3 (Asp175) (#9661) , or cyclin D1  at 37\u2009\u00b0C. Bound antibody was detected using DABMap technology (VMSI) and sections were counterstained with hematoxylin. IHC stained slides were scanned on a NanoZoomer XR whole slide imager  at 200x magnification, and were used to quantify the tumor and IHC positive staining areas. Segmentation of tumor regions and DAB positive pixels was performed by a custom algorithm using standard morphological operations and global RGB color thresholds running on Matlab 2019a .A two-tailed Student\u2019s t-test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Statistical analysis was carried out using Prism v9.3.1 (GraphPad) and Microsoft Excel. Data are expressed as the mean\u2009\u00b1\u2009standard deviation (SD) or standard error of means (SEM) as described in the figure legends. Each experiment was repeated at least three times unless otherwise indicated.Further information on research design is available in the\u00a0Supplementary InformationInventory of Supporting InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5Reporting Summary"}
+{"text": "Ferroptosis has been identified to play a crucial role in the progression and treatment of cancers. KAT6B, as a histone acetyltransferase, is involved in multiple cancer development. However, the function of KAT6B in glioma is still elusive. Here, we aimed to evaluate the effect of KAT6B on ferroptosis in glioma cells and explored the potential mechanisms. We observed that the expression of KAT6B was enhanced in clinical glioma samples. The viability of glioma cells was repressed by erastin and the overexpression of KAT6B rescued the phenotype in the cells. Meanwhile, the apoptosis of glioma cells was induced by the treatment of erastin, while the overexpression of KAT6B blocked the effect in the cells. The levels of lipid ROS and iron were promoted by the treatment of erastin and the overexpression of KAT6B could reverse the effect in the cells. Mechanically, we identified that the expression of STAT3 was repressed by the KAT6B knockdown in glioma cells. The KAT6B was able to enrich on the promoter of STAT3 in glioma cells. Meanwhile, ChIP assay showed that the knockdown of KAT6B inhibited the enrichment of histone H3 lysine 23 acetylation (H3K23ac) and RNA polymerase II (RNA pol II) on STAT3 promoter in the cells. Depletion of STAT3 reversed KAT6B-regulated viability, apoptosis, and ferroptosis of glioma cells. Thus, we concluded that KAT6B contributes to glioma progression by repressing ferroptosis  Glioma serves as a prevalent malignancy among brain tumors in adults, which is also highly aggressive and with grave prognosis . Cancer KAT6B is a major histone acetyltransferase, widely reported during the progression of various diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases . KAT6B eIn this work, we studied the function of KAT6B in glioma cells. We aimed to evaluate the effect of KAT6B on ferroptosis in glioma cells and explored the potential mechanisms.n = 35) were collected from glioma patients hospitalized, who received no chemotherapy and radiotherapy before surgery. All tumor samples were clinicopathologically confirmed as glioma . All of glioma tissues were astrocytoma and IDH wildtype. Normal brain tissues were collected from patients undergoing brain tissue resection due to craniocerebral injury. All samples were immediately stored in liquid nitrogen with RNAhold (TransGen). All patients have signed the informed consent forms. All experiments were approved by the Clinical Ethics Committee of our hospital.The glioma samples , maintained in DMEM medium  added with 10% FBS  and 1% penicillin/streptomycin , and were incubated in a 37\u00b0C atmosphere with 5% CO5 cells per well and transfected with 50\u2009nmol oligonucleotides or their corresponding negative controls [The small interference RNA targeting KAT6B and STAT3 was designed and synthesized by Ribobio (China). Recombinant plasmid pcDNA-KAT6B was constructed by cloning KAT6B cDNA into pcDNA3.1 vectors . The U251 and LN229 cells were seeded in a 6-well plate with 4 \u00d7 10controls  by using\u03bcl/well) was added to each well and incubated for another 2\u2009h at 37\u00b0C. The absorbance values were measured at 450\u2009nm using a microplate detector .Cell viability was evaluated by the cell counting kit-8  in accordance with the manufacturer's description. The cells were planted in a 96-well plate, incubated overnight, and treated with corresponding oligonucleotides. At indicated time point, the CCK-8 solution  was added, and samples were incubated for 30\u2009min at 37\u00b0C, 5% CO2 and protected from light. Excess C11-BODIPY was removed by washing the cells twice with PBS. Oxidation of the polyunsaturated butadienyl portion of the dye results in a shift of the fluorescence emission peak from 590\u2009nm to 510\u2009nm in a manner proportional to lipid ROS generation. This shift was analyzed using a flow cytometer [2+ or total iron in each cell line. 2 \u00d7 106 of cells were rapidly homogenized in 4-10 volumes of Iron Assay buffer. Samples were centrifuged at 13,000\u2009g for 10 minutes at 4\u00b0C to remove insoluble material. To measure ferrous iron, 5\u2009\u03bcL of iron assay buffer was added to each well. To measure ferric iron, two sets of wells were set up. Then, 5\u2009\u03bcL of assay buffer was added to the samples in one set of wells and 5\u2009\u03bcL of iron reducer was added to the other set of wells. To measure total iron, add 5\u2009\u03bcL of Iron Reducer to each of the sample wells to reduce Fe3+ to Fe2+. Samples were mixed well using a horizontal shaker or by pipetting, and the reactions were incubated for 30 minutes at room temperature, protected from light. Then, 100\u2009\u03bcL of Iron Probe was added to each well-containing standard or test samples. Samples were mixed well using a horizontal shaker or by pipetting, and the reactions were incubated for 60 minutes at room temperature, protected from light. Finally, the absorbance was measured at 593\u2009nm (A593) [The lipid ROS was measured by flow cytometry analysis in the cells. For Lipid ROS, cells were treated as indicated, and then typsinezed and resuspended in medium plus 10% FBS. Then, 10\u2009ytometer . The lev\u0394\u0394Ct- method.Total RNAs were isolated from tissues or cells using Trizol reagent , reversely transcribed into cDNA by using the High Capacity cDNA Reverse Transcription Kit . The relative RNA levels were quantified by quantitative real-time PCR using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit . GAPDH was adopted as internal control for normalization following the 2The primer sequences are as follows: KAT6B forward: 5\u2032- ATACGAGCGAATGGGTCAGAGTGATTTTGG-3\u2032, reverse: 5\u2032- GTTCACAGAGTTGACATTGTAGGCTGGCG-3\u2032; STAT3 forward: 5\u2032- AACTCTCACGGACGAGGAGCT-3\u2032, reverse: 5\u2032-AGTAGTGAACTGGACGCCGG-3\u2032; GAPDH forward: 5\u2032-AACGGATTTGGTCGTATTGGG-3\u2032, reverse: 5\u2032-CCTGGAAGATGGTGATGGGAT-3\u2032.6 cells were fixed with formaldehyde  for 10\u2009min at room temperature. Fixation was stopped with the addition of 1/10 volume 1.25\u2009M glycine, and the samples were incubated for 5\u2009min at room temperature. The sonication step was performed in a Qsonica sonicator , and 200\u2009\u03bcg of the protein-chromatin complex was used in each immunoprecipitation. Antibody-protein complexes were captured with preblocked dynabeads protein G . ChIP DNA was analyzed by qPCR with SYBR Green (Biorad) on an ABI-7500 (Applied Biosystems) using the primers specified. The antibodies used are as follows: KAT6B , H3K23ac (Active motif), RNA pol II , and normal mouse IgG . The promoter sequence  was shown as follows:ChIP assay was performed by using Pierce Magnetic ChIP Kit  in accordance with the manufacturer's protocols. Antibodies against KAT6B, H3K23ac, and RNA polymerase II were used to precipitate the DNA-protein complex. The level of immunoprecipitated DNA was evaluated by qRT-PCR assay. Briefly, ChIP assays were essentially performed as previously described  with sliTGCCCTGTAGATGCCTCTGTCCCTCATTGGATATGAGGTGGTACGAGCGGTCTGAATCTGTAGACTTAGACAGGCTTCAGGTATCTGGGGAACCATGTGAGTAAAACCTTGTATTGTTTCTGGGGCTAGAATCTAGTCTCCTTCAAACACTCAGAAGGATCTGTGACTGCCTTCAAAGGTTAAGAGTCATTGATTTTTCTCACTAGAAATTAATAAGAACAGCTATCCTTGGGGAGAAGGAATGATGGGGGTAGGGAAAGATTTTCATTTAATATTTTTGACCTTTCTGAACGGTCTGCATTTTCTAAACAGGAACATGTATTAGGTTCATTGAAAAAAAATATGTCAGGGGTTAGCTGAGCAGTGAGATAATGGGTCCTTTTTAGATTTTTGTCTTTATACCTGTTCGTATTAAACAAACACAAATATTAATAACATATTTTTAAAGACCCAAAAAAATAAAAATTAAAAACCCTGATAGTATCAGCACATACACAGAAATCACTCCATTATGCAAAGTTCATCCTCTATTATGAAAGGCAAAATGTCTACATTTCCTATCAACCACTGGCTTCAATTCAGTAAAACTTGCATACCAAGTAGGCAAGGTGGAAAAGAAAAAGGCAGAACATTTCATGTATTTCAATTCAGACGCATAAAAATGTCAAGCCCTACACGTTATCAGCTTTCGTATACACCGTCTTCTGCATTCGCCTGTACGGGCCAATGGGCTAGCTGGTCGGCGTTTGATGCTTGAAGTGATGGAACGGAGTACGGGGTTAAATCCACTACCCTCTCCCCACGCACTCTAGTAATTACTCTATTTCCACGTCATGTTTCCGGGTGTGTGTGTCCCTGCTCACGCAGAAACTGAAGTTCAAAGCAGGCGGAGTCACCCATGTTCTTTTTGTTGTCCCCAGAACCCAATTCAGGAGTTGGGTCCCCAGAGGATCTGGAGATACCTGGGGACTATCTAACTAGCTGATTCCCGCGTGGTAAGAGGCTCTCAACCTCGCCACCACGTGGTGCCAAGGGCCGGGAAAAGGGAGAGCGGGCAGGAGGGAGCTGTATCAGGGGCATTTAAAGTGCCTTGACGTCACGCACTGCCAGGAACTCAGCTGAGTTTTCAGCAGGACATTCCGGTCATCTTCCCTCCCTCCCCCCGGGCTTCTGTGCCCAAGTCCTCGGCTCTTCCCTCGCTGTGGCGGAGGGAGGAGCACCGAACTGTCGGAACAGCCAGCACAGGGGCGTATCAGTCTCCTCTTGGCTCCGCCCTTTCTCCTAGCTGCTCTCCTCATTGGTCAGTGGGCGGGGCTTCGGCTGTACCGCACACGCACTGGGACCTCTGGGTGGCCGAACGAGCTGGCCTTTCATGAATTATGCATGACGGCGTGCCTCGGCCAGGCTGGGGCTGGGCGAGGATTGGCTGAAGGGGCTGTAATTCAGCGGTTTCCGGAGCTGCGGCGGCGCAGACTGGGAGGGGGAGCCGGGGGTTCCGACGTCGCAGCCGAGGGAACAAGCCCCAACCGGATCCTGGACAGGCACCCCGGCTTGGCGCTGTCTCTCCCCCTCGGCTCGGAGAGGCCCTTCGGCCTGAGGGAGCCTCGCCGCCCGTCCCCGGCACACGCGCAGCCCCGGCCTCTCGGCCTCTGCCGGAGAAACAGGTGAAGGGGGTGCAGGGTGGGGCC.t test or one-way ANOVA test. A p value less than 0.05 was considered to be statistically significant.Data in this work are presented as mean \u00b1 SD. Statistical analysis was performed by using SPSS software (version 17.0). Statistical comparison between two or more groups was determined by student's To analyze the correlation of KAT2B with glioma, the expression of KAT2B was analyzed in the clinical glioma samples. We observed that the expression of KAT2B was enhanced in clinical glioma samples . To evalNext, we were interested in the correlation of KAT2B with ferroptosis in glioma cells. To this end, the U251 and LN229 cells were treated with ferroptosis activator erastin or cotreated with erastin and KAT6B overexpressing plasmid. We found that the viability and colony formation numbers of U251 and LN229 cells were repressed by erastin and the overexpression of KAT6B rescued the phenotype in the cells Figures . MeanwhiThen, we evaluated the effect of KAT6B on the levels of ferroptosis markers, including lipid ROS and iron, in the glioma cells. We observed that the levels of lipid ROS and iron were promoted by the treatment of erastin and the overexpression of KAT6B could reverse the effect in the cells Figures .We next tried to explore the underlying mechanism by which KAT6B regulated glioma. We observed that the expression of STAT3 was repressed by the KAT6B knockdown in U251 and LN229 cells . We idenNext, we validated the correlation of KAT6B and STAT3 in the regulation of glioma cells. The effectiveness of STAT3 depletion by shRNA was validated in U251 and LN229 cells Figure . We obseGlioma is a prevalent malignancy among brain tumors, and ferroptosis plays crucial roles in the progression and treatment of cancers. Histone acetyltransferase KAT6B is involved in multiple cancer development but the function of KAT6B in glioma remains unclear. In this study, we uncovered the effect and the potential mechanism of KAT6B on ferroptosis in glioma cells.\u03baB signaling in tongue squamous cell carcinoma [Previous studies have identified the function of KAT6B in cancer development. The inhibition of histone acetyltransferases KAT6A/B contributes to senescence and regulates tumor growth . KAT6B farcinoma . These rMoreover, it has been reported that STAT3 inhibits ferroptosis-mediated IIR-ALI by targeting SLC7A11 . miR-519via epigenetically inducing STAT3 (Therefore, we concluded that KAT6B contributes to glioma progression by repressing ferroptosis ng STAT3 . KAT6B m"}
+{"text": "In the mouse embryo, the sensors responded to cytoskeletal relaxation and stretch applied by micro-aspiration. They reported organ-specific differences and a spatiotemporal tension gradient along the proximodistal axis of the limb bud, raising the possibility that mechanical mechanisms coregulate pattern formation. These mouse strains and software are potentially valuable tools for testing and refining mechanotransduction hypotheses in vivo.Nuclear mechanotransduction is a growing field with exciting implications for the regulation of gene expression and cellular function. Mechanical signals may be transduced to the nuclear interior biochemically or physically through connections between the cell surface and chromatin. To define mechanical stresses upon the nucleus in physiological settings, we generated transgenic mouse strains that harbour FRET-based tension sensors or control constructs in the outer and inner aspects of the nuclear envelope. We knocked-in a published esprin-2G sensor to measure tensions across the LINC complex and generated a new sensor that links the inner nuclear membrane to chromatin. To mitigate challenges inherent to fluorescence lifetime analysis  Summary: We introduce transgenic and software tools to measure tensions across the outer and inner nuclear envelope in mice. These tools facilitate our ability to test mechanotransduction hypotheses in vivo. The cell nucleus experiences physical forces in a manner that has the potential to alter chromatin conformation , gene exin vitro studies  complex that links f-actin, microtubules, and intermediate filaments to the nuclear lamina . Mammaliin vitro  and the INM protein NEMP1 in mice. The former was previously generated for use in vitro , whereasWe wished to generate knock-in mouse strains that harbour tension sensors within the outer and inner aspects of the nuclear envelope. To measure force transmission across the LINC complex, we generated a mouse strain capable of conditionally expressing a previously published tension sensor (NespTS) . The conin vitro. Super resolution stimulated emission-depletion (STED) microscopy revealed appropriate co-localisation of NmpTS and controls with laminB1 (reflecting INM location), and that of NespTS external to laminB1   . We chosLIMvivo) . To maxiLIMvivo) . DiffereLIMvivo) . The proLIMvivo) .There are two principal sources of noise in our setting. A quick fluorescence decay that is likely due to autofluorescence can artificially decrease the measured fluorescent lifetime, and background, or scattered, light can artificially increase the fit value for measured lifetime unless addressed appropriately . In addi\u00a0We tested individual sensor responses in vivo using E9.5 wild-type (+/+Nemp1) distal limb bud mesoderm as a model system due to its accessibility and well-studied patterning characteristics in intact embryos under live conditions. Relative to NmpTS, NmpDO exhibited higher mean lifetime values, as expected. NmpTL and NespHL reported low lifetime values relative to NmpDO and previously published donor only mTFP measurements  was a barrier to reliable FLIM analysis when we applied commercially available fitting software. Similar hurdles were encountered previously with an E-cadherin tension sensor in Drosophila, resulting in apparently poor sensitivity . Testing of constructs' proper subcellular localisation was done by Lipofectamine 2000  transient transfection in 293T cells. EBFP and IRES were removed from the pR26-CAG/BFP-Dest plasmid  and each construct was cloned into this ROSA26 targeting vector via gateway cloning  through pDONOR  for mouseline generation. These constructs were sequenced to select reliable candidates for mouseline generation.NmpTS/DO/TL were generated by PCR amplification of the tension sensor module from the Vinculin Tension Sensor  and ligaChimeras were generated by traditional homologous recombination through aggregation and implantation of electroporated embryonic stem cells (ESCs) in wild-type embryos . Final ESCs were selected by PCR genotyping and copy number analysis (TCAG at the Peter Gilgan Centre for Research and Learning). Founders were confirmed by PCR genotyping and fluorescence imaging of crossings with various Cre lines. Constitutively active lines were generated by crossings with pCX-NLS-Cre 1Nagy) or CMV-Cre  and littermates were compared visually and by weight. These lines were then inbred or outcrossed with CD1 to assess fertility.in vitro. For immunofluorescence of embryonic tissue sections, 550\u2005nm or 488\u2005nm excitation wavelength with 592\u2005nm or 660\u2005nm depletion wavelength were used on a Leica SP8 Lightning Confocal. Huygens software was employed for deconvolution. Confocal images were captured on a Nikon A1R confocal microscope using Nikon NIS software for acquisition and Volocity for image processing. The FRET donor of the sensors is mTFP, so all FLIM data were acquired on a Nikon A1R confocal microscope connected to a PicoQuant pulse system with a 440\u2005nm laser and 520/35\u2005nm detector channel in Picoquant SymPhoTime 64 software. All FLIM images were acquired using transgenic mice in a wild-type background.All imaging was performed in the Sickkids Research Institute Imaging Facility. Super resolution STED microscopy was performed on a Leica SP8 confocal microscope using 458\u2005nm or 514\u2005nm excitation wavelength with 592\u2005nm or 660\u2005nm depletion wavelength for sensor constructs 2 held in place with cheese cloth. In roller culture experiments, embryos were first placed in roller culture consisting of DMEM with 50% rat serum, 5% DMSO, and specified final drug concentrations and rolled for 1\u2005h at 37\u00b0C in a 20% O2, 5% CO2 chamber. Aspiration experiments were performed on the A1R using a 90\u2005\u00b5m diameter glass needle in a previously published device  fixation allowed for imaging of mTFP or Venus within sensor constructs, but longer fixation  of in vivo samples required labelling of sensor constructs with mouse monoclonal \u03b1-GFP .In fixed sample imaging, \u03b1-aminB1 rabbit polyclonal antibody was used to label LaminB1  or \u03b1-O-GlcNAc mouse monoclonal antibody was used to label nuclear pores . In fixed FLIM data were segmented into isolated nuclear membrane regions. Unless clarified otherwise in the figure legends, each dataset presented is nuclei from at least three separate embryos. With the exception of VinTFP which was fitted to entire fields of view, 15 nuclei, or as many clear nuclei as were available, per image were segmented for each region of each embryo. Further, segments with low quality data  were rejected prior to statistical analysis.P-values were calculated using the t-test, applying Welch's correction to comparisons with significantly different standard deviations for datasets expected to be normally distributed and using the Mann\u2013Whitney test for non-normally distributed datasets. For t-tests, four key assumptions  were considered prior to test application. Pregnancy rate comparison P-values were calculated using the \u03c7-square test. Variance comparisons were performed using F-tests. P-values greater than 0.05 were considered insignificant. In all experiments where tissues from the same embryos were compared, the values were normalised to the mean of all embryos and tissues for that experiment.Statistical analysis was done in Prism. With the exception of pregnancy rate comparisons, regression analyses, and variance comparisons, https://www.github.com/HopyanLab/FLIMvivo. The basic FLIMvivo.py script takes a CSV data file and can perform either convolution or tail fitting applying either a mono-exponential or bi-exponential model. Segmentation masks for analysed FLIM images were generated using either the FLIMvivo or the FLIMfit , then calls upon FLIMvivo to fit the extracted data. The script uses FLIMvivo first to fit the full field with a model of a Gaussian instrument response convoluted with a bi-exponential in order to establish an autofluorescence lifetime C. It cheWe model the FLIM-FRET decay as a bi-exponential, with autofluorescent and signal components,For our fitting algorithms we use a maximum likelihood fitting scheme assuming Poisson statistics, minimizing the \u2018negative log-likelihood\u2019 (NLL),Finally, in fitting, we treat the end point (where the data is cut off) as fit variable, chosen by maximizing the ratio of best fit NLL to the square root of the number of data points. This way the data range is uniformly chosen by maximizing a measure of data contribution to our fit metric, thereby eliminating any potential bias introduced by choosing manually.All constructs were genotyped using these primers: Forward: CTCTGCTGCCTCCTGGCTTCT, WT Reverse: CGAGGCGGATCACAAGCAATA, Mutant Reverse: CCGCGAGCTGTGGAAAAAAAAGGG.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTC CCGGGCCCTGGGGCTCGGGAGTCGGGGGC GGTGGGACAGTGC GGC TACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCT GAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAA GTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCCGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAATTCCCTATTCAGTGGCTGTACATCACCTGCAGAAAGGTGTGTAAGGGAGCAGAAAAGCCTGTTCCCCCTCGTCTCCTGACAGAAGAAGAATATCGGATACAAGGAGAGGTAGAAACCCGAAAGGCTTTAGAGGAGCTCCGAGAATTTTGTAACAGTCCAGACTGCTCTGCTTGGAAGACTGTTTCTCGAATCCAGTCTCCAAAAAGATTTGCTGACTTTGTGGAAGGCTCTTCCCACCTCACGCCAAATGAAGTTTCTGTCCATGAGCAGGAGTATGGATTAGGGAGCATTATTGCCCAGGATGAAATCTATGAGGAAGCATCCTCTGAGGAGGAGGACTCATATTCTCGGTGTCCTGCTATCACACAGAACAACTTTCTAACCTGA.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTCCCGGGCCCTGGGGCTCGGGAGTCGGGGGCGGTGGGACAGTGCGGCTACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCTGAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAAGTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCAGGTGGTGCAGGGCCCGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCAGGTCCAGGTGGTGCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTGATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA.ATGGCGGGAGGAATGAAAGTGGCGGTCTCGCCGGCAGTTGGTCCCGGGCCCTGGGGCTCGGGAGTCGGGGGCGGTGGGACAGTGCGGCTACTCTTGATCCTCTCCGGCTGCTTGGTCTACGGCACAGCTGAAACTGATGTAAATGTGGTCATGCTTCAGGAATCCCAAGTTTGTGAAAAGCGTGCCAGCCAACAATTCTGTTACACAAATGTGCTTATCCCAAAATGGCATGATATATGGACACGGATACAGATCCGAGTAAATAGTTCCAGATTGGTTCGAGTCACCCAGGTGGAGAATGAGGAGAAACTGAAGGAGCTAGAGCAGTTTAGTATCTGGAACTTTTTTTCCTCCTTTTTAAAAGAGAAATTGAATGACACCTATGTTAACGTGGGTCTATACAGCACAAAAACCTGCCTCAAAGTTGAGATTATAGAGAAGGACACCAAGTACAGTGTCATTGTGATCCGGAGATTTGATCCCAAACTCTTTCTTGTTTTCCTTCTTGGACTTATGCTATTTTTTTGTGGAGACTTGCTGAGCAGAAGTCAAATTTTCTACTACTCTACTGGGATGACTGTGGGAATTGTGGCCTCTCTGCTAATCATCATTTTTATACTATCTAAGTTTATGCCTAAGAAAAGTCCCATTTACGTCATCCTGGTGGGAGGCTGGTCTTTTTCTCTGTACCTCATTCAACTAGTTTTTAAAAATTTACAAGAGATCTGGAGGTGTTACTGGCAGTATCTTTTAAGTTATGTCCTCACAGTTGGATTCATGAGTTTTGCAGTATGTTACAAGTATGGGCCCTTGGAGAATGAACGAAGTATCAACCTGCTGACCTGGACCTTGCAGCTGATGGGCCTGTGTTTCATGTATTCTGGCATCCAGATACCACATATTGCCCTTGCCATTATCATCATTGCTCTTTGTACTAAGAACCTGGAACACGGATCCATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCGCCTACGGCAACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGAAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGCACAAGCTGCTGCTGGAGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGAGCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTACAAGGAATTCCCTATTCAGTGGCTGTACATCACCTGCAGAAAGGTGTGTAAGGGAGCAGAAAAGCCTGTTCCCCCTCGTCTCCTGACAGAAGAAGAATATCGGATACAAGGAGAGGTAGAAACCCGAAAGGCTTTAGAGGAGCTCCGAGAATTTTGTAACAGTCCAGACTGCTCTGCTTGGAAGACTGTTTCTCGAATCCAGTCTCCAAAAAGATTTGCTGACTTTGTGGAAGGCTCTTCCCACCTCACGCCAAATGAAGTTTCTGTCCATGAGCAGGAGTATGGATTAGGGAGCATTATTGCCCAGGATGAAATCTATGAGGAAGCATCCTCTGAGGAGGAGGACTCATATTCTCGGTGTCCTGCTATCACACAGAACAACTTTCTAACCTGA.10.1242/biolopen.059656_sup1Supplementary informationClick here for additional data file."}
+{"text": "Previous studies have shown that aging promotes myocardial apoptosis. However, the detailed mechanisms remain unclear. Our recent studies revealed that aging not only activates apoptosis, but also activates some anti-apoptotic factors. By quantitative phosphoproteomics, here we demonstrated that aging increases cytochrome c (Cytc) phosphorylation at threonine 50 (T50), a post-translational modification with unknown functional impact. With point mutation and lentivirus transfection, cardiomyocytes were divided into four groups: empty vector group, WT (wild type), T50E (as a phosphomimic variant), and T50A (non-phosphorylatable). TUNEL staining and flow cytometry were used to determine the apoptosis ratio in different groups after hypoxic/reoxygenated (H/R) treatment. The results showed that T50-phosphorylated Cytc suppressed myocardial apoptosis induced by H/R. Furthermore, Western Blot and ELISA measurements revealed that Cytc T50 phosphorylation inhibited caspase-9 and caspase-3 activity without altering caspase-8, BCL-2, BCL-XL, and Bax expression. In our study, we demonstrated that aging increases phosphorylation Cytc at T50 and this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. Acute myocardial infarction is one of the leading causes of death worldwide. Thanks to the rapid development of interventional technology, the number of people dying from acute myocardial infarction has decreased significantly. On the contrary, more and more people are suffering from post-ischemic heart failure . Heart fSubstantial evidence indicates that apoptosis is the leading cause of ischemia-induced heart failure . Our preMore interestingly, our previous work demonstrates that aging not only stimulates pro-apoptotic pathways in cardiomyocytes, but also activates some anti-apoptotic factors , 9, 10. In the present study, we performed proteomics on myocardial tissue obtained from young and old mice. Compared with the young group, the expression of 88 proteins was up-regulated, and 80 were down-regulated in old mice. At a post-translational modification level, phosphorylation levels were up-regulated at 445 sites and down-regulated at 1526 sites in the old group. Phosphorylation level changes were widespread and stable in each sample. More importantly, these changes occurred in many essential apoptotic regulatory proteins, including MAPK14, Akt1, mTORC2, and p21. Upon further analysis of these results, changes in Cytc phosphorylation at T50 caught our attention as Cytc phosphorylation is involved in mitochondrial function . SurprisWe found a new aging-increasing phosphorylation site by proteomics, which may be associated with aging-related myocardial apoptosis regulation.in vivo and is closely related to apoptosis [P-value<0.05 as a cut-off, the change in the amount of differential modification >1.3 was considered as significantly up-regulated and less than 1/1.3 as significantly down-regulated.  and increased apoptosis in T50A group when compared with WT group  is also closely related to apoptosis. Lee et al. isolated Cytc from cow heart and identified the phosphorylation site by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. Tyr-97 is the first phosphorylation site discovered on Cytc . The othIn conclusion, there are several innovations in this study. First, we observed that aging increases phosphorylation Cytc at T50. Second, this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. In addition, this anti-apoptotic effect is mediated mainly through inhibition of downstream caspase-9 and caspase-3 activity. Based on our previous studies , T50-phoIn summary, our study proved a new phosphorylation site that is increased in the process of aging. More importantly, phosphorylation at this site can decrease the apoptosis ratio induced by H/R.Approved by the institutional ethics committee, this study was in compliance with the United States National Institutes of Health guidelines. Male C57 mice (aged eight weeks and 18 months) were anesthetized with sodium pentobarbital. Mouse hearts were harvested by exteriorizing the heart via a left thoracic incision.Heart tissue from young and old mouse were harvested and delivered to PTM-Biolab Co., Ltd.  for phosphoproteomic analysis. The quantitative study of phosphorylated proteomics used TMT labeling and phosphorylated enrichment techniques and high-resolution liquid chromatography-mass spectrometry.http://www.ebi.ac.uk/GOA/). Firstly, converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt GOA database, the InterProScan soft was used to annotate protein\u2019s GO function based on the protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component, and molecular function.Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database .A special antibody was ordered from PTM-Biolab Co., Ltd. an antigen. Specific antibody response was examined with immunohistochemistry staining.Myocardial tissue and AC16 cells were homogenized in an ice-cold lysis buffer. After homogenization, the lysates were centrifuged. The supernatant was saved and separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride-plus membranes. After blocking buffer, the immunoblots were probed with anti-T50 phosphorylation of Cytc, anti-Cytc, anti-Cleaved Caspase-3, anti-Caspase-3, anti-Cleaved Caspase-9, anti-Caspase-9, anti-BCL-2, anti-BCL-XL, anti-Bax, and anti-GAPDH antibodies overnight at 4\u00b0 C, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour.in situ by the use of 3, 3 diaminobenzidine kit .Paraffin sections made from young and aged mouse hearts were used for immunohistochemistry assays to detect protein expression levels of T50 phosphorylation of Cytc proteins. In accordance with the manufacturer\u2019s instruction, tissue sections stained immunohistochemically are determined separately by two pathologists using the indirect streptavidin peroxidase method. The primary antibodies against T50  and horseradish peroxidase-conjugated IgG were used in this study. Then, the proteins were visualized in vitro, we generated T50E for constant phosphorylation and T50A for non-phosphorylation. Three sequences of WT, T50E and T50A are as below. WT:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T50E:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGAGGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T5OA:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGCCGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. With these sequences, construct lentivirus vectors, purchased from Shanghai Yibeirui Bio-pharmaceutical Technology Co., Ltd. .Three sequences of WT, T50E and T50A were synthesized after codon optimization, purchased from Sangon Biotech Co., Ltd. . To study the effect of T50 phosphorylation Human adult ventricular cardiomyocyte cell line AC16 was purchased from the American Type Culture Collection . Cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37\u00b0 C. In this study, four types of lentiviruses were used to infect AC16 cells. Infection efficiency was monitored by fluorescence microscopy 48 hours post lentivirus infection.In vitro cardiomyocyte H/R modelSeventy-two hours after cardiomyocyte transfection, all groups were treated with hypoxia (95% N2 and 5% CO2) for 6 hours , followed by normoxia  for 2 hours.Cardiomyocytes were cultured directly upon coverslips. TUNEL staining was performed per the manufacturer\u2019s instructions . Total nuclei were stained by DAPI . Apoptotic index  was determined via blinded manner.After incubation for two days, the harvested cells were used for apoptotic determination by two methods. First, the apoptotic rate of transfected cells was evaluated by using Annexin V APC/PI apoptosis detection kit (KeyGEN) by following the manufacturer\u2019s instruction and then analyzed using FACScan. All tests are carried out in triplicate. Second, by following the manufacturer\u2019s instructions, Caspase-9 and Caspase-3 levels were detected utilizing human Caspase-9 ELISA kit (BC3890), Caspase-3 ELISA kit (BC3830) and Caspase-8 ELISA kit (BC3880) from Beijing Solarbio Science and Technology Co., Ltd. .The data were analyzed with Prism 8.0 . All values in the text and figures are presented as mean \u00b1 SD. Statistical differences were determined by Student\u2019s t-test for comparison between 2 groups and ANOVA followed by Bonferroni multiple comparison test for comparison among \u22653 groups. Probabilities of.05 or less were considered statistically significant.The data used to support the findings of this study are available from the corresponding author upon request.in vivo or clinical significance should be explored in the future.In this study, phosphoproteomics were performed only in myocardial tissue from young and old mice, which reduces the targeting of the experiment. The relationship between T50 phosphorylation and mitochondrial release or caspase 9 interaction will be explored in the next step. In addition, the functional impact of Cytc T50 phosphorylation"}
+{"text": "Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (\u03b1-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611\u2013626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab\u2019)2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs. Therefore, their isolation should be very specific [We are in the midst of major advances in understanding cancer biology and in development of rationalized biological drugs against specific targets in cancer cells . Howeverspecific .The most common strategy for capturing CTCs is based on the expression of specific markers on their membrane. By using the EpCAM and CK markers, the commercial technology of CellSearch enables the isolation of epithelial derived tumor cells by magnetic based antibodies ,6,7. AltIn order to overcome these drawbacks, new isolation strategies were being developed based on differences in their properties such as size, adherence, density, electric charge of tumor versus normal cells ,15,16,17Currently, there is no validated universal marker that enables a successful identification of large variety of CTCs as an early diagnosis strategy. Here we describe our approach using telomerase peptide presented on the membrane of all cancer cells for the development of an anti-telomerase antibody-based tool for pan cancer cells CTC isolation. Telomerase is the hallmark of the cancer cell. It is highly expressed in almost all types of cancer cells and is repressed in most somatic cells . A panelThe specificity of the newly synthesized \u03b1-hTERT mAb (designed as \u03b1-hTERT) was tested by assessing its ability to bind specifically the 16-amino acid telomerase peptide of which it was designed against (not shown). The binding of the newly synthesized \u03b1-hTERT mAb to various cancer cell lines and fibroblasts (non-cancer cells) was tested using a flow cytometry. The following cells were assayed: Jurkat (T cell leukemia), MCF-7 (breast adenocarcinoma), U266 (multiple myeloma), MEL (melanoma), JeKo-1 (mantle cell lymphoma), A549 (lung carcinoma). Fibroblast cells served as a negative control. The ability of the antibody to bind these cells is presented in The binding of \u03b1-hTERT mAb to cancer cells isolated from the peripheral blood of patients with CLL (ex vivo) was tested as described in Materials and Methods. A total of 57 blood samples at different stages of the disease , including primary diagnosed and treated, were tested. The binding to 4 representative blood samples of CLL and 4 solid tumors samples of different origin are shown in The lack of \u03b1-hTERT mAb\u2019s binding to normal lymphocytes is depicted in 5 ovarian tumors and 9 ascites samples from ovary cancer patients were analyzed. The binding of \u03b1-hTERT mAb to PBMCs of a patient with Merkel carcinoma is presented in Ex vivo binding of the \u03b1-hTERT mAb to cells obtained from three patients with breast cancer is shown in The In addition, we were able to detect the binding of the \u03b1-hTERT mAb to a small subset of cells from two patients with lung carcinomas as presented by dot plots on In order to optimize the binding specificity of the \u03b1-hTERT mAb to the TERT antigen, the F(ab\u2019)2 fragment was cleaved off using ficin. The F(ab\u2019)2 fragment was detected by gel electrophoresis (SDS-PAGE), and its binding to CLL cells was analyzed by flow cytometry. The results of the binding assays of the F(ab\u2019)2 fragment obtained from two different preparations are demonstrated in SEQ ID #15\u2019-DVVMTQTPLTLSVTIGQPASISCKSSQNLLYSDGKTYLNWLLQRPGQSPKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQGTHLPYTFGGGTKLEIK-3\u2019SEQ ID #25\u2019VQLQQSGAELVRPGASVTLSCKASGYIFTDYEKHWVKQTPVHGLEWIGAIDPESGSTVYNQRFKGKATLTADKSSGTAYMELRSLTSEDSAVYFCFLLRLFAYWGQGTLVTVSA -3\u2019The amino acid sequences of the proteins comprising the light chain of the anti-hTERT mAb was assessed by Edman degradation, and the sequencing of the heavy chain was performed using an in-gel protein digestion followed by liquid chromatography-mass spectrophotometry (LC-MS) procedures, as described in Materials and Methods. The light chain  and heavSEQ ID #35\u2019-GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTGCTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGTGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACATTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATGAGTGTTAA-3\u2019SEQ ID #45\u2019-caggtgCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACATATTTACTGATTATGAAAAGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAGTGGATTGGAGCTATTGATCCTGAAAGTGGTAGTACTGTCTACAATCAGAGATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCTTCCGGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGATTCTGCCGTCTATTTCTGCTTTTTACTACGGCTATTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCC-3\u2019The nucleic acid sequencing was performed using the degenerate primer-based sequencing technique described in Materials and Methods. The light chain and heavy chain nucleic acids sequences of \u03b1-hTERT designated herein as SEQ ID #3 and SEQ ID #4, respectively, are:SEQ ID #65\u2019-GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTGCTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTC-3\u2019The variable region of the light (VL) chain comprises the nucleotide sequence of SEQ ID #6:Many signal transduction pathways which mediate malignant transformation have been deciphered. Accordingly, new efficient anti-cancer rationalized drugs were developed . HoweverRecently, a novel technology has been developed for this purpose which is based on liquid biopsies . Liquid The isolation of CTCs is evolving as a promising approach for genetic analysis and therapeutic decisions in cancer and as a possible tool for detection of tumors. The available methods for isolation of CTCs are specific for various types of cancer and therefore the hunting of these CTCs is done by binding to a specific antibody which serves as a cancer marker, which varies among the different malignant cells as well as lacks in others. Herein we describe a somewhat novel approach in which we use the peptide of telomerase, the hallmark of cancer cells, as a bait to trap all cancer cell types based on the definition of the hTERT as a pan-cancer marker . This deTelomerase containing cells present on their membrane telomerase driven peptides. Our strategy is based on one of them, termed GV1001, a 16 amino acid peptide (EARPALLTSRLRFIPK) which was used to construct an antibody for the identification of cancer cells expressing telomerase . Our conSince the \u03b1-hTERT antibody is present specifically on cancer cells membrane, it was shown to be successfully used as a cancer related antigen for developing anti-cancer vaccination in several studies to treat advanced pancreatic cancer, non-small cell lung cancer, melanoma, and other cancers ,30,31,32Next, we showed that CLL cells obtained from patients differing in their CLL cells concentrations, bound our antibody. When we labelled the cells firstly with our antibody and only then with \u03b1CD19 and \u03b1CD5 as CLL markers, we got similar results, thus emphasizing the capability of the antibody to recognize and bind CLL cells . More imOur study has several limitations. First, hTERT is also expressed in actively dividing normal somatic cells, including small intestine, colon, lymph nodes and also hematopoietic stem cells, although to a much lesser extent compared to malignant cells ,34. In t). The fact that the extent of the antibody binding differed in various cell lines may stem from the different expression of the enzyme in these cancer cells. We have subsequently sequenced our specific antibody and provide herein the full sequence of both heavy and light chains of it.Second, the specificity of our mAb was evaluated by a parallel assessment of the binding of our mAB to ex vivo samples obtained from oncological patients and from healthy volunteers. To calculate the specificity and sensitivity of our antibody, we currently collect more samples in order to analyze these parameters by a Receiver Operating Characteristic (ROC) curve in the coming future. Third, we did not report the efficacy of our antibody, since only in CLL cells and not in the cells originated from the solid tumors we could compare the extent of the hTERT antibody binding to the presence of CLL markers on the neoplastic cells. All in all, we have characterized an anti-telomerase specific mAb which was shown to be specific to telomerase positive cells, both in vitro and ex vivo . The facFinally, to improve the specificity of the \u03b1-hTERT mAb we have cleaved it to F(ab\u2019)2 fragment. As expected, the cleaved F(ab\u2019)2 fragment bound cancer cells specifically ex vivo , in lineThese results show that our anti-hTERT antibody-based system is able to identify and isolate circulating tumor cells of various origin.One major pitfall in our study might have been that the antibody would not identify all cancer cells expressing telomerase and therefore the number of isolated CTCs will not be maximal; This point already proven not to be a problem, as our antibody is highly sensitive and specific for cancer cells. However, the advantages in the development of this \u201cCTC trapping\u201d anti \u201cpersonalized medicine\u201d based approach are clear: it is an easy cheap and noninvasive one.Hopefully the approach that we developed will be thoroughly explored in future studies which may result in the development of a more beneficial method to trap CTCs for the benefit of patients with cancer.2. Jurkat and MEL cell media were supplemented with 10% FBS, U-266 was grown in the presence of 15% FBS and JeKo-1 with 20% FBS. Fibroblasts were propagated in DMEM with 20% FBS supplemented with L-glutamine and penicillin/streptomycin/nystatin.Cells were grown under standard growth conditions. The following cells were used: Jurkat (T cell leukemia), MCF-7 (breast adenocarcinoma), U266 (multiple myeloma), MEL (melanoma), JeKo-1 (mantle cell lymphoma), A549 (lung carcinoma). Human mononuclear cells (MNCs) from healthy volunteers and human fibroblasts served as a negative control. All cell lines were originated from the American Type Culture Collection (ATCC). U-266, Jurkat, JeKo-1 and MEL were cultured in RPMI-1640, A549 and MCF-7 in DMEM supplemented with 10\u201320% fetal bovine serum (FBS), 100 units/mL L-glutamine and 1% penicillin/streptomycin/nystatin  at 37 \u00b0C with 5% COPatients\u2019 samples were obtained upon consent via protocol # 0038-12 approved by the IRB of Rabin Medical Center.Peripheral blood samples were collected and stored at room temperature and processed within 24 h of the collection. All participants signed an informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples using standard protocol with Lymphoprep .The hTERT peptide GV1001, EARPALLTSRLRFIPK, was synthesized by Genemed Synthesis . Hybridomas were produced by ProteoGenix . The Screening of the various antibodies containing supernatants was performed by ELISA and Flow Cytometry. The antibodies and isotype controls for flow cytometry were purchased from BioXCell Company .F(ab\u2019)2 fragments of \u03b1-hTERT mAb were prepared according to the protocol provided by Pierce mouse IgG1 Fab and F(ab\u2019)2 preparation kit . The fragments were generated by using ficin in the presence of cysteine and analyzed by polyacrylamide gel electrophoresis (PAGE).The specificity of monoclonal antibodies (mAbs) to the hTERT peptide was assessed by sandwich ELISA, using a standard protocol, wherein the tested mAbs binds to the hTERT peptide and not to the carrier protein (KLH) immobilized to the assay plate. Unbound antibodies were removed by washing with PBS. Subsequently, the bounded antibodies were detected by horse radish peroxidase (HRP)-conjugated anti-mouse IgG antibody and a 3,3\u2032,5,5\u2032-tetramethylbenzidine (TMB) substrate. The optical density was measured at 450 nm by microplate reader .The binding of the \u03b1-hTERT antibodies to a panel of cancer cells from patients was assessed by flow cytometry analysis. Blood, tumor and ascites samples were first stained with primary \u03b1-hTERT peptide mAb or isotype control, then incubated with FITC or AF647-conjugated \u03b1-mouse secondary Ab  for the detection of hTERT positive cells.Chronic lymphocytic leukemia (CLL) cells were marked as CD19+/CD5+ ; ovarian cancer cells were detected by following the marker CCCR5. Antibodies were purchased from ENCO, Jackson, Sigma and Abcam, CA, GB).All cells were prepared for the flow cytometry analysis according to a routinely used procedure. Cell lines: floating cells collected from the growth media by centrifugation, adherent cells were trypsinized. Peripheral blood mononuclear cells (PBMCs) obtained from whole blood were isolated by Lymphoprep .The flow cytometry data was acquired using a BD FACS Calibur flow cytometer , acquisition and analysis were performed using Cell Quest Pro software .PBMCs were centrifuged, the pellet was resuspended in PBS +2% FBS and equally split into tubes. Cells were subsequently stained with \u03b1-hTERT or isotype mAb, washed with Phosphate Buffer Saline (PBS) and incubated with fluorescently labeled secondary anti-mouse Ab . Following washing cells were stained with tumor markers antibodies and re-suspended in PBS for acquisition on a flow cytometer. The same setup and compensation were used for all CLL samples. Cell debris and aggregates were excluded on forward scatter vs. side scatter. Data is presented by dot plots, histograms and graphs.The sequencing of the light chain of the antibody was performed using Edman degradation, in gel protein digestion and liquid chromatography-mass spectrophotometry (LC-MS) conducted by the Mass Spectrometry (MS) biological services unit of the Weizmann Institute of Science, Rehovot.The heavy chain of the purified antibody was cleaved by trypsinization prior to LC-MS analysis.The nucleotide sequence was obtained by using degenerated primers based on predicted sequences designed at the Tel Aviv University. The Kapa HiFi HotStart PCR  was used for the PCR reaction and the procedure was performed according to routinely used protocol. The nucleic acid sequencing was performed by the Instrumentation and Service Center at Tel Aviv University.Immunofluorescence analysis was conducted as described previously . Basical"}
+{"text": "RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3\u2032 end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3\u2032 end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome. Nano3P-seq presents a nanopore-based sequencing tool to profile polyA-tailed and non-polyA-tailed transcripts, as well as capture polyA tail length and composition. Polyadenylation of RNA is one such modification, which is known to affect the stability and translation efficiency of the RNA molecule4 and to play an essential role in determining the fate of RNA molecules in a wide range of biological processes6.RNA molecules are subject to multiple co- and post-transcriptional modifications, shaping them to their final mature form6. Indeed, in the first hours after fertilization, vertebrate embryos undergo major cellular reprogramming, a process known as the maternal-to-zygotic transition (MZT)7. During the MZT, maternally inherited RNA and proteins are responsible for activation of the zygotic genome and are later replaced by the zygotic program9. Because the MZT begins in a transcriptionally silent embryo, this transition relies heavily on post-transcriptional regulatory mechanisms7, including modulation of the polyadenylation status of the RNA molecules10. Therefore, characterizing the dynamics of RNA polyadenylation is key to understanding how these modifications regulate the fate and function of RNA molecules.One context in which polyadenylation has been shown to play a major role in determining RNA fate and decay is vertebrate embryogenesis11. While these methods have been successfully employed to characterize the dynamics of polyA tail lengths in various contexts, they have several caveats: (i) they provide a limited perspective on isoform\u2013tail relationships owing to the short-read-length nature of NGS-based technologies; (ii) they do not provide single-molecule resolution; (iii) they are severely affected by PCR amplification biases; and (iv) they can only measure tail lengths that are shorter than the read length.In the past few years, several transcriptome-wide methods have become available for studying the dynamics of polyadenylated tails (polyA tails) based on next-generation sequencing (NGS), such as PAL-seq or TAIL-seq13. To sequence native RNAs using dRNA-seq, polyA-tailed RNA molecules are ligated to a 3\u2032 adapter that contains an oligo(dT) overhang. Consequently, dRNA-seq libraries capture the full-length polyA tail; however, ligation occurs only on RNA molecules that anneal to the oligo(dT) overhang, thus exclusively capturing polyA transcripts with tail lengths greater than 10 nucleotides. A variation of this method consisting of in vitro poly(I)-tailing the transcriptome before library preparation has been proposed for studying nascent RNAs using dRNA-seq15, thus capturing both polyadenylated and non-polyadenylated mRNAs. However, major limitations to this variation include low sequencing yields compared with standard dRNA-seq (10\u201330%)14 and a lack of tools to distinguish poly(I) and poly(A) signals; therefore, polyA tail length information is lost in these datasets15. An alternative approach for studying the transcriptome using nanopore technologies is direct cDNA sequencing (dcDNA-seq), but this approach cannot sequence the polyA\u2212 transcriptome, nor can it capture polyA tail length information. Overall, both dRNA-seq and dcDNA-seq nanopore library preparation protocols are limited to the sequencing of polyadenylated transcripts and thus cannot provide a comprehensive view of both polyadenylated and deadenylated RNA molecules, in addition to being unable to capture RNA molecules with other types of RNA tails .To overcome these limitations, the direct RNA sequencing (dRNA-seq) platform offered by Oxford Nanopore Technologies (ONT) has been proposed as a means to study both the transcriptome and polyA tail lengths simultaneouslyHere we present a novel method that employs nanopore sequencing to simultaneously obtain per-isoform transcriptome abundance and tail lengths in full-length individual reads, with minimal library preparation steps, which we term Nanopore 3\u2032-end-capture sequencing (Nano3P-seq). Notably, Nano3P-seq uses template switching to initiate reverse transcription and, therefore, does not require 3\u2032 end adapter ligation steps, PCR amplification, or second-strand cDNA synthesis. We demonstrate that Nano3P-seq can capture any type of RNA molecule regardless of its 3\u2032 sequence, including polyA-tailed and non-tailed RNA. Moreover, we show that Nano3P-seq can accurately quantify RNA abundances in both the coding and non-coding transcriptome, as well as quantify the polyA tail lengths and tail composition of individual RNA molecules in a highly reproducible manner.+ RNA molecules    and per-isoform (Pearson\u2019s R\u2009=\u20090.97) 93) Fig.  and per-Next, we employed Nano3P-seq to examine the RNA dynamics that occur during the maternal-to-zygotic transition (MZT) at single-molecule resolution Fig. . To this8, with a decay of mRNA genes previously reported to have a \u2018maternal decay mode\u2019 . PolyA tail length estimates were highly reproducible across independent biological replicates sequenced in independent flow cells for all three time points studied (R\u2009=\u20090.85\u20130.95)   Fig. . We obse95) Fig. , in agre21. We observed that the three groups of mRNAs that are known to decay  showed a significant decrease in mRNA abundance , as previously describednce Fig. , as expence Fig. . SpecifiA major feature that distinguishes nanopore sequencing from NGS is its ability to produce long reads, allowing RNA polyadenylation dynamics to be studied at the isoform level. Therefore, we wondered whether Nano3P-seq could identify differentially polyadenylated transcript isoforms during the MZT.P\u2009<\u20090.05) during the MZT. Notably, we observed that analyses at the per-gene level often revealed a different picture compared with analyses at per-isoform level. For example, in khdrbs1a and syncrip, per-isoform analysis revealed opposite tail length dynamics among isoforms during the MZT, with one isoform decreasing and another isoform increasing in polyA tail length as the MZT progressed  of analyzed transcripts varied significantly in polyA tail length  of analyzed genes presented significant differences in their polyA tail lengths across isoforms Fig. . Altoget34. Some modifications occur in base positions that are involved in Watson\u2013Crick (WC) base pairing, causing a disruption during reverse transcription. Consequently, these modifications can be seen as increased \u2018errors\u2019 and drop-off rates in RNA-seq datasets38. One example is the hypermodified base m1acp3\u03a8, which is present in the eukaryotic small subunit (SSU) rRNA39 crucial for the final processing steps of precursor rRNA (pre-rRNA) into mature SSU rRNA41.RNA molecules are decorated with chemical modifications, which are essential for the stability, maturation, fate, and function of the RNA1acp3\u03a8-modified site was very high in mature rRNAs but not present in pre-rRNAs, suggesting that this modification is only present in mature rRNA populations. The presence of the hypermodification was also accompanied by a drop-off in sequencing coverage at the m1acp3\u03a8-modified site, in agreement with its ability to disrupt the Watson\u2013Crick base pairing. Analysis of the \u2018error\u2019 signatures along the SSU transcripts showed that this position was the only one position to change between precursor and processed rRNA molecules  at this site in the pre-rRNAs, which also causes increased \u2018errors\u2019 in dRNA-seq datasets, but not in Nano3P-seq datasets  and analyzed the sequencing error patterns in the two populations Fig. . We obseles Fig. . These r44. However, these methods cannot be used to detect tail modifications among the vast majority of tail nucleotides, as Illumina sequencing quality strongly deteriorates in homopolymeric stretches and with increased read length. By contrast, Pacific BioSciences (PacBio)-based approaches such as FLAM-seq can sequence through the entire tail, however, they cannot unambiguously identify 3\u2032 terminal modifications45.Recent works using TAIL-seq have reported that a number of terminal modifications in polyA tails, such as polyuridine stretches, play a role in mRNA decayTherefore, we explored whether Nano3P-seq could accurately identify nucleotide composition variations  within polyA tails. To this end, we designed synthetic cDNA molecules with polyA tails ending with A (30A), U (1U), UUU (3U), UUUUU (5U), CCCCC (5C), and GGGGG (5G), as well as a polyA tail that contained several internal G nucleotides at fixed positions (IntG) Fig. ; MethodsNext, we analyzed the mRNA tail composition of zebrafish embryos from different developmental stages . Analysis of tail base abundance revealed that G was the most common non-A base in zebrafish mRNAs and that there was a significant decrease in non-A bases with progression of the MZT  Fig. . Our ana46.Finally, we examined the relationship between tail length and the presence of non-A bases in the tails. Although the median tail length increases during the MZT Figs  and 3g, + transcripts using oligo(dT) beads. Although these two approaches are often used interchangeably, their effects on the transcriptome composition are not equal. Nano3P-seq allows us to compare the effects of these two approaches on both the transcriptome composition and polyA tail length distribution. In terms of its effects on transcriptome composition, we found that ribodepletion captures a larger variety of RNA biotypes compared with polyA selection, including several non-polyA-tailed RNA biotypes, as expected  ribodepletion of the sample using biotinylated oligonucleotides that are complementary to rRNAs or (ii) selective enrichment of polyAted Fig. . Howeverted Fig. , suggest12. In terms of sequencing output, the yields of Nano3P-seq runs were similar\u2014or slightly better\u2014to those observed in dRNA-seq runs, producing ~100,000\u2013200,000 reads in Flongle devices and ~500,000\u20132,000,000 in MinION devices, depending on the RNA input type and quality of the flowcell  and TAIL-seq (Pearson\u2019s R\u2009=\u20090.19) on HeLa cell lines  and to estimate polyA tail lengths . However, a major limitation of NGS-based methods is their inability to assign a given polyA tail length to a specific transcript isoform, which causes a loss of isoform-specific tail length information. In addition, NGS-based methods cannot measure tail lengths greater than the read length, thus biasing our view of polyA tail dynamics to those transcripts that display shorter tail lengths.In the past few years, a variety of NGS-based high-throughput methods have been developed to characterize the 3\u2032 ends of RNA molecules at a transcriptome-wide scale, including methods to reveal polyA tail sites , or molecules with polyA tails shorter than 10 nucleotides, thus biasing the view of the transcriptome toward polyadenylated molecules. Customized dRNA-seq methods involving in vitro poly(G/I)-tailing have been developed to overcome some of these limitations, but a lack of bioinformatic tools to distinguish polyI and polyA signals limits their applicability to study polyA tail length differences across transcripts in these datasets15. In addition, dRNA-seq requires 500\u2009ng RNA as input, whereas Nano3P-seq requires as little as 50\u2009ng, thus decreasing the required input material by tenfold. Nano3P-seq addresses the current limitations by offering a simple and robust solution for studying the coding and non-coding transcriptome simultaneously regardless of the presence or absence of polyA tails or 3\u2032 tail composition, without PCR or ligation biases, and with single-read and single-isoform resolution. Moreover, the use of thermostable group II intron reverse transcriptase (TGIRT) in the Nano3P-seq protocol not only maximizes the production of full-length cDNAs, but also ensures the inclusion of RNA molecules that are highly structured and/or modified, which would often not be captured (or their representation would be significantly biased) using standard viral reverse transcriptases57.Nanopore dRNA-seq has been proposed as an alternative long-read sequencing technology for studying polyA tail lengthsNano3P-seq also provides quantitative measurements of RNA abundances Fig.  and captOverall, our work demonstrates that Nano3P-seq can simultaneously capture both non-polyA-tailed and polyA-tailed transcriptomes, making it possible to accurately quantify the RNA abundances and polyA tail lengths at per-read and per-isoform levels, while minimizing the amount of biases introduced during library preparation. These features set Nano3P-seq as a simple, potent, and robust method that can provide mechanistic insights into the regulation of RNA molecules and improve our understanding of mRNA tailing processes and post-transcriptional control.58  were in vitro transcribed using Ampliscribe T7-Flash Transcription Kit (Lucigen-ASF3507). Curlcake 2 was polyadenylated using Escherichia coli polyA Polymerase (NEB-M0276S). polyA-tailed RNAs were purified using RNAClean XP beads. The quality of the in vitro transcribed (IVT) products as well as the addition of polyA tail to the synthetic constructs was assessed using Agilent 4200 Tapestation  were loaded on a 2.5% agarose gel  and stained with GelRed . Product sizes determined using the GeneRuler 50-base-pair DNA ladder . Subsequently, tail- and gene-specific PCR products of mouse 16S rRNA were purified by gel-elution  and sent for Sanger sequencing with the shared forward primer (5\u2032-GGTCGGTTTCTATCTATTTACGATTTCTC-3\u2032). Resulting chromatograms were analyzed using SnapGene (v.6.0.2). After confirming alignment to the reference sequence, the unclipped chromatograms were used to visualize 3\u2032 ends.Poly(A) Tail-Length Assay Kit  was used according to the manufacturer\u2019s instructions. PCR products corresponding to the tail- and gene-specific primer combinations of mouse 16S rRNA  and human Saccharomyces cerevisiae (strain BY4741) was grown at 30\u2009\u00b0C in standard YPD medium . Cells were then quickly transferred into 50-ml pre-chilled falcon tubes, and centrifuged for 5\u2009min at 3,000g in a 4\u2009\u00b0C pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen. Flash frozen pellets were resuspended in 700\u2009\u00b5l TRIzol (Life Technologies) with 350\u2009\u00b5l acid washed and autoclaved glass beads . The cells were disrupted using a vortex on top speed for 7 cycles of 15\u2009s (the samples were chilled on ice for 30\u2009s between cycles). Afterwards, the samples were incubated at room temperature for 5\u2009min and 200\u2009\u00b5l chloroform was added. After briefly vortexing the suspension, the samples were incubated for 5\u2009min at room temperature. Then they were centrifuged at 14,000g for 15\u2009min at 4\u2009\u00b0C and the upper aqueous phase was transferred to a new tube. RNA was precipitated with 2\u00d7 volume molecular grade absolute ethanol and 0.1\u00d7 volume sodium acetate . The samples were then incubated for 1\u2009h at \u221220\u2009\u00b0C and centrifuged at 14,000g for 15\u2009min at 4\u2009\u00b0C. The pellet was then washed with 70% ethanol and resuspended with nuclease-free water after air drying for 5\u2009min on the benchtop. Purity of the total RNA was measured with the NanoDrop 2000 Spectrophotometer. Total RNA was then treated with Turbo DNase  (2\u2009\u03bcl enzyme for 50\u2009\u03bcl reaction of 200\u2009ng\u2009\u03bcl\u22121 RNA) at 37\u2009\u00b0C for 15\u2009min, with a subsequent RNAClean XP bead cleanup.2 and tissues were snap frozen in liquid nitrogen. Animals were kept on a 12:12\u2009h light:dark cycle and provided with water and food ad libitum.Experiments were performed with male mice aged between 8 and 10\u2009weeks. All mice were euthanized using COMus musculus) brain, we followed previously published protocols (10.17504/protocols.io.3fkgjkw) with minor changes. A quarter of a C57BL6/J mouse brain was used for this protocol, and all samples and reagents were kept on ice during the protocol. Brain tissue was mined with a razor blade into smaller pieces. Cold Nuclei EZ Lysis Buffer ) was added to the tissue in 1.5-ml eppendorf tube. The sample was homogenized using a 1-ml dounce (stroking ~10\u201320 times), and the homogenate was transferred into a 2-ml eppendorf tube. One milliliter of cold Nuclei EZ Lysis Buffer was added and mixed, followed by 4\u2009min incubation on ice. During the incubation, the sample was gently mixed a couple of times using a pipette. Homogenate was filtered using a 70-\u03bcm strainer mesh, and the flowthrough was collected in a polystyrene round-bottom FACS tube and subsequently transferred into a new 2-ml tube. The sample was centrifuged at 500g for 5\u2009min at 4\u2009\u00b0C and the supernatant was removed. The nuclei/mitochondria enriched sample was resuspended in another 1.5\u2009ml EZ Lysis buffer and incubated for 5\u2009min on ice. The sample was centrifuged at 500g for 5\u2009min 4\u2009\u00b0C and the supernatant was discarded (cytoplasm). Five-hundred microliters of nuclei wash and resuspension buffer ) was added to the sample and incubated for 5\u2009min without resuspending to allow buffer interchange. After incubation, 1\u2009ml nuclei wash and resuspension buffer was added and the sample was resuspended. The sample was centrifuged at 500g for 5\u2009min at 4\u2009\u00b0C. The supernatant was removed and only ~50\u2009\u03bcl was left. Using 1.4\u2009ml nuclei wash and resuspension buffer, the sample was resuspended and transferred to a 1.5-ml eppendorf tube. The last washing step was repeated and the pellet was resuspended in 300\u2009\u03bcL nuclei wash and resuspension buffer. RNA was extracted using TRIzol (Life Technologies) following the manufacturer\u2019s protocol.To isolate nuclear/mitochondrial-enriched RNA from the mouse (Danio rerio) embryos were obtained through natural mating of the TU-AB strain of mixed ages (5\u201318\u2009months). Mating pairs were randomly chosen from a pool of 60 males and 60 females allocated for each day of the month. Embryos and adult fish were maintained at 28\u2009\u00b0C.Wild-type zebrafish  and total RNA was extracted using the manufacturer\u2019s protocol. Total RNA concentration was calculated by nanodrop.For polyA-selected RNA samples, polyadenylated RNAs were isolated with oligo(dT) magnetic beads  according to the manufacturer\u2019s protocol and eluted in 30\u2009\u00b5l before nanodrop quantification.\u22121) to 37\u2009\u00b0C for hybridization. In the meantime, Dynabeads MyOne Streptavidin C1  beads were resuspended by carefully vortexing at medium speed. Eighty microliters of Dynabeads bead resuspension (10\u2009mg\u2009ml\u22121) was transferred into a tube, which then was placed on a magnetic rack. After aspirating the supernatant, 100\u2009\u03bcl of bead resuspension buffer  was added to the sample and beads were resuspended in this buffer by agitating the tube. Sample was placed on a magnet and the supernatant was aspirated. This step was performed twice. Beads were then resuspended in 100\u2009\u03bcl bead wash buffer (0.1\u2009M NaCl) and placed on magnet to aspirate the supernatant. Beads were then resuspended in a 160\u2009\u03bcl depletion buffer . This suspension was then divided into two tubes of 80\u2009\u03bcl, which will be used consecutively. Twenty microliters of hybridized riboPOOL and total RNA was briefly centrifuged to spin down droplets and it was pipetted into the tube containing 80\u2009\u03bcl of beads in depletion buffer . The tube containing the mix was agitated to resuspend the solution well. Then the mix was incubated at 37\u2009\u00b0C for 15\u2009min, followed by a 50\u2009\u00b0C incubation for 5\u2009min. Immediately before use, the second tube containing 80\u2009\u03bcl of beads was placed on a magnetic rack and the supernatant was aspirated. After the incubation at 50\u2009\u00b0C, the first depletion reaction was placed on a magnet and the supernatant was transferred into the tube containing the other set of beads. The mix was incubated again at 37\u2009\u00b0C for 15\u2009min, followed by a 50\u2009\u00b0C incubation for 5\u2009min. After briefly spinning down the droplets, the mix was placed on a magnet for 2\u2009min. The supernatant was transferred into a different tube and cleaned up using RNA Clean & Concentrator-5 .Ribodepletion was performed on zebrafish total RNA using riboPOOL oligos  following the manufacturer\u2019s protocol. In brief, 5\u2009\u03bcg total RNA in 14\u2009\u03bcl was mixed with 1\u2009\u03bcl resuspended riboPOOL oligos, 5\u2009\u03bcl hybridization buffer and 0.5\u2009\u03bcl SUPERase\u2022In RNase Inhibitor . The mix was incubated for 10\u2009min at 68\u2009\u00b0C, followed by a slow cool down (3\u2009\u00b0C\u2009minMycoplasma-tested in-house) were cultured in DMEM high glucose  with 10% fetal bovine serum supplement . Pellets were obtained from 6 million cells and 1\u2009ml TRIzol (Life Technologies) was added to each pellet, Sample was incubated at room temperature for a few minutes after adding 200\u2009\u03bcl chloroform and vortexing briefly. After the incubation, the solution was centrifuged at 4\u2009\u00b0C with 14,000g. Aqueous phase from the previous step was transferred to another eppendorf tube and equal amount of absolute ethanol  was added to the solution. This suspension was then transferred to an RNeasy Mini Spin Column from RNeasy Mini Kit , and total RNA was isolated following the manufacturer\u2019s protocol. Then, polyadenylated RNAs were isolated with Dynabeads Oligo(dT) 25  according to the manufacturer\u2019s protocol and eluted in 20\u2009\u00b5L. Concentration and quality of RNA was evaluated using a Nanodrop 2000 Spectrophotometer and an Agilent 4200 TapeStation, respectively.HeLa cell lines  in order to pre-anneal the oligos. Then, 50\u2009ng RNA was mixed with 2\u2009\u00b5l pre-annealed R_RNA\u2009+\u2009D_DNA oligo, 1\u2009\u03bcl 100\u2009mM dithiothreitol, 4\u2009\u00b5l 5\u00d7 TGIRT Buffer , 1\u2009\u00b5l RNAse Inhibitor Murine , 1\u2009\u00b5l TGIRT (InGex) and nuclease-free water up to 19\u2009\u00b5l. We should note that if 50\u2009ng are used as input, only 1\u2009\u00b5l TGIRT is needed, whereas if 100\u2009ng is used as input, 2\u2009\u00b5l TGIRT enzyme is needed. The reverse transcription mix was initially incubated at room temperature for 30\u2009min before adding 1\u2009\u00b5l 10\u2009mM dNTP mix. Then the mix was incubated at 60\u2009\u00b0C for 60\u2009min and inactivated by heating at 75\u2009\u00b0C for 15\u2009min before moving to ice. RNAse Cocktail  was added to the mix to digest the RNA, and the mix was incubated at 37\u2009\u00b0C for 10\u2009min. The reaction was then cleaned up using 0.8\u00d7 AMPure XP Beads . To be able to ligate the sequencing adapters to the first cDNA strand, 1\u2009\u00b5l 10\u2009\u00b5M CompA_DNA (5\u2032 GAAGATAGAGCGACAGGCAAGTGATCGGAAGA 3\u2032) was annealed to the 15\u2009\u00b5l cDNA in a tube with 2.25\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 2.25\u2009\u00b5l 0.5\u2009M NaCl and 2\u2009\u00b5l nuclease-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to anneal the complementary to the first-strand cDNA. Then, 22.5\u2009\u00b5l first-strand cDNA was mixed with 5\u2009\u00b5l Adapter Mix (AMX), 22.5\u2009\u00b5l Rnase-free water and 50\u2009\u00b5l Blunt/TA Ligase Mix  and incubated in room temperature for 10\u2009min. The reaction was cleaned up using 0.8\u00d7 AMPure XP beads, using WSB (Washing buffer) buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell. Libraries were run on either Flongle or MinION flow cells with MinKNOW acquisition software version v.3.5.5. A detailed step-by-step Nano3P-seq protocol is provided as a The protocol is based on the direct cDNA Sequencing ONT protocol (DCB_9091_v109_revC_04Feb2019), with several modifications to be able to perform TGIRT template switching. Before starting the library preparation, 1\u2009\u00b5l 10\u00b5M R_RNA (oligo: 5\u2032 rGrArArGrArUrArGrArGrCrGrArCrArGrGrCrArArGrUrGrArUrCrGrGrArArG/3SpC3/3\u2032) and 1\u2009\u00b5l 10\u2009\u00b5M D_DNA (5\u2032/5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCN 3\u2032) were mixed with 1\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 1\u2009\u00b5l 0.5\u2009M NaCl, 0.5\u2009\u03bcl RNAse Inhibitor Murine  and 5.5\u2009\u03bcl RNase-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to pre-anneal the oligonucleotides. Then, 50\u2009ng polyA-tailed RNA was mixed with 1\u2009\u00b5l pre-annealed VNP\u2009+\u2009CompA, 1\u2009\u03bcl 100\u2009mM dithiothreitol, 4\u2009\u00b5l 5\u00d7 TGIRT Buffer , 1\u2009\u00b5l RNAse Inhibitor Murine , 1\u2009\u00b5l TGIRT and nuclease-free water up to 19\u2009\u00b5l. The reverse transcription mix was initially incubated at room temperature for 30\u2009min before adding 1\u2009\u00b5l 10\u2009mM dNTP mix. Then the mix was incubated at 60\u2009\u00b0C for 60\u2009min and inactivated by heating at 75\u2009\u00b0C for 15\u2009min before moving onto ice. Furthermore, RNAse Cocktail  was added to the mix to digest the RNA and the mix was incubated at 37\u2009\u00b0C for 10\u2009min. Then the reaction was cleaned up using 0.8\u00d7 AMPure XP Beads . To be able to ligate the sequencing adapters the the first strand, 1\u2009\u00b5l 10\u2009\u00b5M CompA was again annealed to the 15\u2009\u00b5l cDNA in a tube with 2.25\u2009\u00b5l 0.1\u2009M Tris pH 7.5, 2.25\u2009\u00b5l 0.5\u2009M NaCl and 2\u2009\u00b5l nuclease-free water. The mix was incubated at 94\u2009\u00b0C for 1\u2009min and the temperature was ramped down to 25\u2009\u00b0C (\u22120.1\u2009\u00b0C\u2009s\u22121) to anneal the complementary to the first-strand cDNA. Furthermore, 22.5\u2009\u00b5l first-strand cDNA was mixed with 5\u2009\u00b5l Adapter Mix (AMX), 22.5\u2009\u03bcl Rnase-free water and 50\u2009\u00b5l Blunt/TA Ligase Mix  and incubated at room temperature for 10\u2009min. The reaction was cleaned up using 0.8\u00d7 AMPure XP beads, using WSB Buffer for washing. The sample was then eluted in elution buffer and mixed with sequencing buffer and loading beads before loading onto a primed R9.4.1 flowcell and run on a MinION sequencer with MinKNOW acquisition software version v.3.5.5.Some adjustments were made to the original Direct cDNA-Sequencing ONT protocol (SQK-DCS109), to be able to use TGIRT (InGex) as reverse transcription enzyme for nanopore sequencing, as this enzyme does not produce CCC overhang, which is typically exploited by the dcDNA-seq library preparation protocol Fig. . In brieA total of 12 synthetic cDNA standards were synthesized as ultramers by IDT (Integrated DNA Technologies) to assess the tail length estimation and tail composition quantification accuracy of Nano3P-seq.Synthetic cDNA standards designed to assess accuracy in tail length estimation:cDNA_pA_standard_0: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGCAACCCTATATAGAAcDNA_pA_standard_15: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGAAAAATGCTTGTTTGcDNA_pA_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_standard_60: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGcDNA_pA_standard_90: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGcDNA_pA_standard_120: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTSynthetic cDNA standards designed to assess accuracy in tail composition analyses:cDNA_p29A_pU1_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p27A_pU3_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAATTTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pU5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCAAAAATTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pC5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_p25A_pG5_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATGcDNA_pA_internalG_standard_30: /5Phos/CTTCCGATCACTTGCCTGTCGCTCTATCTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTGTAAATAGAAATAGACTAGCTCCACTTTTAAGAATTATTTATGCAATTAAATACATGGGTGACCAAAAGAGCGGGCGGATACACGCGTCACCACAAGCAGAATAAAAGGTAAACCTGAAATTGTTTTAACATAAAATG18 with minimap2 with \u2018-ax splice -k14 -uf --MD\u2019 parameters59. For zebrafish dRNA-seq samples, reads were base-called with Guppy v.4.0. Base-called reads were first mapped to maternal and somatic zebrafish ribosomal RNA sequences taken from42 and then to the genome (GRCz11) with minimap259 with \u2018-ax splice -k14 -uf --MD\u2019 parameters. Mapped reads were intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) using bedtools v.2.29.1 intersect option60.dRNA-seq library preparations were prepared following manufacturer\u2019s recommendations, using 450\u2009ng (in the case of polyA-enriched RNA zebrafish run) or 500\u2009ng (in the case of in vitro transcribed \u2018sequins\u2019) as input material. Samples were sequenced in an R 9.4.1 MinION flowcell using a GridION sequencing device in the case of \u2018sequins\u2019, and in a R 9.4.1 PromethION flowcell using a PromethION sequencing device in the case of zebrafish RNA. For sequins, reads were base-called using stand-alone Guppy v.3.0.3 with default parameters and then the base-called reads were mapped to sequin sequences59 with the following \u2018-ax splice -k14 -uf --MD\u2019 parameters when mapping to genome and \u2018-ax map-ont -k14 --MD\u2019 when mapping to transcriptome, unless stated otherwise.All the Nano3P-seq runs were base-called and demultiplexed using stand-alone Guppy v.6.0.1 with default parameters. All runs were mapped using minimap258, and mapped reads were then intersected with annotations of Curlcake 1 and 2 sequences to filter out the incomplete reads using bedtools v.2.29.1 intersect option. For yeast total RNA, we first mapped the base-called reads to S. cerevisiae ribosomal RNAs  and then mapped the rest of the reads to the S. cerevisiae genome (SacCer3). Mapped reads were then intersected with SacCer64 annotation exon ends, to filter out incomplete reads. For the analysis of nuclear/mitochondrial-enriched mouse brain RNA spiked in with sequins18, we first mapped the base-called reads to Mus musculus ribosomal RNAs  and then mapped the rest of the rest of the reads to the M. musculus genome (GRCm38), supplemented with sequin chromosome (chrIS). Mapped reads were then intersected with ENSEMBL version 102 annotation (Mus_musculus.GRCm38.102.gtf) and sequin annotation (RNAsequins.v2.2.gtf) exon ends, to filter the incomplete reads. For zebrafish RNA (polyA-selected and ribodepleted), we first mapped the base-called reads to ribosomal RNAs and then mapped the rest of the reads to the genome (GRCz11). Mapped read starts were then intersected with ENSEMBL version 103 annotation (Danio_rerio.GRCz11.103.2.gtf) exon ends, to filter the incomplete reads. For HeLa mRNA, we first mapped the base-called reads to human ribosomal RNAs ; then, the rest of the reads (unmapped) were mapped to the Homo sapiens genome (GRCh38). Mapped read starts were then intersected with ENSEMBL version 107 annotation (Homo_sapiens.GRCh38.107.gtf) exon ends, to filter out incomplete reads . For cDNA standards, base-called reads were mapped to cDNA sequence reference using minimap2, except for the cDNA_pA_standard_120, which was mapped using bwa short-read aligner61 with the following parameters \u2018mem -xont2d\u2019. A different aligner was used for cDNA_pA_standard_120 because minimap2, which is a long-read mapping algorithm, did not yield any mapped reads for this standard owing to its short length62 (30 nucleotides once the pA tail length has been soft-clipped). We should note that the use of a different aligner should not affect the polyA tail length estimations of the reads, as these are done at the level of current intensity . We should note that fewer reads were base-called and mapped to cDNA_pA_standard_120, cDNA_pA_standard_90 and cDNA_pA_standard_60, in comparison to the other cDNA standards , base-called reads were mapped to Curlcake 1 and 2 sequenceshttps://github.com/ablab/IsoQuant) was used with Danio_rerio.GRCz11.103.2.gtf annotation using the following parameters \u2018--genedb gtf_file --complete_genedb --bam bam_file --data_type nanopore -o output\u2019. A complete step-by-step command line of the bioinformatic analysis done on Nano3P-seq datasets can be found in the GitHub repository . All reference sequences used to map the runs mentioned above are included in the Nano3P-seq GitHub repository.For the assignment of reads to isoforms, IsoQuant package ; rather, they estimate polyA tail lengths by comparing the relative duration of the current signal corresponding to the polyA tail region to the total duration of the sequenced read.For dRNA-seq reads, polyA tail length estimation was performed using NanoTail, a module from Master of Poreshttps://github.com/adnaniazi/tailfindr/tree/nano3p-seq). All code used to estimate polyA tail lengths and post-process Nano3P-seq data can be found at https://github.com/novoalab/Nano3P_Seq.For Nano3P-seq reads, polyA tail length estimation was performed using the Nano3P-seq version of tailfindR (https://github.com/rrwick/Porechop) with the following parameters \u2018--extra_end_trim 0 --end_threshold 50\u2019, to remove the adapter sequences. Because Porechop sometimes failed at removal of the adapter sequences, only reads containing more than 80% A bases in their tail composition were kept for downstream analyses, thus ensuring that untrimmed reads are not included in downstream analyses .Base-called reads mapped to the zebrafish genome and cDNA standards for tail composition quantification were first trimmed using the Porechop tool . Mice maintenance was approved by the Garvan/St Vincent\u2019s Hospital Animal Ethics Committee, in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (Project No. 16/14 and 16/26). All animals were entered into the study in a randomized order and operators were blinded to genotype and treatments.Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41592-022-01714-w.Supplementary InformationSupplementary Protocol, Supplementary Tables 1\u20133, and Supplementary Notes 1\u20132.Reporting SummaryPeer Review FileSupplementary SoftwareCode corresponding to TailfindR Nano3P-seq branch found in GitHub repository (attached as a separate zip file). This version incorporates the custom adapter sequences that are used in Nano3P-seq library preparation protocols."}
+{"text": "The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP\u2019s suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25. The type I interferon (IFN) response is one of the first lines of cellular defense against invading pathogens. The IFN-induced zinc finger antiviral protein (ZAP) is a potent inhibitor of diverse RNA and DNA viruses . ZAP encEarlier efforts to elucidate ZAP RNA binding activity showed that ZAP binds RNA with its four N-terminal CCCH ZnFs, and mutations of ZnFs 2 and 4 most dramatically reduce ZAP antiviral activity . The firWhile much attention has been given to characterizing ZAP RNA binding, less has been given to its critical co-factors such as TRIM25. TRIM25 was identified as a ZAP co-factor in the context of inhibiting alphavirus translation . HoweverWhile some have attempted to illuminate the contribution of ZAP or TRIM25 RNA binding to the ZAP-TRIM25 interaction, these studies have focused on only a few select mutations. Still, most agree that RNA binding in either ZAP or TRIM25 is not required for the ZAP-TRIM25 interaction. One group demonstrated not only that RNase A treatment has little effect on the ZAPL-TRIM25 interaction, but also that an example ZAPL RNA binding mutant retains and even increases its interaction with TRIM25 . AnotherIn light of the recent novel structural insights, we asked how different ZAP and TRIM25 RNA binding mutations affect the ability of ZAP and TRIM25 to interact with one another and to restrict SINV and JEV translation. We curated a panel of ZAP and TRIM25 RNA binding mutants from prior studies, including mutants with a range of RNA binding and antiviral capabilities . We firsTo investigate the role of ZAP and TRIM25 RNA binding in the context of viral translation, we generated a panel of constructs with mutations previously demonstrated to impact RNA binding. For ZAP, each of the following mutations was introduced in both ZAPS and ZAPL to probe potential isoform differences in RNA binding and antiviral function. These mutations fall into two general categories: 1) ZnF mutants that individually disrupt each CCCH motif and 2) CpG RNA binding cavity mutants. We made four individual mutations to disrupt each N-terminal CCCH ZnF: H86K, C88R, C168R, and H191R, which are located in ZnF 1, 2, 3, and 4, respectively . Two putFor TRIM25, we generated two constructs, one in which we replaced the lysine-rich motif in the L2 linker with alanines (TRIM25 7KA) and one in which we deleted the previously identified RNA binding domain in the PRY/SPRY domain (TRIM25 \u0394RBD) .in vitro RNA pull-down assay. We incubated lysates of cells transfected with ZAP or TRIM25 mutants with biotin-labeled SINV (Toto1101 strain) genomic RNA, allowing for RNA and bound protein to be immunoprecipitated using streptavidin beads and probed for the presence of bound ZAP or TRIM25. As a negative control, we also assessed the ability of the WT constructs to bind firefly luciferase (Fluc) RNA. We quantified the resultant ZAP and TRIM25 bound to RNA and normalized to input ZAP and TRIM25 protein levels with ImageJ   . Becausee (Rluc) . We attee (Rluc) .We found that the ZAP and TRIM25 RNA binding mutants inhibit JEV translation to varying degrees Figure\u00a05Upon assaying TRIM25 variants, we found that TRIM25 \u0394RBD inhibits JEV translation similarly to TRIM25 WT  and its parental 293T lines . TRIM25 Infections with SINV expressing firefly luciferase (Toto1101/Luc) and temperature-sensitive SINV (Toto1101/Luc:ts6) have been previously described . Each inBamHI and HindIII restriction sites. ZAPS and ZAPL were cloned into pcDNA3.1-3XFLAG from pTRIP-TagRFP-hZAPS and pTRIP-TagRFP-hZAPL \u201d, to replace wild-type sequence in TRIM25. The 7KA mutated sequence is bolded in the below gene block, and nonessential nucleotides on the 5\u2019 and 3\u2019 ends are written in lowercase.Point mutations in ZAPS and ZAPL were generated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and all plasmids were verified by sequencing (Genewiz). Primers for mutations were synthesized by Integrated DNA Technologies  was genealanines , and utiTGTACAGTCAGATCAACGGGGCGTCGAGAGCACTGGATGATGTGAGAAACAGGCAGCAGGATGTGCGGATGACTGCAAACAGAAAGGTGGAGCAGCTACAACAAGAATACACGGAAATGAAGGCTCTCTTGGACGCCTCAGAGACCACCTCGACAAGGAAGATAAAGGAAGAGGAGAAGAGGGTCAACAGCAAGTTTGACACCATTTATCAGATTCTCCTCAAGAAGAAGAGTGAGATCCAGACCTTGAAGGAGGAGATTGAACAGAGCCTGACCAAGAGGGATGAGTTCGAGTTTCTGGAGAAAGCATCAAAACTGCGAGGAATCTCAACAAAGCCAGTCTACATCCCCGAGGTGGAACTGAACCACAAGCTGATAAAAGGCATCCACCAGAGCACCATAGACCTCAAAAACGAGCTGAAGCAGTGCATCGGGCGGCTCCAGGAGCCCACCCCCAGTTCAGGTGACCCTGGAGAGCATGACCCAGCGTCCACACACAAATCCACACGCCCTGTGGCAGCAGTCTCCGCAGAGGAAGCAGCATCCGCAGCACCTCCCCCTGTCCCTGCCTTACCCAGCAAGCTTCCCACGTTTGGAGCCCCGGAACAGTTAGTGGATTTAAAACAAGCTGGCTTGGAGGCTGCAGCCAAAGCCACCAGCTCACATCCGAACTCAACATCTCTCAAGGCCAAGGTGCTGGAGACCTTCCTGGCCAAGTCCAGACCTGAGCTCCTGGAGTATTACATTAAAGTCATCCTGGACTACAACACCGCCCACAACAAAGTGGCTCTGTCAGAGTGCTATACAGTAGCTTCTGTGGCTGAGATGCCTCAGAACTACCGGCCGCATCCCCAGAGGTTCACATACTGCTCTCAGGTGCTGGGCCTGCACTGCTACAAGAAGGGGATCCgttt-3\u20195\u2019gtttX-tremeGENE9 DNA Transfection Reagent (Roche Life Science) was used to transfect cells at a ratio of 3 \u03bcL to 1 \u03bcg DNA according to the manufacturer\u2019s instructions. To keep the total plasmid amount in co-transfections constant, empty vectors pcDNA3.1-myc or 3XFLAG were transfected as necessary .XhoI linearization of pToto1101 (in vitro by Sp6 RNA polymerase (New England Biolabs) in the presence of the cap analog [m7G(5\u2019)ppp(5\u2019)G] (New England Biolabs). Fluc DNA templates for transcription were amplified from the pGL3-Control plasmid (Promega). Fluc RNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Biotin-labeled RNAs were generated by adding 10mM biotin-16-UTP (Roche Life Science) to in vitro transcription reactions. JEV replicon DNA templates for transcription were generated by XhoI linearization and transcribed in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). Transcribed RNAs were purified using the Quick-RNA Miniprep Kit (Zymo Research) and biotinylation was confirmed by streptavidin dot blot  and incubated with RNA probes in a final volume of 100 \u03bcL RNA binding buffer supplemented with 1 unit/\u03bcL RNAseOUT (Thermo Fisher Scientific), 1 \u03bcg/\u03bcL heparin (Sigma-Aldrich), and 100 ng/\u03bcL yeast tRNA (ThermoFisher) for 30\u00a0min at 30\u00b0C. Lysate-RNA mixtures were then incubated with 300 \u03bcL of Dynabeads M-280 Streptavidin (Invitrogen) for 30\u00a0min at room temperature on a shaker. Protein-RNA complexes were washed three times by RNA binding buffer, and proteins were eluted by incubation with 30 \u03bcL of 4x Laemmli Sample Buffer (Bio-Rad) for 5\u00a0min at 95\u00b0C. The proteins were further analyzed by immunoblot and quantified by ImageJ as previously described  and 4-15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:2,500 anti-myc , 1:20,000 anti-FLAG (Sigma-Aldrich), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch) or 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific). Proteins were visualized on a ChemiDoc (Bio-Rad) using ProSignal Pico ECL Reagent (Genesee Scientific). ImageJ was used to quantify western blots as previously described . BrieflyTo assess ZAP or TRIM25 co-immunoprecipitation (co-IP) with RNA binding mutants of TRIM25 or ZAP, respectively, cells were transfected in 6-well plates, collected, and then lysed by rotating in FLAG IP buffer  supplemented with a complete protease inhibitor cocktail (Roche Life Science) at 4\u00b0C for 30\u00a0min, before spinning down at 14,000 rpm at 4\u00b0C for 15\u00a0min. To equilibrate beads prior to use, anti-FLAG beads  or anti-myc beads  were washed 3 times in FLAG IP buffer. Three hundred \u03bcL of whole cell lysate (WCL) were incubated with 30 \u03bcL of anti-FLAG or -myc beads rotating at 4\u02daC for 45 minutes. FLAG IP buffer was used to wash immunoprecipitates 3 times before eluting bound proteins with SDS loading buffer, and boiling for 5 minutes for immunoblot analysis. Western blot ImageJ analysis was performed as previously described . BrieflyTransIT-mRNA Transfection Kit (Mirus Bio). Cells were lysed 4 hours post-transfection of replicon RNA and luciferase activity was measured by Dual-Luciferase Reporter Assay (Promega). Each independent experiment included triplicate wells of biological replicates per condition.Following transfection of ZAP or TRIM25 constructs into ZAP or TRIM25 KO 293T for 48 hours, JEV replicon RNA was transfected by Statistical analyses in The original contributions presented in the study are included in the article/EY, LN, and ML conceptualized and designed the study. CW and RK assisted in cloning ZAP and TRIM25 RNA binding mutants. LN performed RNA binding and JEV replicon experiments, while EY performed co-IP and SINV replication and translation experiments. LN performed the correlation analysis. EY and LN co-wrote the first draft of the manuscript. ML provided critical feedback. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported in part by NIH R01AI158704 (ML), UCLA AIDS Institute and Charity Treks 2019 Seed Grant (ML), Ruth L. Kirschstein Multidisciplinary Training Grant in Microbial Pathogenesis , Ruth L. Kirschstein Cellular and Molecular Biology Training Program , Warsaw Fellowship (EY), and Whitcome Fellowship .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Currently, a total of 81 corticalis group species are known worldwide, amongst them 67 were recorded from China. However, the diversity of this group in China is still insufficiently known.The Clubionaxianningsp. nov. is described as a new species of the C.corticalis species-group collected from Hubei Province, China. Clubiona Latreille, 1804 currently contains 518 catalogued species that are found worldwide, except for the Polar Regions and South America .OP675437; YHCLU0273, \u2640, GenBank OP675436.A DNA barcode was also obtained for species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019) . For addAll measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follows All specimens are deposited Museum of Guizhou Normal University, Guiyang, Guizhou, China.Zhong & Yusp. n.B0CC6F23-8DFB-5B4A-BAD0-E8AC5114D6878DFF1CC4-1C1A-46A6-8300-FF36FFFA2644Type status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0272; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675437GenBank: ; occurrenceID: 1312422A-3320-5AE6-9BA9-5DCB3013B14F; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenType status:Holotype. Occurrence: recordedBy: Yang Zhong, Xusheng Gong, Qianle Lu; individualID: YHCLU0273; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (ETOH); associatedSequences: OP675436GenBank: ; occurrenceID: 085B9104-F139-5EC1-9913-5A5DB9C7C9C6; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: xianning; scientificNameAuthorship: Zhong & Yu; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; municipality: Xianning; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2022-05; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: basisOfRecord: PreservedSpecimenMale 5'TTCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' .5'TCTGGTCAGCTATAGTTGGTACAGCTATAAGAGTTATAATTCGTATAGAATTAGGTCAATCTGGAGCTTTTTTAGGTGATGATCATTTGTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGGTTTGGAAATTGATTAGTTCCATTAATATTAGGGGCAGGTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGACTTTTACCACCTTCATTAATATTATTAGTTATATCATCTATGGCTGAGATGGGAGTTGGGGCTGGATGAACAGTTTATCCCCCTCTTGCTTCTTTAGTAGGTCATACGGGAAGAGCAATGGATTTTGCTATTTTTTCATTACATTTAGCTGGGGCTTCTTCTATTATAGGAGCTGTTAATTTTATTACTACTATTATGAATATACGATCTTTTGGAATAATAATGGAAAAGATTTCATTATTTGTTTGGTCTGTTTTAATTACAGCTATTTTATTATTATTATCTTTGCCAGTTTTAGCCGGGGCTATTACTATATTATTAACTGATCGTAATTTTAATACGTCTTTTTTTGACCCTGCTGGGGGAGGTGATCCTATTTTATTTCAACATTTATTTTGATTTTTTGGTCACCC3' , differ from C.altissimus by: (1) atrium large, nearly as wide as epigynal plate  (cf. Fig. C.xianningsp. nov. also can by separated from C.caohai and C.altissimus by their habitus: abdomen without distinct colour pattern in C.xianningsp. nov. (Fig. C.caohai and C.altissimus (Male of the new species resembles that of Yu, 2020 : fig. 2DYu, 2020 : fig. 2Bov. Fig. E\u2013H, but The specific name refers to the type locality and is a noun in apposition.Known from the Mt. Jiugong, Hubei Province, China Fig. ."}
+{"text": "P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with tumor, node, and metastasis (TNM) stage I (P < 0.05). Expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). Of the lung cancer tissues of patients with different TNM stages, only six patients had high expression, and the remaining 104 patients had low expression. Moreover, electrophoresis revealed that circHIPK3 can only be amplified in cDNA, but not in gDNA. Gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably. In summary, the circular RNA circHIPK3 may have a notably low expression in lung cancer tissues, whose low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib.To study the mechanism of circular ribonucleic acid (RNA) circHIPK3 involved in the resistance of lung cancer cells to gefitinib, 110 patients with lung cancer were recruited as the research objects, and the tumor tissue and para-cancerous tissue of each patient's surgical specimens were collected and paraffinized to detect the expression of circHIPK3 in different tissues. Gefitinib drug-resistant cell line of lung cancer was constructed with gefitinib to detect cell apoptosis under different conditions. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3\u2009cm was dramatically inferior to that in patients with tumor diameter less than 3\u2009cm ( Lung cancer is a malignant tumor, mainly originated from the mucosal epithelium of the bronchus, which is classified into small cell carcinoma and nonsmall cell carcinoma according to the histological changes. In recent years, with the serious air pollution and the gradual deterioration of the environment, the global mortality rate of lung cancer is on the rise, seriously threatening people's health . AccordicircRNA is a new type of RNA molecule that is characterized by a covalent closed loop and widely exists in eukaryotes . circRNAIn recent years, related studies found that using siRNA to knockdown circHIPK3 can inhibit cell proliferation. In addition, circHIPK3 can adsorb miR-124 in liver cancer and promote the proliferation of liver cancer cells by regulating the expression of target genes. However, its regulation and expression mechanism in lung cancer is still unclear. Therefore, this study was developed to explore the mechanism of the circular RNA circHIPK3 involved in the resistance of lung cancer cells to gefitinib.In this study, 110 lung cancer patients admitted to X Hospital from September 2019 to September 2020 were collected as the research objects. Paraffin specimens were collected from 58 male patients and 52 female patients. The study has been approved by the Medical Ethics Committee. Patients and their families understood the study content and methods of specimen acquisition and agreed to sign corresponding informed consent forms.Inclusion criteria: (i) patients diagnosed with lung cancer by pathology and imaging; (ii) patients aged between 45 and 75 years; (iii) there was no metastasis of lung lesions, mediastinal lymph node enlargement, or pleural hypertrophy; (iv) patients who were not recently treated with other drugs or antibiotics in the study; (v) patients with normal coagulation function and platelets.Exclusion criteria: (i) patients with diseases of other systems or organs; (ii) patients who had not received cooperative treatment due to personal or other factors; (iii) patients with incomplete clinical data and history information , etc.).\u03bcm/slice) and stored in an enzyme-free centrifuge tube  containing xylene .Paraffin sections of tumor tissue and para-cancer tissue  from surgical specimens of each included subject were collected. The collected paraffin samples were sliced with uniform thickness  were added. After the tissue was dissolved, it was bathed in water at 95\u00b0C for 10\u2009min and centrifuged at 8,600\u2009rpm at room temperature for 5\u2009min. The supernatant was absorbed and transferred to a new Rnase-free centrifuge tube. 220\u2009\u03bcL buffer RB was added, shaken, and mixed, then added with anhydrous ethanol, and mixed again.Sectioning: the collected paraffin specimens were sectionalized with a thickness of 10\u2009\u03bcL solution and precipitate were absorbed and added to the adsorption column, centrifuged at high speed for 1\u2009min. The waste solution was filtered and repeated several times until the solution and precipitate passed through the adsorption column. 80\u2009\u03bcL DNase I working solution was added to the center of the adsorption column and left for 15\u2009min at room temperature. 500\u2009\u03bcL protein-free RW1 was added and centrifuged, then added with rinse solution, centrifuged again, and the waste solution was discarded. After drying, the adsorption column was transferred to a new RNase-free centrifuge tube. 35\u2009\u03bcL RNase-free ddH2O was added and centrifuged at 8,600\u2009rpm for 5\u2009min to obtain RNA solution. RNase R was used to process the extracted total RNA to avoid artifacts. RNase R processing reaction system: 2\u2009\u03bcg RNA, 1\u2009\u03bcL RiboLock, 2\u2009\u03bcL 10x RNase R reaction buffer, and 1\u2009\u03bcL RNase R, with sterile water supplementing the system to 20\u2009\u03bcL [\u03bcL 5\u2009\u00d7\u2009PrimeScript Buffer, 0.5\u2009\u03bcL enzyme mix I, 0.5\u2009\u03bcL random 6 mers, and 7\u2009\u03bcL total RNA were added to a 0.2\u2009mL enzyme-free EP tube to configure a 10\u2009\u03bcL system. The EP tube was closed and shaken for 15\u2009s to mix, centrifuged, and put in the reverse transcription machine. The reverse transcription conditions: 37\u00b0C for 15\u2009min, 85\u00b0C for 5\u2009sec. After the reaction, the synthesized cDNA was transferred to -20\u00b0C refrigerator for storage. The length of circHIPK3 searched on NCBI was 1,099\u2009bp, and the specific base sequence was as follows: GTATGGCCTCACAAGTCTTGGTCTACCCACCATATGTTTATCAAACTCAGTCAAGTGCCTTTTGTAGTGTGAAGAAACTCAAAGTAGAGCCAAGCAGTTGTGTATTCCAGGAAAGAAACTATCCACGGACCTATGTGAATGGTAGAAACTTTGGAAATTCTCATCCTCCCACTAAGGGTAGTGCTTTTCAGACAAAGATACCATTTAATAGACCTCGAGGACACAACTTTTCATTGCGACAAGTGCTGTTGTTTTGAAAAACACTGCAGGTGCTACAAAGGTCATAGCAGCTCAGGCACAGCAAGCTCACGTGCAGGCACCTCAGATTGGGGCGTGGCGAAACAGATTGCATTTCCTAGAAGGCCCCCAGCGATGTGGATTGAAGCGCAAGAGTGAGGAGTTGGATAATCATAGCAGCGCAATGCAGATTGTCGATGAATTGTCCATACTTCCTGCAATGTTGCAAACCAACATGGGAAATCCAGTGACAGTTGTGACAGCTACCACAGGATCAAAACAGAATTGTACCACTGGAGAAGGTGACTATCAGTTAGTACAGCATGAAGTCTTATGCTCCATGAAAAATACTTACGAAGTCCTTGATTTTCTTGGTCGAGGCACGTTTGGCCAGGTAGTTAAATGCTGGAAAAGAGGGACAAATGAAATTGTAGCAATCAAAATTTTGAAGAATCATCCTTCTTATGCCCGTCAAGGTCAAATAGAAGTGAGCATATTAGCAAGGCTCAGTACTGAAAATGCTGATGAATATAACTTTGTACGAGCTTATGAATGCTTTCAGCACCGTAACCATACTTGTTTAGTCTTTGAGATGCTGGAACAAAACTTGTATGACTTTCTGAAACAAAATAAATTTAGTCCCCTGCCACTAAAAGTGATTCGGCCCATTCTTCAACAAGTGGCCACTGCACTGAAAAAATTGAAAAGTCTTGGTTTAATTCATGCTGATCTCAAGCCAGAGAATATTATGTTGGTGGATCCTGTTCGGCAGCCTTACAGGGTTAAAGTAATAGACTTTGGGTCGGCCAGTCATGTATCAAAGACTGTTTGT TCAACATATCTACAATCTCGGTACTACAG.700\u2009to 20\u2009\u03bcL . The insThe designed upstream and downstream primers were as follows: circHIPK3-F: TAGACTTTGGGTCGGCCAGT; circHIPK3-R: TGGAATACACAACTGCTTGGC.The expression of circHIPK3 was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). circHIPK3 is derived from the HIPK3 gene and consists of exon 2  head-to-tail splicing. Head-to-tail splicing can be generated by trans-splicing or genome rearrangement. The sequence is a circular sequence, which is assembled from the first position of the corresponding linear RNA, and a new unique sequence is generated at the reverse splicing site. The circRNA specific primers were designed with this as the target sequence, so as to specifically recognize circRNA and distinguish circular sequence from linear RNA. The identification and location of circHIPK3 was shown in Serum-free cell freezing medium (RPMI) 1640 cell culture medium consisted of 10% fetal bovine serum (FBS) , 100\u2009U/mL penicillin  and 100\u2009mg/mL streptomycin . The cell cryopreservation tube was taken out of the liquid nitrogen and quickly melted in a constant temperature water bath at 37\u00b0C. The thawed cell cryopreservation solution was transferred to a centrifuge tube containing cell culture medium and centrifuged at high speed for 5\u2009min. Then, after cell culture medium was added to the lower sediment to resuspend the cells, they were transferred to RPMI medium and incubated at constant temperature. The medium was changed once the cells grew adherently.After the cell coverage in the culture dish reached more than 80%, passaging began. After the cell culture solution was aspirated, trypsin digestion solution was added, and cells were observed under a microscope. After culturing until the cells became round, culture medium was added to stop the digestion, and the cells were resuspended and passed to a culture dish.50) of gefitinib on sensitive cells was 1.78\u2009mol/L. Then, the culture medium containing 17.5\u2009\u03bcmol/L gefitinib was used for 24\u2009h, and the medium containing half of the inhibitory concentration gefitinib was immediately replaced for 5-10\u2009d, until the cells grew steadily and were subcultured for 3 times. IC50 was determined again. The medium containing 17.5\u2009\u03bcmol/L gefitinib was cultured for 24\u2009h, and the medium with gradually increased inhibitory concentration of gefitinib was replaced until the cells grew statically in the medium containing 17.5\u2009\u03bcmol/L gefitinib and were continuously passed for 3 times. Then, the IC50 of gefitinib was 34.5\u2009\u03bcmol/L and was named A549/GR.Gefitinib was dissolved in dimethyl sulfoxide (DMSO) (Abis (Shanghai) Biotechnology Co., Ltd.). The methyl tetrazolium (MTT) assay was used to determine the median lethal concentration of gefitinib on lung cancer cells . In this study, human lung adenocarcinoma cell line A549 was used to induce gefitinib-resistant cell lines. Resistant cells were induced by a combination of high-dose shocks and gradually increased doses. MTT assay was used to determine the 50% inhibitory concentration  was added to each well for 4\u2009h, and the culture was terminated. The supernatant cultured in the hole should be carefully sucked and discarded. For suspended cells, the supernatant should be discarded after centrifugation. 150\u2009\u03bcL dimethyl sulfoxide (DMSO) was added to each well and shaken to dissolve completely. At 490\u2009nm, the light absorption value (OD) of each hole was measured by enzyme-linked immunosorbent assay (ELISA) and recorded. The cell growth curve was plotted with time as abscissa and light absorption value as ordinate.Single cell suspension was prepared with culture medium containing 10% fetal bovine serum and inoculated into 96-well plates with 1,000-10,000 cells per well, and volume of each well was 200\u2009P < 0.05.SPSS 19.0 was employed for data statistics and analysis. Mean \u00b1 standard\u2009deviation (P > 0.05). There was also no considerable difference in the relative expression of circHIPK3 between patients less than 60 years old and patients no less than 60 years old (P > 0.05).P < 0.05). The relative expression of circHIPK3 in patients with T stage II/III tumors was dramatically inferior to that in patients with T stage I (P < 0.05).P < 0.05). The relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05).The circular RNA circHIPK3 is widely and highly expressed in lung cancer cells, mainly located in the cytoplasm, but also in the nucleus. The expression of circHIPK3 in cancer tissues and normal lung tissues adjacent to cancer is notably different, so it is used for the diagnosis of lung cancer. With pathological and imaging findings as diagnostic criteria, The 110 lung cancer patients included in this study were graded into stage I, stage II, and stage III according to the TNM staging. Among them, 39 patients were in TNM stage I, 36 were in stage II, and 35 were in stage III. The circular RNA circHIPK3 was amplified by designing primers and then electrophoresed on agarose gel plate to get the electrophoresis, as shown in The surface of normal cells is composed of lipids distributed asymmetrically on the inner and outer lobes of the plasma membrane. Phosphatidylserine is mostly distributed in the inner lobes of the plasma membrane and exposed to the cytoplasm . HoweverGefitinib can specifically block a certain pathway of lung cancer cells, block the conduction, infiltration, and metastasis of lung cancer cells, locate and kill cancer cells, and cause little damage to normal human cells , 26. It P < 0.05). The relative expression of circHIPK3 in patients with TNM stage II/III was dramatically inferior to that in patients with TNM stage I (P < 0.05). In addition, the relative expression of circHIPK3 in patients with lymph node metastasis was dramatically inferior to that in patients without lymph node metastasis (P < 0.05). The results indicated that the relative expression of circHIPK3 in the tumor tissues of patients with lung cancer was closely related to the diameter of the patient's tumor, the TNM stage, and the occurrence of lymph node metastasis. However, there was no high correlation with the patient's age, gender, and other information. Among lung cancer tissues of patients with different TNM stages, only 6 patients had high expression, and the remaining 104 patients had low expression. Therefore, circHIPK3 may have a notably low expression in lung cancer tissues and related cell lines, which had a high correlation with the tumor stage of patients. Flow cytometry apoptosis detection results found that gefitinib-mediated apoptosis rate of lung cancer drug-resistant cell lines decreased notably, which was similar to Zhou et al.'s (2020) [In this study, the tumor tissues and para-cancerous tissues from surgical specimens of 110 study subjects were collected to detect differences in the expression of circHIPK3 in different tissues. Moreover, a lung cancer gefitinib drug-resistant cell line was constructed, and cell apoptosis was detected under different conditions, to study the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. As a result, the relative expression of circHIPK3 in patients with tumor diameter no less than 3\u2009cm was dramatically inferior to that in patients with tumor diameter less than 3\u2009cm (s (2020)  researchIn this study, tumor tissues and adjacent tissues were collected from surgical specimens of lung cancer patients to detect differences in the expression of circHIPK3 in different patients and different tumor tissues. In addition, a lung cancer gefitinib drug-resistant cell line was constructed to detect cell apoptosis under different conditions. The results showed that the circular RNA circHIPK3 may have a considerably low expression in lung cancer tissues, and its low expression had a certain enhancement effect on the drug resistance of lung adenocarcinoma cells to gefitinib. However, the sample size selected in this study is small, and the representativeness is low. Therefore, the selection of test sample size will be increased in subsequent experiments. Further study is required to clarify the mechanism of circRNA involved in the resistance of lung cancer cells to gefitinib. In short, this study provides a certain theoretical basis and data support for the drug treatment of lung cancer."}
+{"text": "In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing. The mechanism by which RNAPII transcribes the DNA in the chromatosome with H1 has remained enigmatic. Here the authors present the cryo-EM structures of the RNAPII-chromatosome complexes, and explain how RNAPII is regulated by H1 in chromatin. NCPs are connected by linker DNA segments, and appear as a beads-on-a-string fiber2.Chromatin compacts genomic DNA in eukaryotes, and the nucleosome is an elemental architecture. The nucleosome is composed of linker DNAs and the nucleosome core particle (NCP), which folds an approximately 150 base-pair DNA segment into a histone octamer, comprising the four core histones H2A, H2B, H3, and H4. In the NCP, the DNA is symmetrically wrapped 1.7-turns around the histone octamer, and the central region of the nucleosomal DNA is located on the dyad axis3, and promote further compaction of chromatin7. H1 consists of three domains, the N-terminal disordered region (\u223c35 residues), the central globular domain (\u223c80 residues), and the C-terminal disordered region (\u223c100 residues), and in the chromatosome, these domains mainly bind to a linker DNA, the DNA around the dyad axis, and another linker DNA, respectively11. This tripartite nucleosome binding by H1 is termed \u201con-dyad\u201d binding, and it compacts the nucleosome architecture by restricting the linker DNA orientation and flexibility14. H1 also binds the nucleosome in an \u201coff-dyad\u201d mode, in which the central globular domain mainly binds to a linker DNA16.Linker histones, such as H1, are major chromatin components that specifically bind to the nucleosomal DNA forming the chromatosome17. H1 binding to the nucleosome induces structural changes in chromatin, thus regulating transcription processes catalyzed by RNA polymerase II (RNAPII)5. For instance, a biochemical study with Drosophila egg extracts indicated that H1 binding to reconstituted chromatin represses RNAPII transcription18. A mouse knock-out study revealed that embryos with an H1 ratio reduced to approximately 50% cease development at mid-gestation19. H1 reportedly functions in gene regulation through chromatin compaction and 3D genome organization20. Consistently, acute depletion of H1 leads to perturbations of gene expression in the constitutive heterochromatin in mouse embryonic stem cells21. H1 alleles have been identified as genes with driver mutations in lymphoma, revealing that H1 is a bona fide tumor suppressor22. These facts indicate that H1 plays pivotal roles in development, differentiation, and tumorigenesis by appropriately reducing transcription efficiency according to the cellular status.H1 is the most abundant nucleosome binding protein, and exists in a 50\u2013130% amount relative to nucleosomes5. H1.2 is the major somatic linker histone. Genome-wide studies revealed that the genomic H1.2 binding loci negatively correlate with the RNAPII transcription activation, suggesting the repressor function of H1.225. In contrast, genetic and biochemical studies demonstrated that H1.2 potentially upregulates gene expression30. Therefore, H1.2 may function to both repress and activate RNAPII transcription5, probably depending on cell types and genomic loci. These different outcomes may be the results of complex transcription regulation at the levels of transcription initiation and elongation, but the direct effects of H1 on transcription are not well understood.Eleven non-allelic human H1 isoforms have been described, and they may have specific functions in altering the cellular transcription status, primarily by repressing transcription by RNAPII32. H1 causes RNAPII pausing at the 3\u2032 splice site, and regulates alternative splicing32. These observations suggested the specific involvement of linker histones in the regulation of transcription elongation, but the molecular details have remained elusive. In the present study, we investigate the basic properties of the chromatosome in transcription elongation by performing in vitro transcription experiments and cryo-EM analyses, which reveal how H1 accomplishes nucleosome repression in transcription elongation.Linker histones are associated with the gene-body regions of actively transcribed genesKomagataella pastoris was loaded onto the bubble, and the transcription reaction was conducted in the presence of the transcription elongation factor TFIIS , lacking half of the C-terminal region (amino acid residues 151-212)  Fig.\u00a0. Interes12) Fig.\u00a0. The RNA12) Fig.\u00a0, probablSus scrofa domesticus36  analysis  of the RNAPII transcribing the entry linker DNA , the exit linker DNA of the chromatosome sterically conflicts with the transcribing RNAPII. In the natural chromatin context, this exit linker DNA is followed by another nucleosome (or chromatosome). This led us to test whether a second, neighboring nucleosome/chromatosome affects the RNAPII pausing. To do so, we performed the chromatosome transcription assay with the di-nucleosome template in the presence of H1.2 Fig.\u00a0. In thisner Fig.\u00a0. The strner Fig.\u00a0. Therefo40. It binds and seals the RNAPII DNA-binding cleft41, and enhances the transcription elongation efficiency in the nucleosome43. Interestingly, when the Spt4/5-bound RNAPII structure was superimposed on the current RNAPII-chromatosome complex, Spt4/5 overlapped with the exit linker DNA in the form I complex  is one of the transcription elongation factors that is tightly associated with transcribing RNAPIIlex Fig.\u00a0. This filex Fig.\u00a0. This sulex Fig.\u00a0, lane 4.This study has revealed that a chromatosome poses a higher transcriptional barrier than an H1-free nucleosome in vitro. H1 causes RNAPII pausing before it enters the chromatosome. This is because H1 brings the entry and exit linker DNAs close together, and the RNAPII on the entry linker DNA sterically conflicts with the exit linker DNA, hindering its progression. As shown in the form I complex, the exit linker DNA directly interacts with the RNAPII DNA-binding cleft, thus inducing the RNAPII pausing Fig.\u00a0. The for14. Therefore, the bound H1 may persist on the nucleosome while the RNAPII invades the nucleosomal DNA region  strain  and the JM109(DE3) strain (for H4) as N-terminally hexa-histidine (His6)-tagged proteins, and isolated under denaturing conditions. The His6-tagged proteins were purified by chromatography with nickel-nitrilotriacetic acid agarose (Ni-NTA) resin (QIAGEN). The His6-tag portion was then removed by thrombin protease, and the histones were purified by cation exchange chromatography on a MonoS column under denaturing conditions. The purified histones were dialyzed against water and lyophilized.Human histones H2A, H2B, H3.1, and H4 were expressed in E. coli cells and purified by the method described previously49. Briefly, H1.2 and H1.2 deltaC were produced in the BL21-CodonPlus(DE3)-RIL strain, as C-terminally His6-SUMO-tagged proteins. The His6-SUMO-tagged proteins were purified by Ni-NTA agarose (QIAGEN) chromatography. The His6-SUMO tag portion was then removed by PreScission protease treatment, and H1.2 and H1.2 deltaC were purified by MonoS cation exchange column chromatography. Purified H1.2 and H1.2 deltaC were stored at \u221280\u2009\u00b0C. K. pastoris RNAPII, composed of 12 subunits, was purified as described previously50. Briefly, K. pastoris encoding a TAP-tagged Rpb2 was cultured, and the tagged-RNAPII was purified by affinity column chromatography on anti-FLAG M2 affinity gel (Sigma-Aldrich), and anion-exchange column chromatography. K. pastoris TFIIS and Spt4/5 were purified as described previously40. These proteins were produced as His6-tagged proteins in the E. coli KRX strain. The His6-tagged proteins were then purified by chromatography on a COSMOGEL His-Accept column , and eluted by cleavage of the His6-tag portion by HRV-3C protease treatment. The eluted proteins were purified by Resource S cation-exchange column chromatography.Human H1.2 and H1.2 deltaC (H1.2(1-150)) were expressed in S. scrofa RNAPII preparation, fresh S. scrofa thymus was homogenized using a 2L blender (Waring) in buffer A (50\u2009mM Tris-HCl (pH 7.9), 1\u2009mM EDTA, 10 \u03bcM ZnCl2, 10% glycerol, 1\u2009mM DTT, 1\u2009mM phenylmethylsulfonyl fluoride, 1\u2009mM benzamidine, 0.6\u2009\u00b5M leupeptin, 2\u2009\u00b5M pepstatin A). The homogenized material was centrifuged at 12,200\u2009\u00d7\u2009g for 20\u2009min at 4\u2009\u00b0C, and the supernatant was filtered through Miracloth (Millipore). A 5% solution of polyethyleneimine (adjusted to pH 7.9 at 25\u2009\u00b0C) was added to a final concentration of 0.1%. After mixing for 10\u2009min at 4\u2009\u00b0C, the precipitate was collected by centrifugation at 15,750 xg for 20\u2009min. The pellet was washed twice with buffer A, and the RNAP II fraction was extracted from the pellet with buffer A containing 0.15\u20130.2\u2009M ammonium sulfate. A Q Sepharose (Cytiva) slurry was added to the eluted fraction, and the ammonium sulfate concentration was adjusted to 0.15\u2009M. After rotation at 4\u2009\u00b0C for 30\u2009min, the resin was washed with 6 volumes of buffer A containing 0.15\u2009M ammonium sulfate, followed by 3 volumes of buffer A containing 0.2\u2009M ammonium sulfate. RNAPII was eluted with buffer A containing 0.4\u2009M ammonium sulfate. The eluate was precipitated by 50% ammonium sulfate overnight. The precipitate was collected by centrifugation at 22,900\u2009\u00d7\u2009g for 20\u2009min at 4\u2009\u00b0C, resuspended in buffer A, and then dialyzed against 1\u2009L of buffer B (50\u2009mM Tris-HCl (pH 7.9), 150\u2009mM NaCl, 1\u2009mM EDTA, 10\u2009\u00b5M ZnCl2, 2\u2009mM DTT). The dialyzed sample was subjected to anion-exchange chromatography on a Mono Q (Cytiva) column. The RNAPII-containing fraction was concentrated with an Amicon Ultra-15 centrifugal filter unit (Merck) and purified by size exclusion chromatography on a Superose 6 column (Cytiva), equilibrated with buffer C (5\u2009mM HEPES-NaOH (pH 7.3), 150\u2009mM NaCl, 10\u2009\u00b5M ZnCl2, 10\u2009mM DTT). Peak fractions containing RNAPII were collected and stored as a 10% glycerol stock at \u221280\u2009\u00b0C. Judging from the electrophoretic band intensities, the purity of RNAPII in this fraction is about 30%  buffer, containing 150\u2009mM NaCl, 1\u2009mM DTT, 5% glycerol, and 10\u2009\u03bcM zinc chloride, flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C.For the E. coli DH5 strain. The 193\u2009bp Widom 601 fragment was excised from the plasmid DNA by EcoRV (Takara), and purified by polyethylene glycol precipitation. The ends of the DNA fragments were dephosphorylated by calf intestinal alkaline phosphatase (Takara). The 193\u2009bp Widom 601 DNA fragment was also produced by polymerase chain reaction. The purified 193\u2009bp Widom 601 DNA fragment was cleaved with HinfI, resulting in the 161 base-pair DNA with a 3\u2009bp overhang at the 5\u2032 end (Takara). This DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH) or native polyacrylamide gel electrophoresis purification using a Prep Cell model 491 apparatus (Bio-Rad).For the preparation of the DNA fragment used in the mono-chromatosome transcription, the plasmid DNA containing the 193\u2009bp Widom 601 sequence was prepared from the The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5\u2032:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5\u2032:ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the double-stranded oligonucleotide 50-mer (purchased from Eurofins) by T4 DNA ligase (NIPPON GENE). The sequences of both strands of the double-stranded oligonucleotide 50-mer are as follows:non-template strand: 5\u2032:CCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGtemplate strand: 5\u2032:ATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAA.For the preparation of the DNA fragment used in the di-chromatosome transcription, the 368\u2009bp DNA fragment containing the Widom 601 and 603 sequences connected with 48\u2009bp linker DNA was produced by polymerase chain reaction. The purified DNA fragment was cleaved with HinfI, resulting in the 354\u2009bp DNA with a 3\u2009bp overhang at the 5\u2032 end (Takara). This DNA fragment was further purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).The sequences of both strands of the resulting DNA fragment are as follows:non-template strand: 5\u2032:AATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5\u2032:ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGG.The purified DNA fragment was ligated with the same double-stranded oligonucleotide 50-mer (purchased from Eurofins) as described above by T4 DNA ligase (NIPPON GENE).48. Briefly, the freeze-dried histones were mixed at an equal molar ratio under denaturing conditions. The histone octamer was refolded by dialysis against refolding buffer, containing 10\u2009mM Tris-HCl (pH 7.5), 1\u2009mM EDTA, 2\u2009M NaCl, and 5\u2009mM 2-mercaptoethanol. The histone octamer was purified by chromatography on a Superdex 200 gel filtration column . The resulting octamer was flash-frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C. The histone octamer and the 161 or 354\u2009bp DNA fragment were mixed, and the nucleosome was reconstituted by the salt dialysis method. The double-stranded oligonucleotide 47-mer containing a 3\u2009bp overhang at the 5\u2032 end was ligated to the cohesive DNA end of the reconstituted nucleosome. The oligonucleotide also contained a 9 base mismatched region (bubble) at the region 14\u201322\u2009bps from the 3\u2032 end. The resulting nucleosome with a bubble region was purified by non-denaturing gel electrophoresis, using a Prep Cell apparatus (Bio-Rad).The histone octamer was reconstituted as described previouslyThe sequences of both strands of the oligonucleotide 47-mer containing a bubble region are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGtemplate strand: 5\u2032:AGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the mono-chromatosome transcription experiments are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGCCTTGTGTCGCGAAATTCATAGATtemplate strand: 5\u2032ATCTATGAATTTCGCGACACAAGGCCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAA.The complete DNA sequences of both strands for the di-chromatosome transcription experiments are as follows:non-template strand: 5\u2032TTCTTAAATACCATGGCCATCTTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACGGTCCACTTGTGTTTGGTGTGTTTGGGAATCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCAGGTCATTCCGGACGTGTTTGTCCTCTGCCTTTAAAGCAATAGGAGCTTACCCCCAGGGACTTGAAGTAATAAGGACGGAGGGCCTCTTTCAACATCGATGCACGGTGGTTAGCCTTGGATTGCGCTCTACCGTGCGCTAAGCGTACTTAGAAGCCCGAGTGACGACTTCACACGGTAGGTGGGCGCGCGAACTGAGCCTTGTGTCGCGAAATTCATGATtemplate strand: 5\u2032ATCATGAATTTCGCGACACAAGGCTCAGTTCGCGCGCCCACCTACCGTGTGAAGTCGTCACTCGGGCTTCTAAGTACGCTTAGCGCACGGTAGAGCGCAATCCAAGGCTAACCACCGTGCATCGATGTTGAAAGAGGCCCTCCGTCCTTATTACTTCAAGTCCCTGGGGGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGACCTGGATGTATATATCTGACACGTGCCTGGAGACTAGGGAGTAATCCCCTTGGCGGTTAAAACGCGGGGGACAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTACGACCAATTGAGCGGCCTCGGCACCGGATTCCCAAACACACCAAACACAAGTGGACCGTAAGCTCCTATTGCTTTAAAGGCAGAGGACAAACACGTCCGGAATGAGAGCTAATTTGGTATTTAAGAAK. pastoris RNAPII or approximately the corresponding amount of the partially purified S. scrofa RNAPII), TFIIS (0.14\u2009\u03bcM of K. pastoris TFIIS or 0.11\u2009\u03bcM of H. sapiens TFIIS), and the DY647-labeled primer RNA (5\u2032-DY647- AUAAUUAGCUC-3\u2032) (0.57\u2009\u03bcM) (Dharmacon), in the presence or absence of Spt4/5 (0.57\u2009\u03bcM), in a 7\u2009\u03bcL reaction at 30\u2009\u00b0C for 30\u2009min. For the experiment with K. pastoris RNAPII, the reaction solution contained 37\u2009mM HEPES-KOH (pH 7.5), 7.1\u2009mM MgCl2, 43\u2009mM potassium acetate, 0.29\u2009\u03bcM zinc acetate , 0.37\u2009mM HEPES-NaOH (pH 7.5), 7.1\u2009mM MgCl2, 37\u2009mM potassium acetate, 0.29\u2009\u03bcM zinc acetate, 29\u2009\u03bcM Tris(2-carboxyethyl)phosphine, 0.46\u2009mM DTT, 2.0% glycerol, 6.14\u2009mM NaCl, 0.29\u2009\u03bcM zinc chloride, 0.57\u2009mM UTP, 0.57\u2009mM GTP, and 0.57\u2009mM ATP. The reactions were then incubated for 15\u2009min at 4\u2009\u00b0C for transcription with K. pastoris RNAPII or 30\u2009\u00b0C for transcription with S. scrofa RNAPII. H1.2 was added to the reaction mixture in nucleosome:H1.2 ratios of 1:0, 1:3, 1:4.5, 1:6, and 1:9, and was bound to the nucleosome for 30\u2009min by an incubation at 4\u2009\u00b0C for transcription with K. pastoris RNAPII or at 30\u2009\u00b0C for transcription with S. scrofa RNAPII. H1.2 was dissolved in 10\u2009mM Tris-HCl buffer (pH 7.5), 0.05\u2009mM phenylmethylsulfonyl fluoride (PMSF), 0.25\u2009mM EDTA, 75\u2009mM NaCl, 10% glycerol, 10\u2009mM HEPES-KOH (pH 7.5), 0.5\u2009mM dithiothreitol, 50\u2009mM KCl, and 1\u2009mM 2-mercaptoethanol. CTP  was then added to re-start the RNAPII progression. After the reaction  was incubated with RNAPII phosphine, 0.14\u2009mM DTT, 0.7% glycerol, 0.57\u2009mM UTP, 0.57\u2009mM GTP, and 0.57\u2009mM ATP, at 30\u2009\u00b0C for 30\u2009min, followed by an incubation at 4\u2009\u00b0C for 15\u2009min. Afterwards, H1.2 was added to the reaction mixture at a nucleosome:H1.2 ratio of 1:6, and was bound to the nucleosome by an incubation at 4\u2009\u00b0C for 30\u2009min. CTP  was then added to re-start the RNAPII progression. After a 5\u2009min reaction, 216\u2009\u03bcL of 0.5\u2009M EDTA was added to the reaction mixture. Under these conditions, a ~60-nt RNA product predominantly accumulated in the presence of H1.2. The resulting RNAPII-chromatosome complexes were fractionated by the GraFix method51. A gradient was prepared with low buffer (20\u2009mM HEPES-KOH (pH 7.5), 50\u2009mM potassium acetate, 0.2\u2009\u03bcM zinc acetate, 0.1\u2009mM Tris(2-carboxyethyl)phosphine, and 10% (w/v) sucrose) and high buffer (20\u2009mM HEPES-KOH (pH 7.5), 50\u2009mM potassium acetate, 0.2\u2009\u03bcM zinc acetate, 0.1\u2009mM Tris(2-carboxyethyl)phosphine, 25% (w/v) sucrose, and 0.1% glutaraldehyde). The sample was applied to the top of the gradient and centrifuged at 4\u2009\u00b0C for 16\u2009h at 124,779 \u00d7 g, using an SW41 rotor (Beckman Coulter). After centrifugation, the fractions containing the RNAPII-chromatosome complexes were collected and dialyzed twice against 20\u2009mM HEPES-NaOH (pH 7.5) buffer, containing 0.2\u2009\u03bcM zinc acetate and 0.1\u2009mM Tris(2-carboxyethyl)phosphine. The resulting RNAPII-chromatosome complexes were then concentrated with an Amicon Ultra 100\u2009K centrifugal filter unit (Millipore), until the DNA concentration of the sample reached 78.4\u2009\u03bcg/mL. The samples were applied to glow-discharged Quantifoil grids . Each grid was blotted at 4\u2009\u00b0C and 100% humidity in a Vitrobot Mark IV (Thermo Fisher Scientific) , and then immediately plunged into liquid ethane.The 6-FAM-labeled nucleosome (0.14\u2009\u03bcM) was mixed with RNAPII (0.14\u2009\u03bcM), TFIIS (0.14\u2009\u03bcM), and the DY647-labeled primer RNA (5\u2032-DY647- AUAAUUAGCUC-3\u2032) (0.57\u2009\u03bcM) (Dharmacon) in 1.5\u2009mL of reaction solution, containing 37\u2009mM HEPES-KOH (pH 7.5), 7.1\u2009mM MgCl\u2212/\u00c5, using the SerialEM software52.Cryo-EM images of the RNAPII-chromatosome complexes were collected by a Krios G3i electron microscope (Thermo Fisher Scientific), equipped with a K3 direct electron detector with a 25\u2009eV slit width of the BioQuantum energy filter (Gatan), and operated at 300\u2009kV at a pixel size of 1.07 \u00c5. Each image of the RNAPII-chromatosome complex was recorded with a 7\u2009s exposure time, and then fractionated into 40 frames with a total dose of 56.2 e53 and Gctf54, respectively. Preliminary RNAPII-chromatosome maps were then obtained after particle picking with gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and quick 2D and 3D classifications with Relion3. Using the particle coordinates from these preliminary processing results, the neural networks for Topaz55 were trained, and then another round of particle picking was performed with Topaz. Finally, the coordinates from the Topaz picking and the initial gautomatch picking were merged, and served as the starting set for further image processing with Relion3.First, the motion correction and CTF estimation were performed with Relion3During the initial stage of image processing, the dataset was split in two batches that were processed independently. As the dataset was highly heterogeneous, refinements were mostly focused on the RNAPII region of the molecule, and to remove bad particles and particles centered on the nucleosome, 2D and 3D classifications were performed occasionally. After the initial 3D refinement using the RNAPII mask, the Bayesian polishing and CTF refinement were performed. After another 3D refinement, 3D classification was performed to remove RNAPII particles lacking downstream DNA. A 2.8\u2009\u00c5 resolution RNAPII map was obtained at this stage, and then another Bayesian polishing was performed to downscale particles to 1.49\u2009\u00c5/pix. Finally, 3D classifications with the mask enveloping RNAPII and nucleosome were done to select particles containing both RNAPII and nucleosome.56.Next, the particles from the two batches were merged and subjected to 3D classification, which revealed two different complexes of RNAPII transcribing a chromatosome (forms I and II). For each complex, RNAPII subtraction and 3D classification around the nucleosome were performed to select classes with high quality chromatosome density. A significant amount of continuous motion remained between the RNAPII and chromatosome, so local refinements for the RNAPII region and the chromatosome region were performed, in addition to the overall reconstruction. Composite maps were also calculated for each complex structure, using the phenix.combine_focused_maps tool in the Phenix package40, modeled manually with WinCoot (COOT57 for windows), and then refined against the consensus RNAPII reconstruction using phenix.real_space_refine tool in the Phenix package56. The atomic model for the chromatosome was based on previous crystal structures (PDB: 3LZ0 and 4QLC)58, modeled manually, and then refined against the nucleosome reconstruction of the form I complex. The RNAPII and nucleosome models were then fit into their respective overall reconstructions, and the interfaces between the two bodies and the connecting DNA were manually edited with WinCoot and ISOLDE59. Figures were prepared using UCSF Chimera60, ChimeraX61, and PyMOL.First, the RNAPII and the chromatosome were independently modeled. The atomic model for RNAPII was based on the previous crystal structure (PDB: 5XOG)Further information on research design is available in the\u00a0Supplementary informationPeer Review FileReporting Summary"}
+{"text": "Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis, and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002 and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignonicomb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbertspp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris  and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyispp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.The Neotropical members formerly included in  Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002, and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignoni comb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbert spp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris  and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyi spp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.Neotropical species formerly included in c oxidase subunit I (COI) gene using standard insect primers LepF1 (5\u2032-ATTCAACCAATCATAAAGATATTGG-3\u2032) and LepR1 (5\u2032-TAAACTTCTGGATGTCCAAAAAATCA-3\u2032) (Molecular work was carried out at the CBG using standard protocols. A leg from each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol . ExtractATCA-3\u2032) . If inithttp://www.boldsystems.org/index.php/IDS_OpenIdEngine); (2) paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library ; (3) the search results page shows the top hits based on percentage similarity starting with the closest matches ; (4) use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.The BOLD database can be used to identify specimens using the following steps: (1) navigate to the identification tab of the BOLD Systems database  can also be employed to differentiate them. Members of Lytopylus differ most significantly in that they lack vein Cub in the hind wing. See couplet 25 in the key below.Notauli absent; hind coxal cavities open; tarsal claws with basal lobes; second submarginal cell quadrate, never petiolate; foretibia lacking sclerotized spines/pegs; hind wing Cub strong and emanating from an angle on the basal cell. Most similar morphologically to Head. Lateral carina on frons (as found in members of Alabagrus) absent; interantennal space slightly raised above antennal sockets; gena not extended ventroposteriorly into sharp prominence; mandible dorsoventrally flattened (twisted); labial palpus with 4 segments, third segment slightly more than \u00bd length of apical segment. Mesosoma. Propleuron lacking a sharp bump; notauli absent; mesoscutum smooth with a median pit (presumably a remnant of notauli), postscutellar depression absent; propodeum mostly smooth, sometimes with weak smooth sculpture medially; sclerite between hind coxal cavities and metasomal foramen absent. Precoxal groove absent or smooth and weakly impressed. Legs. Foretibia lacking dull pegs (unlike Earinus); mid- and hind tibia with blunt apical or preapical pegs; all tarsal claws with a rounded basal lobe. Wings. Forewing RS+Ma vein mostly present but not usually completely tubular; second submarginal cell large, quadrate and usually  higher than long; RS of forewing complete to wing margin; hind wing r and r-m cross veins absent; hind wing vein Cub strong and emanating from an angle on the basal cell. Metasoma. First median tergite smooth, longer than apical width, lateral longitudinal carina absent or weak and short; remaining terga smooth; ovipositor ranging from as long as the body to twice the length of the body, but this is based on small sample of a few dozen species.Unknown.This is a species-rich genus with hundreds of species, based on specimens identified by MS. It is widespread in Chile and southern Argentina. A few species are found at high altitudes as far north as Ecuador and Colombia.Chilearinus in a broader concept of Earinus. Agathidinae from Chile. Since members of Chilearinus are by far the most species-rich of Chilean agathidines, and since his descriptions do not contradict membership in the genus, these species are probably members of Chilearinus, i.e., Agathislaevithorax, Agathisrubricata, and Agathisareolata. They certainly are not members of Agathis since this genus does not extend into the southern regions of South America. These specimens should be in the Hymenoptera collection of Maximilian Spinola whose collection is housed in the Museo Regionale di Scienze Naturali (MRSN) in Turin (Torino). One of us (MS) could not locate these specimens during a visit to MSRN in 1985, but a specimen of Chilearinus, Microgasterrubricollis Spinola, 1851, was present. Microgaster may seem an odd place for placement of what we now consider an agathidine, but such was the classification at the time. It is clear from the following that Spinola knew the species was closely related to Earinus, \u201cEste Microgastro habria pertenecido \u00e1 [sic] la primera seccion del G. Microdus, N. V. Es., y al sub-g\u00e9nero Earinus Wesm.\u201d .\u2640, Chile, Regi\u00f3n IX, PN Nahualbuta, COI barcode. BOLD sample ID H1145. BOLD BIN code BOLD:AAV0870. GenBank Accession Code OL702761.AATTTTATATTTTATATTTGGAATTTGATCGGGAATTTTAGGTTTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGGGGTAATTTTATTGGTAATGATCAAATTTATAATAGAATTGTNGCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTGGGGGGGCCAGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGGTCTTTAATTAATGTTGGGGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTAGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGTGCTATAAATTTTATTACAACTATTTTAAATATGTGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACGGCAATTTTATTATTATTATCTTTGCCAGTTTTAGCTGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGAGGAGATCCAATTTTATATCAACATTTATTTSee key.None.Named in acknowledgment of the covid pandemic occurring during the production of this manuscript.Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.AB9C892C-E5CA-5F6A-BE83-F0ED72EAEB87http://zoobank.org/AF4C4A3B-EBD8-4305-AF39-DC9176C868A837.493\u00b0S, 72.582\u00b0W, 1168 m, 8.ii.2005, Heraty, .\u2640, Chile, Regi\u00f3n IX, PN Nahualbuta, COI barcode. BOLD sample ID H12114. BOLD BIN: BOLD:AEM7846. GenBank Accession Code OL702760.TTTTAGGATTATCAATAAGTTTAATTATTCGAATAGAATTAAGAGTAGGTGGTAATTTTATTGGTAATGATCAAATTTATAATAGGATTGTNACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCATTAATATTAGGGGGTCCAGATATAGCCTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGATCTTTAATTAATGTTGGAGTAGGTACTGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGTCATAGTGGTATATCTGTAGATTTGGCTATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGGGCTATAAATTTTATTACAACTATTTTAAATATATGAATAATTAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTGATTACAGCAATTTTATTATTATTATCTTTACCAGTTTTAGCTGGGGCTATTACTATATTATTAACAGATCGTAATTTAAATACTAGATTTTTTGATCCTTCTGGAGGGGGAGATCCAATTTTATATCAACATTTATTTTGATTTTTSee key.None.A conjunction of Paul Hebert and Dan Janzen in recognition of their enormous contributions towards the conservation of nature.Taxon classificationAnimaliaHymenopteraBraconidae\ufeffWesmael, 18373EB02370-41B3-561B-B842-5117B904EE03Earinus, i.e., E.erythropoda Cameron, 1887, E.limitaris , and E.zeirapherae Walley, 1935, and here we describe two more, Earinusaustinbakeri sp. nov. and Earinuswalleyi sp. nov. In the Nearctic, Earinus is common and widespread with the southernmost record being the sole recognized specimen of E.erythropoda from northern Sonora state, Mexico. Earinus differs from Chilearinus in the possession of pegs/spines in the foretibia and the characters given in the key.In the Americas, there are three previously recognized species of Hymenoptera Institute  and borrowed specimens, there are probably between eight and 12 species in the Nearctic region. They are extremely similar in color, but there are obvious differences among specimens in body dimensions, degree of punctation, color of the hind coxae, ocellar configuration, ovipositor length, length and density of setae on the ovipositor sheath, and dimensions of the first metasomal tergum. Unfortunately, these are not sufficient to allow confident delineation of species limits. For example, the differences in the key between E.limitaris and E.erythropoda are trivial. There are numerous specimens scattered over the Nearctic region that will key to E.erythropoda, but they might all be E.limitaris, or the two nominal species may be conspecific, or there may be multiple cryptic species. Likewise, there are probably a number of undescribed Nearctic species that will key to either E.zeirapherae or E.austinbakeri. In other words, the key is sufficient to discriminate among the barcoded species and E.zeirapherae but not among these and the undescribed species. The key is presented in part to satisfy the code of Zoological Nomenclature to act as a diagnosis for E.austinbakeri and E.walleyi. Only dense sampling of COI barcodes and perhaps other genes will supply the information necessary to delimit Nearctic Earinus species.Based on the collection in the Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.107889AA-0F66-5D06-A568-CA774826DA26http://zoobank.org/D169A981-8A48-4E53-B1D1-CB072D89814744.2829\u00b0N, 77.7963\u00b0W, 131 m, 05\u201320.Jun.2014 . BOLD sample ID BIOUG33065-A05, BOLD BIN code BOLD:ADL5164. GenBank Accession Code OM158425.\u2640, Canada, Ontario, Ferris Provincial Park, Consensus barcode based on four specimens.ATTTTATATTTTATATTTGGGATTTGATCYGGAATTGTGGGKTTATCAATAAGTTTAATTATTCGTATGGARTTAAGAGTAGGGGGBAATTTAATTGGKAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATRCCAATTATAATTGGTGGGTTTGGTAATTGGTTAATTCCTTTAATATTAGGRGGTCCCGATATRGCTTTCCCTCGAATGAAYAATATAAGRTTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTAAGATCTTTAATTAATATTGGGGTTGGAACTGGTTGAACGGTYTATCCTCCTTTATCATTRAATATAAGTCATAGTGGTATATCTGTTGATTTGGCTATTTTYTCTTTACATATTGCGGGRATTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATATGAATAATAAATATTAAAGTTGATAAAATGTCTTTATTRATTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCTTTACCTGTTTTAGCRGGRGCAATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCTGGAGGTGGGGATCCAATTTTATATCAACATTTATTTE.austinbakeri but differing by the characters given in the key as well as having the ovipositor sheath more setose. The COI barcodes of the two species differ by 6.29% (p-distance), reinforcing the conclusion that they are different species.Very similar to http://www.boldsystems.org).BIOUG01028-C01, BIOUG01028-F12, BIOUG32793-A05. These are sample IDs; the data for these specimens can be found by searching for these codes on BOLD .E.limitaris, or several more species may have similar morphologies. COI barcode data are needed. Several line drawings, modified from The sole identified specimen is the holotype. It differs little from many specimens that are widespread in the United States. It could be that they all belong to Taxon classificationAnimaliaHymenopteraBraconidae\ufeffEAE38190-F29E-5D79-A78E-22DCB293E7E9Bassuslimitaris Say, 1835.38\u00b055'N, 78\u00b049'W, 30.viii\u201319.ix.2005 . BOLD sample ID H1141. BOLD BIN code BOLD:AAU8493. GenBank Accession Code OM237775.\u2642, USA, West Virginia, Hardy County, 3 mi. NE Mathias, COI barcode based on 9 specimens.Consensus AATTTTATATTTTATATTTGGAATTTGATCAGGAATTTTAGGTTTATCAATAAGATTAATTATTCGAATAGAATTAAGDATAGGTGGTAATTTRATTGGTAATGATCAAATTTATAATAGTGTTGTTYCTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATGATTGGRGGRTTTGGRAATTGATTAGTTCCTTTAATATTGGGRGGTCCTGATATAGCTTTYCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTATTATTATTAATTTTGAGTTCTTTAATTAATATTGGGGTRGGGACTGGKTGAACAGTTTATCCTCCRTTATCTTTAAATATAAGRCATAGTGGAATATCAGTTGATTTAGCTATTTTTTCATTACATATYGCAGGAATTTCTTCAATTATAGGGGCAATAAATTTTATTACTACTATYATAAATATATGAATAATAAATATTAAAATTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACTGCTATTTTATTATTATTATCATTRCCAGTTTTAGCTGGRGCAATTACTATATTATTAACAGATCGAAATTTRAATACAAGATTTTTTGATCCTTCTGGAGGGGGGGATCCAATTTTATATCAACATTTATTTSee key.http://www.boldsystems.org).ASGLE-0444, ASGLE-0446, ASGLE-0449, ASGLE-0451, ASGLE-0452, ASGLE-0445, BIOUG01022-D11, BIOUG32892-B07. These are sample IDs; data on them can be found by searching for these codes on BOLD  which differs by only 2.54% (p-distance) from E.limitaris , as well as either Missouri or Indiana, or both. It is unknown if Taxon classificationAnimaliaHymenopteraBraconidae\ufeffSharkeysp. nov.8DA974A7-E3F6-5E6F-A2B2-FF42AC77EBAAhttp://zoobank.org/BDFBEADA-2082-46A5-B648-EB181E09CBB558.3734\u00b0N, 94.1342\u00b0W, 3\u20137.vii.2007, Malaise trap . BOLD sample ID. 07PROBE-20853, BOLD BIN code BOLD:AAF9894. GenBank Accession Code FJ413805.\u2640, Canada, Manitoba, Churchill pump house, 15 km S Churchill, Goose Creek Road, Consensus barcode based on four specimens.TATTTTATATTTTATATTTGGAATTTGATCAGGTATTGTAGGTTTATCAATAAGATTAATTATTCGAATGGAATTAAGAGTGGGRGGTAATTTAATTGGRAATGATCAAATTTATAATAGTATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGGGGRTTTGGTAATTGATTARTCCCATTAATATTGGGAGGTCCTGATATAGCTTTCCCTCGTATAAATAATATGAGATTTTGATTATTAATCCCYTCTTTATTAATATTAATTTTAAGATCTTTAATTAATATTGGAGTAGGGACTGGTTGGACAGTTTATCCTCCKTTATCATTAAATATAAGTCATAGTGGAATATCTGTTGATTTGGCTATTTTTTCTTTACATATTGCGGGRGTTTCTTCTATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATRTGAATAATAAATATTAAAATTGATAAAATGTCTTTATTAATTTGATCAATTTTAATTACTGCTATTTTATTATTATTRTCTTTACCAGTTTTAGCAGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATACAAGATTTTTTGATCCTTCYGGAGGGGGTGACCCAATTTTATATCAACATTTATTTE.zeirapherae, differing by the characters given in the key as well as having the ovipositor sheath less setose. The COI barcodes of the two species differ by 6.29% (p-distance) all but ensuring that they are different species.Very similar to http://www.boldsystems.org).All are from the same locality as the holotype, 07PROBE-23096, 07PROBE-23097, 09PROBE-A0304. These are specimen IDs; more data on the specimens can be found by searching for these codes on BOLD , former research scientist at the Canadian National Collection and author of Taxon classificationAnimaliaHymenopteraBraconidae\ufeffWalley, 1935766B1C04-99FA-52A3-8733-9263AE5C92E1\u2640, Grand River, Nova Scotia, 11.May.1932 (M. L. Prebble) No. 3847 .Tortricidae: Aclerishudsoniana, Choristoneurarosaceana, Rhyacioniaadana, Zeirapheracanadensis, Zeirapheragriseana, and Zeirapheraratzeburgiana. Since there are many species, including E.austinbakeri and E.walleyi, that are morphologically similar to E.zeirapherae, all hosts that do not belong to the genus Zeiraphera need confirmation.The following are all reported as hosts by Zeirapheraratzburgiana. Contrary to the image of the holotype in Figure The holotype Fig.  is from (Modified from"}
+{"text": "However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum\u00a0(ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180\u2013microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions The endoplasmic reticulum proteins CLIMP63, kinectin and p180 bind preferentially to subsets of microtubules with different post-translational modifications, thereby linking the \u2018tubulin code\u2019 to the intracellular distribution of membrane organelles. Although this code has been implicated in cargo selection and directed organelle movement2, how it is decoded to mediate transport and control distribution remains largely unknown.Eukaryotes compartmentalize cellular functions within distinct organelles, and regulation of organelle position is critical for cell health. Organelles are transported bidirectionally by motor and adaptor proteins along microtubules7. The ER is a compelling candidate for exploiting the complexity of the tubulin code, since it spreads throughout the cytoplasm in association with microtubules and makes abundant organelle contacts10. Most studies of ER shaping and organelle contacts have emphasized peripheral tubular ER. How the denser perinuclear ER is shaped and asymmetrically distributed remains largely unknown, although three ER membrane-bound proteins\u2014CLIMP63, p180 and KTN1\u2014localize prominently to perinuclear ER and are considered sheet-forming proteins11. Even so, depletion of CLIMP63 may paradoxically lead to\u00a0the expansion of ER matrices or\u00a0sheets in the periphery12, and perinuclear ER matrices or\u00a0sheets remain abundant even upon simultaneous knockdown of all three proteins, prefiguring more complex functional roles11.Endoplasmic reticulum (ER) comprises structurally and functionally divergent membrane compartments that include interconnected tubules, perinuclear matrices and sheets, and the nuclear envelope12, peripheral ER in CLIMP63-knockout cells is populated with increased numbers of dense matrices\u00a0or\u00a0sheets\u2014a \u2018dispersed\u2019 phenotype. KTN1 knockout also disperses ER, whereas p180-knockout cells exhibit a contrasting \u2018clustered\u2019 ER phenotype, with the peripheral network remaining tubular and perinuclear ER collapsing asymmetrically into a smaller area at one side of the nucleus  to visualize their microtubule associations in cells ) that shape the tubules. The polygonal network is generated via atlastin-mediated tethering and fusion of tubules at three-way junctions and distributed via cytoskeletal interactions2. Modifications are dynamic and rapidly reversible, but evidence for how they affect microtubule-related functions has been limited2. We have shown here that KTN1 preferentially binds perinuclear polyglutamylated microtubules with long glutamate chains, whereas p180 binds glutamylated microtubules with either short or long chains. By contrast, CLIMP63 has a higher threshold for response to microtubule glutamylation. We cannot exclude that increased affinity\u00a0at higher glutamate numbers for TTLL7-modified microtubules stems from additional chains that TTLL7 initiates on tubulin tails, and not only from introduction of longer chains. Conversely, p180 is more sensitive to any increase in glutamate numbers on the tubulin tail and robustly binds both mono- and polyglutamylated microtubules. This differential effect on microtubule binding according to glutamylation state has previously been observed for the microtubule-severing ATPase spastin29 and may represent a general feature of this modification, enabling fine tuning of molecular interactions. Thus, a small difference in the number of glutamates added to tubulin side chains may exert a substantial qualitative effect on ER distribution. Other ER-localized, microtubule-binding proteins30 are likely to contribute to overall cellular ER positioning. Indeed, even in p180 and KTN1 double-knockout cells, TTLL7 overexpression still disperses ER, suggesting the involvement of other ER proteins. Moreover, tubular ER selectively moves along acetylated microtubules27, further indicating that ER distribution is broadly sensitive to microtubule modifications.Microtubule diversity can be achieved via different tubulin gene products, differential interactions with microtubule-associated proteins and numerous post-translational modifications31, and axonal microtubules are highly glutamylated2. Thus, p180 may affect microtubule remodelling by differentially recognizing glutamylated axonal microtubules. Of note, although dysregulation of ER shaping and microtubule polyglutamylation lead to different neurodegenerative diseases33, these diseases share some\u00a0similar cellular phenotypes, including mitochondrial distribution defects and axon degeneration, suggesting possible convergence.The ability of cells to dynamically control ER distribution through differential microtubule modifications has important functional implications. For instance, p180 regulates microtubule remodelling in axons1, and their ability to selectively distribute organelles relies on a tubulin code. Our results indicate that ER distribution is mediated via specific membrane-bound proteins with differential binding to different levels and types of microtubule glutamylation, broadly affecting distributions of most other organelles. ER thus interprets the tubulin code to regulate movement and positioning of cellular organelles. Rather than imbuing each organelle with its own sensing and response mechanisms, cells achieve organizational efficiency by using ER as a first-line sensor and responder. This role is exemplified during nutrient starvation, when cells increase CLIMP63 protein levels to move ER towards the perinuclear region, which also clusters lysosomes for efficient autophagic degradation. Then, cells harness enhanced p180\u2013microtubule binding to redistribute ER and lysosomes for a proper reset. There are likely to be other ER proteins that also decipher the tubulin code, with important implications for ER function in health and disease.When ER positioning is disrupted, distributions of other organelles are affected. Microtubules have key roles in organelle distribution21. pCMV6_p180s-myc-Flag (RC218816), pCMV6_KTN1-myc-Flag (RC219832), pCMV6_TTLL4-myc-Flag (RC205206) and pCMV6_CCP1-myc-Flag (RC220826) were obtained from Origene Technologies. pcDNA3.1_TTLL6-Flag (OHu07095), pcDNA3.1_CCP5-Flag (Ohu28493), pcDNA3.1_CCP6-Flag (OHu24335) and pcDNA3.1_p180L (OHu24745) were obtained from GenScript. Mutants of CLIMP63, p180s and KTN1 were generated in a pcDNA3.1(+) vector with a C-terminal HA-epitope tag. Specifically, Ser-to-Glu mutants  of CLIMP63 were synthesized by GenScript. pcNDA3.1-KTN1, pN1-KTN1-mApple, pC1-sp-mScarlet-KTN1, pN1-KTN1-mNeonGreen, 2\u00d7Strep-p180s_29-381-mNeonGreen, 2\u00d7Strep-p180s_29-381-mNeonGreen and CLIMP63-mNeonGreen-2\u00d7Strep were also constructed using standard cloning procedures. 5\u2032 and 3\u2032 UTR of KTN1 were synthesized by Integrated DNA Technologies and ligated into the pN1-KTN1-mNeonGreen vector. Human TTLL4, TTLL6, TTLL7, CCP1, CCP5 and CCP6 inserts were also cloned into pCMV14-3\u00d7Flag and pN1-mApple. DNA oligonucleotides were synthesized by Integrated DNA Technologies.GFP-mCherry-LC3 was a gift from Juan S. Bonifacino. mEmerald-Sec61\u03b2, pcDNA3.1_CLIMP63-HA, mApple-SiT (Golgi apparatus), mKO-SKL (peroxisome), YFP-KDEL (ER) and CFP-LAMP1 (lysosome) were constructed as described previously34. Camptothecin (S1288), DC661 (S8808), etoposide (S1225) and MG132 (S2619) were purchased from Selleckchem. LysoSensor Green DND 189 (L7535), G418 (11811098) and puromycin (A1113802) were from Thermo Fisher Scientific. Centrinone B  was from Tocris. GTP (G8877), ATP, Taxol (T7402), tunicamycin (SML1287), Earle\u2019s Balanced Salts  and Duolink In Situ PLA kit  were from Sigma-Aldrich. The Cathepsin L Activity Assay Kit (Fluorometric) (ab65306) was obtained from Abcam.LD540 was provided by C. Thiele2. HEK 293T cells were transfected with Avalanche-Everyday Transfection Reagent . U2OS and COS7 cells were electroporated using Cell Line Nucleofector Kit V  and Cell Line Nucleofector Kit R  following the manufacturer\u2019s instructions. Amounts of key plasmids transfected are (per 1\u2009\u00d7\u2009106 U2OS cells or 5\u2009\u00d7\u2009105 COS7 cells): 0.3\u2009\u03bcg CLIMP63-HA, 0.5\u2009\u03bcg p180s-HA, 2\u2009\u03bcg KTN1-mApple, 1\u2009\u03bcg CLIMP63-mEmerald (for overexpression), 2\u2009\u03bcg p180s-mEmerald, 4\u2009\u03bcg p180L-mEmerald, 4\u2009\u03bcg KTN1-mEmerald, as well as (in various vectors) 1\u2009\u03bcg TTLL4, 1\u2009\u03bcg TTLL6, 0.3\u2009\u03bcg TTLL7, 4\u2009\u03bcg CCP1, 0.5\u2009\u03bcg CCP5, and 1\u2009\u03bcg CCP6.All cell lines were obtained from the American Type Culture Collection: HEK 293T (CRL-11268), COS7 (CRL-1651), HeLa (CCL-2), RPE1 (CRL-4000) and U2OS (HTB-96) cells. HEK 293T, COS7 and HeLa cells were cultured in Dulbecco\u2019s Modified Eagle Medium , RPE1 cells were cultured in DMEM/F12 (1:1) Medium , and U2OS cells were cultured in McCoy\u2019s 5A medium (Thermo Fisher Scientific16600108); all were supplemented with 10% fetal bovine serum  and 1\u00d7penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific 15240112) at 37\u2009\u00b0C with 5%\u2009CO6 U2OS cells using Lonza Cell Line Nucleofector Kit V.For RNAi knock down of AKAP450, two siRNAs targeting ATATGAACACAGCTTATGA and AACTTTGAAGTTAACTATCAA were synthesized by Eurofins Genomics. Cells were transfected using Avalanche-Omni Transfection Reagent  with 20\u2009pmol siRNA for 3 days. For RNAi knockdown of CCP5, ON-TARGETplus siRNA sets targeting human CCP5 were purchased from Horizon Discovery (LQ-009468-00-0005), and 60\u2009pmol siRNAs were transfected per 1\u2009\u00d7\u200910Primary antibodies used: mouse monoclonal anti-AKAP450 , rabbit polyclonal anti-Atlastin2 , rabbit polyclonal anti-Atlastin3 , rabbit monoclonal anti-Catalase , mouse monoclonal anti-Climp63 , mouse monoclonal anti-Flag M2 , rabbit polyclonal anti-GFP , mouse monoclonal anti-GM130 , rabbit polyclonal anti-GM130 , mouse monoclonal anti-HA , rabbit polyclonal anti-kinectin , rabbit monoclonal anti-kinectin , mouse monoclonal anti-Lamp1 , rabbit polyclonal anti-LC3 , rabbit polyclonal anti-Lunapark , mouse monoclonal anti-Myc , rabbit polyclonal anti-p180 , rabbit polyclonal anti-Pericentrin , rabbit polyclonal anti-polyglutamylation (polyE) , mouse monoclonal anti-glutamylation clone GT335 , rabbit polyclonal anti-REEP2 , rabbit polyclonal anti-REEP3 , rabbitpolyclonal anti-REEP4 , rabbit polyclonal anti-REEP5 , rabbit polyclonal anti-reticulon3 , rabbit polyclonal anti-reticulon4 , rabbit polyclonal anti-RPL3 , rabbit polyclonal anti-TOM20 , mouse monoclonal anti-TOM20 , rabbit polyclonal anti-TRAP\u03b1 , rat monoclonal anti-\u03b1-tubulin Alexa Fluor 647 , mouse monoclonal anti-\u03b1-tubulin , mouse monoclonal anti-\u03b2-tubulin . Alexa Fluor 405/488/568/633 conjugated goat anti-rabbit/mouse IgG (H+L) highly cross-adsorbed secondary antibodies were from Thermo Fisher Scientific. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Santa Cruz Biotechnology.7 and selected using 200\u20131,000\u2009\u03bcg\u00a0\u03bcl\u22121  G418 for two weeks; green-positive cells were sorted into mono-clones by flow cytometry using a MoFlo Astrios cell sorter (Beckman Coulter) and cultured in the presence of 200\u2009\u03bcg\u00a0\u03bcl\u22121 G418 for 2\u20133 weeks. Proliferated clones were verified by immunoblotting and fluorescence imaging.To generate U2OS cells stably expressing mEmerald-Sec61\u03b2, cells were transfected with the mEmerald-Sec61\u03b235. The targets used were: CLIMP63:GCCGCGCCCGCCATGCCCTCGG; p180 in U2OS:\u00a0GGTGTCGACTTTCTCCATGAAGG; p180 in COS7: GACACCAGGAAGATGCCAATGG; KTN1:\u00a0GAAAAGCCAGAAGAAGAGG and GTTAGGGAAAGAAAAAAGAAGG.All CRISPR\u2013Cas9 knockout assays used eSpCas9(1.1)CCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGtccggactcagatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccCCCTCGGCCAAACAAAGGGGCTCCAAGGGCGGCCACG;For knock-in of CLIMP63, the same target as in CLIMP63 knockout was used, and a PCR fragment with 37\u2009bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows:\u00a0GGCCTCCTCGGCTTGGCCGCCGTCGAGCCCGCCGTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAGCCCGCCGTCTACTTCAAGGAGCAGTTTCTGGAC. Note that amino acids 18\u201320 (EPA) were appended to both sides, acting as a linker..\u00a0To generate mEmerald-calreticulin knock-in COS7 cells, wild-type Cas9 with a gRNA targeting the end of the signal sequence of calreticulin (GAGCCCGCCGTCTACTTCAAGG) was selected, and a PCR fragment with 36\u2009bp homology arms on each side of the mEmerald-coding sequence was used as a homologous recombination template as follows: 15 before further analysis.To deplete the centrosome, cells were treated with 125\u2009\u03bcM CNB for 1 week as describedCells were quickly rinsed with PBS, directly lysed with sample buffer , and boiled for 5\u2009min. Proteins were then resolved by SDS\u2013PAGE using Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad Laboratories) following the manufacturer\u2019s instructions. Membranes were blocked with 4% milk in TBST , and incubated with primary antibody (diluted in blocking buffer) at 4\u2009\u00b0C overnight. After washing with TBST, membranes were incubated with secondary antibody at room temperature for 2\u2009h, followed by intensive washing with TBST. Immunoreactive proteins were visualized with GE Healthcare LS ECL Prime Western Blotting Detection Reagent (RPN2236) and imaged using a ChemiDoc XRS+ (Bio-Rad). Band intensities were quantified using Fiji software (NIH).\u22121 LD540 in PBS for 5\u2009min. Cells were mounted using Fluoromount-G (SouthernBiotech) and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63\u2009\u00d7\u20091.4 NA Plan-Apochromat oil objective (Carl Zeiss). Images were acquired using ZEN software (Carl Zeiss) and processed with ZEN software or Fiji (NIH).Cells were fixed with 4% paraformaldehyde in PBS (Lonza) for 30\u2009min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10\u2009min. Alternatively, for immunostaining of glutamylation (GT335) and polyglutamylation (polyE), cells were fixed and permeabilized with cold methanol for 5\u2009min at \u221220\u2009\u00b0C. Then, after blocking with 3%\u2009BSA for 30\u2009min, cells were immunostained with polyE antibody at 4\u2009\u00b0C overnight, then with polyE and GT335 together at 4\u2009\u00b0C overnight, followed by secondary antibody staining at room temperature for 1\u2009h, and finally with anti-\u03b1-tubulin Alexa Fluor 647 at room temperature for 2.5\u2009h. For staining of lipid droplets with LD540 dye, cells were incubated with 0.1\u2009\u03bcg\u00a0mlr,\u03b8)-space representation of the cell\u2019s fluorescence distribution. For analysis referring to \u2018normalized\u2019 data, we account for the shape of the cytoplasm by finding the radius at each angle where the nuclear envelope and the edge of the cell are located. The fluorescence data were then rescaled to a normalized axis with the cytoplasm between the nuclear envelope and the cell periphery scaled from 0 to 100%. The nucleoplasm is scaled to stretch between \u221225 and 0, as a control. .Three-dimensional images were acquired using a Zeiss LSM880 confocal microscope in Airyscan mode and reconstructed using ZEN software (Zeiss Microscopy). Summed intensity projections were generated using floating point notation to carry precision. A custom macro in Fiji-ImageJ was used to define the centre of the nucleus and remove the signal of neighbouring cells to avoid perturbing the results. From the manually defined centre, a radius was drawn out past the furthest point on the cell and swept through 360\u00b0 in 0.1\u00b0 steps, taking a line profile each time and rescaling the data to correct for artifacts generated by the square shape of the pixels. The resulting data represents an  for 30\u2009min on ice. Cell lysates were centrifugated twice at 20,000g for 20\u2009min at 4\u2009\u00b0C. The supernatant was supplemented with 1\u2009mM GTP and 40\u2009\u03bcM Taxol and incubated at 4\u2009\u00b0C or 37\u2009\u00b0C for 30\u2009min for tubulin polymerization before centrifugation at 20,000g for 30\u2009min at 4\u2009\u00b0C or 37\u2009\u00b0C, respectively. The resulting pellets (P) and supernatants (S) were collected and subjected to immunoblot analysis. In some experiments, only the pellets and supernatants of the 37\u2009\u00b0C samples are shown.To test the microtubule-binding affinities of CLIMP63, p180 and KTN1, cells were lysed in PIPES buffer  was performed according to the manufacturer\u2019s instructions. Samples were observed under a Zeiss LSM880 confocal microscope with a 20\u2009\u00d7\u20091.0 NA objective using the Airyscan function. The total intensity of the PLA signal per cell was quantified using Fiji software.g at 4\u2009\u00b0C for 30\u2009min. Supernatants were combined with Strep-Tactin XT beads (IBA Lifesciences) and rotated gently for 3\u2009h. After extensive washing with lysis buffer (PBS plus 500\u2009mM NaCl and 1% Triton X-100) and then wash buffer (IBA Lifesciences), bound proteins were eluted with Strep-Tactin XT Elution Buffer (IBA Lifesciences). Eluted proteins were subjected to multiple rounds of PBS dilution and concentration using 10 kDa protein concentrators (Sigma-Aldrich), before being aliquoted and frozen in liquid nitrogen.Deletion fragments of p180  and KTN1  as well as full-length CLIMP63 were expressed as fusions with mNeonGreen-2\u00d7Strep in HEK 293T cells. 48\u2009h post-transfection, cells were lysed in PBS (Lonza) plus 500\u2009mM NaCl, 1% Triton X-100, and protease inhibitors and then centrifugated at 30,00036. TTLL4 and TTLL6 were expressed in Escherichia coli and purified as previously described18. TTLL7 was also expressed in E. coli and purified as previously described19\u00a0. Taxol-stabilized microtubules were polymerized out of 98.5% unmodified tubulin and 1.5% biotinylated brain tubulin29 (Cytoskeleton T333P). Unmodified microtubules were modified using TTLL4, TTLL7 or TTLL6 at 1:10 molar ratio of enzyme to tubulin at room temperature in 20\u2009mM HEPES (pH 7.0), 50\u2009mM NaCl, 10\u2009mM MgCl2, 1\u2009mM glutamate, 1\u2009mM ATP, 0.5\u2009mM TCEP, and 10\u2009\u03bcM Taxol for 4.5\u2009h for TTLL4, between 20\u2009min and 2\u2009h for TTLL7, and between 7.5 and 22\u2009h for TTLL6. Control microtubules were incubated with the enzymes under the same conditions but with aspartate, which is not a substrate for TTLL glutamylases, instead of glutamate. Enzymes were removed through a high-salt wash as previously described29. The extent of glutamylation was determined by liquid chromatography\u2013electrospray mass spectrometry29 (LC\u2013MS). The spectra display the characteristic distributions of masses with peaks separated by 129 Da, which corresponds to one glutamate . Next, a solution containing 60\u2009mM Pipes (pH 6.8), 0.7\u2009mM MgCl2, 0.7\u2009mM EGTA, 50\u2009mM KCl, 10\u2009mM 2-mercaptoethanol, 10\u2009\u03bcM Taxol, 1% F127 Pluronic, 1.4\u2009mg/ml casein, 20\u2009mM glucose, glucose oxidase, and catalase was flushed into the chamber, followed by the same solution containing 4.7\u2009nM mNeon-labeled p180, KTN1 or CLIMP63. Images were acquired after allowing for equilibration for 5\u2009min at room temperature using total internal reflection fluorescence (TIRF) microscopy at an exposure of 100\u2009ms for the GFP channel. Unlabelled microtubules were visualized using interference reflection microscopy38. Multiple fields of view were imaged. Background corrected line scan average intensities were measured using Fiji software. Multiple chambers were quantified for each condition.For microtubule-binding assays, microtubules were immobilized in chambers made of silanized glassTotal mRNA were extracted using TRIzol (Thermo Fisher Scientific 15596018) and Direct-zol RNA Miniprep , then reverse-transcribed using the SuperScript IV First-Strand Synthesis System . Real-time PCR primers, designed by a free online tool developed by Integrated DNA technologies, were as follows: CCP5-RT: GACTGCCAGGAACTGCTAAA and AGGAGCTCCCGATGGTAATA; GAPDH-RT: GGTGTGAACCATGAGAAGTATGA and GAGTCCTTCCACGATACCAAAG.Real-time PCR was performed using Applied Biosystems PowerUp SYBR Green Master Mix  with Applied Biosystems QuantStudio 6 Flex real-time PCR instrument. Data were collected and analysed in QuantStudio Real-time PCR Software and Microsoft Excel using the 22. Images were acquired with a Zeiss LSM880 confocal microscope equipped with a 32-channel multi-anode spectral detector (Carl Zeiss) using a 63\u00d7/1.4 NA objective lens, at 37\u2009\u00b0C and with 5%\u2009CO2. Fluorophores were excited simultaneously using 458, 514 and 594\u2009nm lasers and a 458/514/594\u2009nm beam splitter, with images collected onto a linear array of 32 photomultiplier tube elements in \u03bb mode at 9.7\u2009nm bins from 468 to 687\u2009nm. Spectra were defined by imaging singly labelled cells for each of the fluorophore reporters, using the same acquisition and laser settings as for multiply labeled cells. Multispectral images were unmixed using the linear unmixing package in ZEN (Carl Zeiss).Multispectral imaging was performed as described previouslyz-projection was performed using maximum projection before quantification. The mCherry-positive vesicles indicate autophagosomes already fused with lysosomes, as the GFP signal would be quenched by the acidic environment of lysosomes; vesicles with both GFP and mCherry fluorescence indicate autophagosomes not yet fused with lysosomes. Quantifications of these two types of vesicles were performed manually using Fiji software.For autophagosome\u2013lysosome fusion assessments, U2OS cells were transfected with GFP-mCherry-LC3 for 24\u2009h, treated with EBSS for 2\u2009h before fixation with 4% paraformaldeyde in PBS, and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63\u2009\u00d7\u20091.4 NA Plan-Apochromat oil objective (Carl Zeiss). A For lysosome acidification assays, U2OS cells were labeled with 1 \u03bcM LysoSensor Green DND 189 for 4\u2009min and immediately imaged within one minute with a Zeiss Axio microscope using a 20\u00d7/0.4 NA objective. Images were captured with ZEN software, and total intensities of each cell were quantified in Fiji.6 cells were assayed in each sample.Cathepsin L activity assays were carried out using the Abcam Cathepsin L Activity Assay kit  following the manufacturer\u2019s instructions; 1\u2009\u00d7\u200910P values are shown on top of the corresponding columns, as determined by one-way ANOVA followed by Dunnett\u2019s multiple comparisons test, Mann\u2013Whitney test, Kruskal\u2013Wallis test or by unpaired two-sided t-test as indicated in the figure legends. When representative images are shown, at least three repeats were performed except for Extended Data Figs. No statistical method was used to predetermine sample size. All groups were randomly assigned and every group represents a distincttreatment or condition. Data were not analysed in a double-blinded manner. All comparisons were performed using Graphpad Prism or Microsoft Excel software. Data are expressed as means \u00b1 s.d., Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-04204-9.Supplementary InformationThis file contains Supplementary Text and Supplementary Figs 1\u20137.Reporting SummaryPeer Review FileSupplementary Video 1Wild-type or CLIMP63, p180 KO U2OS cells stably expressing mEmerald\u2013Sec61\u03b2 were labelled with Lysotracker Red, starved with EBSS, and recorded for 2.5\u00a0h with imaging at 1 min intervals. Scale bar, 10 \u00b5m."}
+{"text": "This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell\u2019s transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, \u2018RM-scTCR-Seq\u2019, which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR\u2019s with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track  More icities) .To date, clonotype tracking technologies have been developed for both mouse and human TCRs, facilitating studies of T cell clonal dynamics at an unprecedented level of molecular sensitivity \u201311. HoweTo address these limitations, nonhuman primate (NHP) models (particularly using rhesus macaques (RM)) have been essential , and havThe identification and tracking of TCR clonotypes are of critical importance for each of the clinical settings described above, in order to understand the molecular mechanisms driving immune protection or disease pathogenesis. For acute GVHD (aGVHD), dissecting the link between T cell clonal architecture and disease is particularly relevant, given that donor-derived alloreactive T cells are the main culprits in the aGVHD-mediated destruction of the skin, intestine, and liver \u201334. Accuin vitro and in vivo identification and tracking of RM derived T cell clonotypes. Pairing of in vivo identified alloreactive clonotypes with their transcriptional profile revealed a highly activated cytotoxic CD8 T cell signature during NHP aGVHD.We now describe the design and validation of RM-specific primers compatible with the human 10x Genomics single cell sequencing platform, which accurately amplifies the RM TCR alpha and beta regions. Amplified fragments aligned to our custom-assembled and annotated RM TCR reference permit, for the first time, the This study was conducted in strict accordance with (USDA) United States Department of Agriculture regulations and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. It was approved by the Massachusetts General Hospital and Biomere Animal Care and Use Committees. T cells were obtained from healthy colony RM or from animals who underwent allogenic HCT in the setting of previously published studies .137Cs) radiation. Responder PBMCs were stained with cell trace violet  as per manufacturer\u2019s instructions. 2\u00d7105 T cell-enriched responder PBMCs along with an equal number of stimulator PBMCs were added to each well in a 96-well plate (Corning) in X-vivo-15 medium (BioWhitaker) supplemented with 10% FBS (Irvine Scietific) and incubated at 37\u00b0C for a total of 5 days. Cell culture media change was performed on day 3 of the culture. At the end of 5 days, cells were stained with an extracellular antibody for CD3 (clone SP34-2), CD20 (clone 2H7) and CD14  for 20min at 4\u00b0C, and high-, medium- and non-proliferating CD3+ T cells (identified based on the dilution of CTV) were sorted and processed for single cell sequencing using the Chromium Next GEM Single Cell 5\u2019 Reagent Kit v1 with optimized RM primers as described below.RM PBMCs were isolated from whole blood by Ficoll gradient centrifugation, and then used for MLR assays either immediately, or after liquid nitrogen cryopreservation in 10% dimethyl sulfoxide (DMSO)/90% fetal bovine serum (FBS). At the time of the MLR, stimulator PBMCs were irradiated with 3500 cGy of , we also validated primers and PCR conditions compatible with the newer Next GEM Single Cell 5\u2019 v2 Sample Index Plate TT  (see below). https://assets.ctfassets.net/an68im79xiti/31W4aZOJ8C2ipxzTrcQIWn/72c5c2bc3dd7784f44f5218ca345ce04/CG000086_ChromiumSingleCellV_D_J_ReagentKits_UG_RevM.pdf). Primer sequences are listed in Single Cell V(D)J v1: GEMs were generated using v2 Gel Beads and the v1 Target Enrichment kit substituting the custom primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the i7 Multiplex Kit plate . TCR alpha and beta primers were designed to perform in a nested PCR approach to optimize target enrichment of the respective constant regions Table\u00a01.PCR was performed with an initial denaturation temperature of 98\u00b0C for 45 seconds, followed by denaturation at 98\u00b0C for 20 seconds. Annealing was optimal at 67\u00b0C for 30 seconds with extension of 72\u00b0C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72\u00b0C for 1 min.https://assets.ctfassets.net/an68im79xiti/57JaTECQNBPSpyDz8oucdi/92da9f62521ea1f5c781ba234bd0a0f5/CG000331_ChromiumNextGEMSingleCell5-v2_UserGuide_RevC.pdf). TRAC inner and outer primers were 2996bp apart, and the beta chain inner and outer primers were 354bp apart. Primer sequences are listed in Next GEM Single Cell 5\u2019 v2: GEMs were also generated using v2 reagent kits for GEM generation and library preparation while substituting the custom RM primers for the off-the-shelf human/mouse T Cell Mix 1 and 2 premixed primers. Sample Indexing was performed using the Dual Index Kit TT (10x Genomics PN-1000215). Primers Table\u00a02 PCR was performed with an initial denaturation temperature of 98\u00b0C for 45 seconds, followed by denaturation at 98\u00b0C for 20 seconds. Annealing was optimal at 62\u00b0C for 30 seconds with extension of 72\u00b0C for 1 min. A total of 20 PCR cycles were performed with a final extension step at 72\u00b0C for 1 min. Sequencing results utilizing v2 primers are listed in The reference transcriptome Data S4 1. We first filtered Ensembl\u2019s gtf file for v104 of the macaca mulatta gene annotation using cellranger\u2019s mkgtf command with the following attributes:\u2013attribute=gene_biotype:protein_coding \u2013attribute=gene_biotype:lincRNA \u2013attribute=gene_biotype:miRNA \u2013attribute=gene_biotype:antisense \u2013attribute=gene_biotype:IG_LV_gene \u2013attribute=gene_biotype:IG_V_gene \u2013attribute=gene_biotype:IG_V_pseudogene \u2013attribute=gene_biotype:IG_D_gene \u2013attribute=gene_biotype:IG_J_gene \u2013attribute=gene_biotype:IG_J_pseudogene \u2013attribute=gene_biotype:IG_C_gene \u2013attribute=gene_biotype:IG_C_pseudogene \u2013attribute=gene_biotype:TR_V_gene \u2013attribute=gene_biotype:TR_V_pseudogene \u2013attribute=gene_biotype:TR_D_gene \u2013attribute=gene_biotype:TR_J_gene \u2013attribute=gene_biotype:TR_J_pseudogene \u2013attribute=gene_biotype:TR_C_gene2. We next edited cellranger\u2019s mkref program to use more than one thread using the command:sed -i \u201888s/num_threads/#num_threads/\u2019/cellranger-6.0.0/bin/rna/mkref3. We then created the reference transcriptome using cellranger\u2019s mkref command: cellranger-6.0.0/bin/rna/mkref \u2013genome=MMulatta_Ensembl_v104 \u2013fasta=MMulatta_Ensembl_v104_combined.fasta \u2013genes=MMulatta_Ensembl_v104_combined_filtered.gtf \u2013nthreads=32 \u2013memgb=1281. We used version 6.0.2 of Cellranger to create the initial VDJ reference based on IMGT , 36.2. We used the following command to generate the initial reference: cellranger-6.0.2/lib/bin/fetch-imgt \u2013genome vdj_IMGT_rhesus \u2013species \u201cMacaca mulatta\u201d.3. This version of the VDJ reference did not include constant regions, so we added the following sequences - obtained from IMGT and Ensembl - to the reference:>340|TRAC*01 IMGT|TRAC|C-REGION|TR|TRA|None|01ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCGATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>341|TRAC*02 TRAC_205_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|02ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>342|TRAC*03 TRAC_201_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|03ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>343|TRAC*04 TRAC_202_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|04ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>344|TRAC*05 TRAC_203_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|05ACTGGGGTAAACAACCTCTTCTTTGGGACTGGAACAAGACTCACCGTTCTTCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGGCTCTAAATCCAATGACACCTCTGTCTGCCTATTTACTGATTTTGATTCTGTAATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>345|TRAC*06 TRAC_204_Ensembl_CDS|TRAC|C-REGION|TR|TRA|None|06ATGAATGTGTCACAAAGCAAGGATTCTGACGTGCATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTTAAGAGCAACGGTGCTGTGGCCTGGAGCAACAAATCCAATTTTGCATGTACAAGCGCCTTCAAGGACAGCGTTATTCCAGCAGACACCTTCTTCCCCGGCACAGAAAGTGTCTGTGATGCCAACCTGGTTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA>346|TRBC1*01 NW_001114291|TRBC1|C-REGION|TR|TRB|None|01AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAGAGGAAGGATTTC>347|TRBC1*02 IMGT000073|TRBC1|C-REGION|TR|TRB|None|02AGGACCTGAAAAAGGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCGCCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCAGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCCGAGGTCTGGGGTAGAGCAGACTGTGGCTTCACCTCAGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTGGGGAAGGCCTCCCTGTATGCTGTGCTGGTCAGTGCCCTCATGTTGATGGCCATGGTCAAGAGGAAGGATTTC>348|TRBC2*03 IMGT000073|TRBC2|C-REGION|TR|TRB|None|01AGGACCTGAAAAAAGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACGCTGGTGTGCCTGGCCACAGCTTCTACCCCGACCACGTGGAGTTGAGCTGGTGGGTGAACGGGAAAGAGGTGCACAGTGGGGTCAGCACGGACCCACAGCCCCTCAAGGAGCAGCCCACCCTCGAGGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCACAACCCCCGCAACCACTTCCGCTGCCAAGTCCAGTTCTATGGGCTCTCGGAGGATGACGAGTGGACCGAGGACAGGGACAAGCCCATCACCCAAAAGATCAGCGCTGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCTGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCThis resulted in an initial set of 348 sequences. We then checked these sequences for premature stop codons, frameshifts, and other potential problems using 10x\u2019s enclone software, v0.5.42. This identified 44 problematic sequences which were removed, leaving us with a final total of 304 VDJ segments.Samples were aligned using the Cellranger multi pipeline (v6.0.2), with VDJ libraries listed as \u201cvdj-t\u201d to indicate T cell libraries. Samples were aggregated with the Cellranger aggr pipeline (v6.0.2) with normalize=none. Our reference transcriptome was created with the Cellranger makeref command, and used genome assembly Mmul_10 as the reference genome , and EnsWe used FASTQC to assess the quality of our sequencing data. After alignment of this data, we loaded the filtered_feature_bc_matrix.h5 file from Cellranger multi into Seurat and removed genes which were present in less than 5 cells. This resulted in a dataset with 38,147 cells by 16,163 genes. Following the recommendations in Lucken and Theis  to normaWe then limited the dataset to samples from the PBMC, the MLR experiments, and the GVHD target organs. We then removed genes present in less than 5 cells, resulting in a dataset with 23,618 cells by 15,195 genes. This dataset was normalized using the same pipeline previously described in this section, retaining 22 PC\u2019s from the PCA.Clonotypes used in the gene expression analysis were identified using cellranger\u2019s default settings in \u201ccellranger vdj\u201d for the scTCR-Seq libraries. Clonotypes used for the Shannon Diversity metric calculations  were identified by grouping together T cells on the basis of the CDR3 region in the alpha chain or beta chain. This is to provide a more stringent test of the specificity of clonotype calling, as Cellranger\u2019s default settings do not allow for clonotypes to be shared across different donors. The Morisita index was calculated using the \u201cvegan\u201d package in R.As discussed in the Results section, the clonotypes used for the alpha chain and beta chain Morisita heatmaps . The primers targeting the beta chain were specifically designed to amplify both TRBC1 and TRBC2. Primer length, CG content and annealing temperatures were adjusted to be used with the forward primers supplied in the human 5\u2019 10x Genomics kit. Cycling conditions were modified from the original 10x protocol to allow optimal amplification of the targeted region in RM 2. We haRM-scTCR-Seq primers annealed to the alpha and beta RM constant region and amplified transcripts covering the complete alpha and beta loci including the variable V and J regions for the alpha as well as V, D and J regions for the beta locus.  and can We assessed the quality of our primers using summary statistics from 10x\u2019s \u2018Cellranger\u2019 software , and found that our primers consistently amplify high-quality reads from the TCR alpha and beta chains. For this assessment, we prepared peripheral blood mononuclear cells (PBMCs) from three representative RMs  and used these PBMCs to perform RM-scTCR-Seq. We observed that 49-52% of the aligned reads mapped to the alpha chain and 22-25% of the reads aligned to the beta chain Table S2In addition to demonstrating that the sequencing reads mapped to known TCR loci, we were also able to reconstruct the alpha and beta chains from these reads Table S2These data demonstrate, for the first time, efficient amplification of the RM TCR alpha and beta region by utilizing optimized RM primer pairs compatible with the human 5\u2019 10x Genomics single cell sequencing platform. Amplified reads enabled high quality beta chain reconstruction in almost all, and reconstruction of full TCRs  in a significant number of sequenced cells.in vitro assays or in vivo disease models enables the evaluation of the clonal repertoire, clonal dynamics, and the tracking of specific T cells over time. To validate our single cell TCR sequencing and analysis pipeline, we first utilized an in vitro allo-proliferation assay: the mixed lymphocyte reaction (MLR) (Identification of T cell clonotypes using on (MLR) \u201347. To pon (MLR)  (Table on (MLR) .Using the RM-scTCR-Seq pipeline, we were also able to track individual clonotypes throughout the three MLR peaks  (To further assess our ability to accurately track T cell clonotypes samples) , 50 betw). Importantly we were also able to identify clonotypes that existed in both the Blood sample and the high-proliferating population  or beta chain MI (MI <0.001), demonstrate the specificity of the RM-scTCR-Seq approach  or not (termed `MLR-`). Each MLR+ cluster incorporated at least 37 cells Table\u00a03.ted gene , 52 was d VISION  to scored VISION  as well d VISION .in vivo utility of the RM-scTCR-Seq pipeline.These results demonstrate the feasibility of combining RM-scTCR-Seq and scRNA-Seq to identify and interrogate alloreactive T cell clonotypes in aGVHD target organs in RM, providing proof-of-concept of the TCR repertoire analysis has become a fundamental tool to understand the immune responses to infections and vaccines, as well as the immunopathogenesis of rejection after solid organ transplantation, and GVHD after HCT , 54\u201358. Prior to the work described herein, two major limitations significantly hindered the development of T cell clonotype tracking in RM. They were (1): The lack of optimized, single cell sequencing-compatible primers covering both the TCR alpha and beta region for RM; (2) The poor genomic annotation of the alpha and beta loci in RM, which resulted in inadequate reconstruction of amplified VDJ PCR reads. To address both of these technical barriers, we have designed a robust set of RM-specific TCR primer pairs which anneal to the constant region of the alpha and beta TCR loci, and which are compatible with the commonly used single cell sequencing platform from 10x Genomics. Optimizing PCR cycling and temperature conditions resulted in efficient amplification of the alpha and beta regions of the RM TCR and generated productive TCR reconstructions. Additionally, we created an updated reference for the TCR alpha and beta chain region, enhancing our ability to annotate the assembled RM TCR\u2019s.www.10xgenomics.com/resourses/dataset). The difference in the alignment rate may be explained by differing levels of annotation for these organisms: 10x provides human references which have been refined over time, but this resource does not exist for RM, which necessitated our creation of a new reference (see Methods) by combining elements of the Ensembl and IMGT references, and then carefully filtering out sequences identified by 10x\u2019s enclone software that contained errors . As annotation of the RM TCR improves, we anticipate some gains in read mapping. As mentioned earlier, we have noticed that UTR regions are annotated in the human and mouse VDJ references, and we expect that addition of these sequences to the RM reference will improve alignment rates. We additionally tested whether our reads adequately covered all genomic V(D)J regions and confirmed that in the 30 primary VDJ samples analyzed, reads mapped to 91% (179 of 196) of all V(D)J regions from our reference J gene. We note that this mapping efficiency is somewhat less than the ~91% of reads mapping to the human VDJ reference, in a human dataset provided by 10x  and the National Cancer Center. VT is supported by an ASTCT New Investigator Award and the CIBMTR/Be The Match Foundation Amy Strelzer Manasevit Research Program Award. AS is supported by the Searle Scholars Program, the Beckman Young Investigator Program, a Sloan Fellowship in Chemistry, and the NIH . LK is supported by NIH grants U19 Al1051731, R01 HL095791, P01 HL158504, and by a Leukemia and Lymphoma Society TRP grant.AS reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Third Rock Ventures, Ochre Bio, FL82, and Dahlia Biosciences unrelated to this work. AS has received research support from Merck, Novartis, Leo Pharma, Janssen, the Bill and Melinda Gates Foundation, the Moore Foundation, the Pew-Stewart Trust, Foundation MIT, the Chan Zuckerberg Initiative, Novo Nordisk and the FDA unrelated to this work. LK is on the scientific advisory board for HiFiBio and Mammoth Biosciences. She reports research funding from Kymab Limited, Magenta Therapeutics, BlueBird Bio, and Regeneron Pharmaceuticals. She reports consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gillead Sciences, Vertex Pharmaceuticals, and Takeda Pharmaceuticals. LK reports grants and personal fees from Bristol Myers Squibb that are managed under an agreement with Harvard Medical School.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input\u2013output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization The BRAIN Initiative Cell Census Network has constructed a multimodal cell census and atlas of the mammalian primary motor\u00a0cortex in a landmark effort towards understanding brain cell-type diversity,\u00a0neural circuit organization and brain function. Unique among body organs, the human brain is a vast network of information processing units, comprising billions of neurons interconnected through trillions of synapses. Diverse neuronal and non-neuronal cells display a wide range of molecular, anatomical, and physiological properties that together shape the network dynamics and computations underlying mental activities and behaviour. Brain networks self-assemble during development, leveraging genomic information shaped by evolution to build a set of stereotyped network scaffolds that are largely identical among individuals; life experiences then customize neural circuits in each individual. An essential step towards understanding the architecture, development, function and diseases of the brain is to discover and map its constituent elements of neurons\u00a0and other cell types.5. Neurons are remarkably complex and heterogeneous, both locally and in their long-range axonal projections, which can span the entire brain and connect to many target regions. Many conventional techniques analyse one neuron at a time, and often study only one or two cellular phenotypes in an incomplete way . As a result, despite major advances in past decades, phenotypic analyses of neuron types have remained severely limited in resolution, robustness, comprehensiveness and throughput. Complexities in the relationship between different cellular phenotypes  have fuelled long-standing debates on neuronal classification6.The notion of a\u00a0\u2018neuron type\u2019, with similar properties among its members,\u00a0as the basic unit of brain circuits has been an important concept for over a century; however, rigorous and quantitative definitions have remained surprisingly elusive11. Similarly, single-cell DNA methylation and chromatin accessibility studies have begun to reveal cell-type-specific genome-wide epigenetic landscapes and gene regulatory networks in the brain15. Notably, the scalability and high information content of these methods enable comprehensive quantitative analysis and classification of all cell types, which are readily applicable to brain tissues across species and provide a quantitative means of comparative analysis17.Single-cell genomics technologies provide unprecedented resolution and throughput to measure the transcriptomic and epigenomic profiles of individual cells and have rapidly influenced many areas of biology including neuroscience, promising to catalyse a transformation from phenotypic description and classification to a mechanistic and explanatory molecular genetic framework for the cellular basis of brain organization. The application of single-cell RNA sequencing (scRNA-seq) to the neocortex and other brain regions has revealed a complex but tractable hierarchical organization of transcriptomic cell types that are consistent overall with knowledge from decades of anatomical, physiological and developmental studies but with an unmatched level of granularity19. Imaging-based single-cell transcriptomics and its combination with functional imaging, and integration of electrophysiology and single-cell sequencing, enable mapping of the spatial organization and key phenotypic properties of molecularly defined cell types24. Finally, molecular classification of cell types enables genetic access to specific cell types using transgenic mice27 and, more recently, enhancer-based viral vectors32. All of these methods have been applied to brain tissues in independent studies, but not yet in a coordinated fashion to establish how different modalities correspond with one another, and whether a molecular genetic framework is explanatory for other functionally important cellular phenotypes.Other recent technological advances provide the resolution and throughput to analyse whole-brain neuronal morphology and comprehensive projection mapping33. A key concept is the Brain Cell Census, similar conceptually to a population census, that defines the constituent neuronal and non-neuronal cell types and their proportions, spatial distributions and defining phenotypic characteristics. This cell-type classification, organized as a taxonomy, should aim for consensus across modalities and across mammalian species for conserved types. Beyond the cell census, a Brain Cell Atlas would be embedded in a 3D common coordinate framework (CCF) of the brain34, in which the precise location and distribution of all cell types and their multi-modal features are registered and displayed. This spatial framework facilitates integration, interpretation and navigation of various types of information for understanding brain network organization and function.The overarching goal of the BRAIN Initiative Cell Census Network (BICCN) is to leverage these technologies to generate an open-access reference brain cell atlas that integrates molecular, spatial, morphological, connectional and functional data for describing cell types in mouse, human and non-human primate36. We describe a synthesis of eleven companion studies through a coordinated multi-laboratory effort. In these studies, we derive a cross-species consensus molecular taxonomy of cell types using scRNA-seq or single-nucleus RNA sequencing (snRNA-seq), DNA methylation and chromatin accessibility data40. In mouse, we map the spatial cellular organization by multiplexed error-robust fluorescence in situ hybridization (MERFISH)41, characterize morphological and electrophysiological properties by multimodal profiling using patch clamp recording, biocytin staining and scRNA-seq (Patch-seq)43, describe the cellular input\u2013output wiring diagrams by anterograde and retrograde tracing44, identify glutamatergic neuron axon projection patterns by Epi-retro-seq45, Retro-MERFISH41 and single-neuron complete morphology reconstruction46, and describe transgenic driver lines targeting glutamatergic cell types on the basis of marker genes and lineages47. Finally, we integrate this information into a cohesive description of cell types in MOp. These datasets are organized by the BRAIN Cell Data Center (BCDC) and made public through the BICCN web portal (https://www.biccn.org). Key concepts and terms are described in Extended Data Table Here we present the cell census and atlas of cell types in the primary motor cortex  of mouse, marmoset and human , suggesting another level of regulation in defining single-cell connectional specificity.Cell-type transcriptional and epigenetic signatures guide the generation of genetic tools for targeting glutamatergic pyramidal neuron types and fate mapping their progenitor types.Multi-site coordination within BICCN and data archives enabled a high degree of standardization, computational integration and creation of open data resources for community dissemination of data, tools and knowledge.Major findings:37. The combined sc/snRNA-seq datasets contained a\u00a0large number of cells profiled using both droplet-based and deep full-length sequencing methods  and LIGER, to combine the transcriptomic and epigenomic datasets and derive an integrated molecular taxonomy consisting of 56 neuronal cell types 37  and transcriptional regulatory networks. Similarly, we established M1 cell-type taxonomies for human (127 t-types) and marmoset (94 t-types) by unsupervised clustering of snRNA-seq data, followed by integration with epigenomic datasets38.A mouse MOp molecular taxonomy was derived from seven scRNA-seq and snRNA-seq (sc/snRNA-seq) datasets and single-nucleus methylcytosine sequencing (snmC-seq2) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) datasets)37 Fig. . This inLamp5, Sncg and Vip) and three medial ganglionic eminence (MGE)-derived subclasses ), layer and projection pattern in mouse for glutamatergic excitatory neurons , extratelencephalic (ET), corticothalamic (CT), near-projecting (NP) and layer 6b (L6b)), and non-neuronal functional subclasses  .To establish a consensus classification of MOp and M1 cell types among mouse, human and marmoset, we integrated snRNA-seq datasets across species and identified 45 conserved t-types, including 24 GABAergic (\u03b3-aminobutyric acid-producing), 13 glutamatergic and 8 non-neuronal types , whereas others aligned several-to-several . This may reflect over- or under-clustering, limitations in aligning highly similar cell types, or species-specific expansion of cell-type diversity.The resolution of this cross-species consensus taxonomy was lower than that derived from each species alone, owing to variation in gene expression across species. The degree of species alignments varied across consensus types Fig. ; some tyn\u2009=\u200940 nuclei) were under-sampled compared with marmoset (n\u2009=\u2009463) and mouse , and average expression was not adequately estimated38.We expected that cell types from more recent common ancestors would share more similar gene expression profiles. Indeed, transcriptomic profiles of consensus cell types were more correlated between human and marmoset, and had 25\u201350% fewer differentially expressed genes than between primate and mouse Fig. . The oneGlutamatergic subclasses expressed 50\u2013450 marker genes and, unexpectedly, the majority of markers were species-enriched Fig. . This ev51, to identify cell types in situ and map their spatial organization. We selected a panel of 258 genes  on the basis of prior knowledge of marker genes for major cortical cell types and genes identified using sc/snRNA-seq data, and we imaged approximately 300,000 individual cells across MOp and adjacent areas41.We used MERFISH, a single-cell transcriptome imaging methodLamp5, Sncg, Vip, Sst and Pvalb subclasses) and good correspondence at cluster level41.Clustering analysis of the MERFISH-derived single-cell expression profiles resulted in a total of 95 cell clusters in MOp  Fig. , which s41. Notably, IT cells, the largest branch of neurons in the MOp, formed a largely continuous gradient of cells with correlated gradual changes between their expression profiles and their cortical depths41  identified projection targets of different neuron types in the MOp41 Fig. . Retrogr 41 Fig. . Overall42. We mapped these cells to the mouse MOp transcriptomic taxonomy37  and obtained their transcriptomes using Smart-seq2 sequencingy37 Fig. . Cells wy37 Fig. , therebyy37 Fig. .Fig. 3CoSst interneurons were often characterized by large membrane time constants, pronounced hyperpolarization sag, and rebound firing after stimulation offset. However, within each subclass, there was substantial variation in morpho-electric properties between t-types. This variation was not random but organized such that transcriptomically similar t-types had more similar morpho-electric properties than distant t-types. For example, excitatory t-types from the IT subclasses with more similar transcriptomes were also located at adjacent cortical depths, suggesting that distance in t-space co-varied with anatomical distance42, even within a layer , and cortico-subcortical projecting neurons in L5 ET. Many cortico-thalamic projecting neurons were also observed in L6 CT  levels to integrate the L5 ET Epi-retro-seq cells with the ALM Retro-seq cells and observed enrichment of MY-projecting cells in the same cluster45.Enrichment of L5 ET neurons with Epi-retro-seq (40.2% versus 5.62% in unbiased profiling of MOp using snmC-seq2) enabled investigation of subtypes of L5 ET neurons known to project to multiple subcortical targets in TH, VTA+SN, pons and MYers Fig. . MY-projers Fig. , in agre12. We identified 511 differentially CH-methylated genes (CH-DMGs) and 58,680 CG-DMRs across the L5 ET clusters  are reliable markers of regulatory elements such as enhancersers Fig. . We alsoers Fig. . For exa25. Here we present a genetic toolkit for dissecting and fate-mapping glutamatergic pyramidal neuron (PyN) subpopulations largely on the basis of their developmental genetic programs.Genetic access to specific neural subpopulations and progenitors is necessary for multi-modal analyses to validate t-types, fate-map their developmental trajectories, and study their function in circuit operation55 12.5 RGs in the dorsal neuroepithelium, distributed along a medial-high and lateral-low gradient, consistent with their mRNA expression at this stage60. These RGs generated PyNs across all cortical layers, suggesting multipotency  generate PyNs either directly or indirectly through intermediate progenitors\u00a0(IPs)55 Fig. . Temporanes Fig.  that faincy Fig. .Fig. 5GePlxnd1 (ITPlxnd1), L5b and L6 for ET-Fezf2 (ETFezf2), L6 for CT-Tle4 (CTTle4). Anterograde projection tracing in MOp of adult animals demonstrated that ITPlxnd1 projected to multiple ipsilateral and contralateral cortical areas and to STR/caudate putamen (CP); ETFezf2 projected robustly to several ipsilateral cortical sites, CP and numerous subcortical targets including TH, MY and corticospinal tract; CTTle4 projected specifically to a set of thalamic nuclei47  neurons resided in L5a and projected ipsilaterally to multiple cortical areas, contralaterally to homotypic and heterotypic areas, and bilaterally to CP  neurons resided in L2; although they also projected to ipsilateral cortical and striatal targets, and to homotypic contralateral cortex, they extended minimal projections to heterotypic contralateral cortex and CP ) and retrograde (cholera toxin b (CTb)) tract tracing44  region using classic anterograde , ET  and CT (Ntsr1 and Tle4) subclasses with distinct laminar distributions62.We generated a fine-grained areal and laminar distribution map of multiple MOp-ul projection neuron populations using retrograde tracing44 , ventral anterior-lateral complex (VAL) and ventral medial nucleus (VM)) and cortical areas such as MOs and SSp.Viral tracers were used to systematically examine MOp-ul cell subclass-specific inputs and outputs46, augmented with publicly available single-cell reconstructions from the Janelia Mouselight project18. Additional analysis was also conducted using BARseq64. This analysis revealed a rich diversity of projection patterns within the IT, ET and CT subclasses , Rspo1 for L4/5 IT (1), Htr2c for L4/5 IT (2-3), and Rorb for L4/5 IT and L5 IT  and uniform manifold approximation and projection (UMAP) embedding of all IT neurons (excluding the highly distinct L6 IT Car3 cells) from 11 mouse molecular datasets, including 6 sc/snRNA-seq datasets, and the snmC-seq2, snATAC-seq, Epi-retro-seq, MERFISH and Patch-seq data Fig. . This repo1 Fig. , a L4 cevel Fig. , probabl9 after co-clustering all the SMART-seq glutamatergic transcriptomes from both regions ons Fig. . In UMAP42, cells from the L4/5 IT_1 type had no or minimal apical dendrites without tufts in L1, in contrast to cells from the L2/3 IT, L4/5 IT_2 and L5 IT types, which had tufted apical dendrites  exhibited two local morphological features typical of L4 neurons from sensory cortices  type and Npnt for SCF L5 ET (2-3) types  display a largely gradual transition across cortical depth or layers   and master transcription factors specific to neuronal subclasses in the combined transcriptomic and epigenomic datasets Fig. . We founUnderstanding the principles of brain circuit organization requires a detailed understanding of its basic components. The current effort combines a wide array of single-cell-based techniques to derive a robust and comprehensive molecular cell-type classification and census of the primary motor cortex of mouse, marmoset and human, coupled with a spatial atlas of cell types and an anatomical input\u2013output wiring diagram in mouse. We demonstrate the robustness and validity of this classification through strong correlations across cellular phenotypes, and strong conservation across species. Together these data comprise a cell atlas of the primary motor cortex that encompasses a comprehensive reference catalogue of cell types, their proportions, spatial distributions, anatomical and physiological characteristics, and molecular genetic profiles, registered into a CCF. This cell atlas establishes a foundation for an integrative study of the architecture and function of cortical circuits akin to reference genomes for studying gene function and genome regulatory architecture. Furthermore, it provides a map of the genes that contribute to cellular phenotypes and their epigenetic regulation. These data resources and associated tools enabling genetic access for manipulative experimentation are publicly available. This body of work provides a roadmap for exploring cellular diversity and organization across brain regions, organ systems and species.10, our multimodal cross-species study of the primary motor cortex suggests that a general principle of cortical cell-type organization is its hierarchical relationship, whereby high-level classes linked by major branches comprise progressively finer subpopulations connected by minor branches. In this scheme, the higher-level classes and subclasses are categorically and concordantly distinct from each other across modalities, are conserved across species, and probably arise from different developmental programs, such as GABAergic neuron derivatives of different zones of the ganglionic eminences or the layer-selective glutamatergic neurons derived sequentially from progenitors of the cortical plate. At the lower branch levels (types or clusters), however, while certain cell types are highly distinct , distinctions and boundaries among many other clusters can be ambiguous and vary among different modalities.Substantiating previous studies69, is the coexistence of discrete and continuous variations of cell features across modalities at the lower branch level. A compelling example is the continuous and concordant variation of transcriptomic, anatomical and physiological properties along cortical depth within multiple cell populations, including the glutamatergic L2/3\u2013L6 IT and GABAergic Sst and Pvalb subclasses. Although some of the variations may result from technical factors, such as differences in the resolution of measurements across data modalities (with transcriptomics providing the highest granularity at present), a major source of these continuous variations may reflect true biology, supported by the coordinated variation across transcriptomic, spatial, morphological and physiological properties as shown by MERFISH and Patch-seq. Therefore, another emerging principle of cell type organization is the coexistence of discrete and continuous variations that underlie cell-type diversity.In this context, another important finding, consistent with and building on multiple other studies11. Region-specific connectivity patterns of similar molecular types may be a major factor defining the functional specificity of the primary motor cortex.Together, the principles of hierarchical organization comprising discrete classes and types as well as continuum within and across subpopulations represent a more nuanced and biologically realistic description of cell-type landscape, with implications in cell classification and census. For example, the multimodal variations at finer granularity may preclude a fully discretized representation of cell types with consistency across cell phenotypes, and may explain some of the discrepancies in estimated numbers of cell types using different approaches. An intriguing question is whether continuous variations of cell features will increase further or become more discretized in the context of neural circuit operation, converging to a set of distinct functional elements from a more continuous cellular landscape. An example of this is regionalization. We identify a MOp-specific input\u2013output wiring diagram\u2014however, transcriptomic cell types are generally shared between MOp and its neighbouring cortical areasOur findings have major implications for understanding the biological basis of cellular identity towards a more rigorous, quantitative and satisfying definition and classification of cell types. First and foremost, our discovery of the compelling correspondence across molecular genetic, anatomical and physiological features of hierarchically organized cell populations, reflecting developmental origins and mainly conserved across mammalian species, demonstrates the biological validity and genomic underpinning of major cell types. These findings establish a unifying and mechanistic framework of cell-type classification that integrates multi-layered molecular genetic information with multi-faceted phenotypic properties. Thus, single-cell transcriptomics and epigenomics can serve as powerful approaches for establishing a foundational framework of cell types, owing to not only their unparalleled scalability but also to their representation of the underlying molecular genetic programs rooted in development and evolution. Physiological, morphological and connectional characterizations assign functional attributes to cells; their concordance with molecular identities provides strong validation to the molecularly defined cell types, whereas their differential variations reveal additional, probably network- and activity-driven factors that contribute to further refinement of cell types.37. Looking ahead, it is important to note that at more refined levels, the number of cell types that can be distinguished will probably change with additional cellular features characterized at greater breadth and depth using new methods and approaches.While the higher levels of the hierarchy comprise ~around 25 subclasses  that are identified with remarkable consistency across multiple species and experimental modalities, many finer levels of cell properties do not neatly segregate into discrete and consistent sets of cell types with perfect correspondence among data modalities. These include aspects of continuous distributions, species specializations and mismatches between molecular and anatomical phenotypes that may result from developmental events no longer represented in the adult. Different methods provide somewhat different granularity of clustering, and thus different numbers of putative cell types. For example, single-cell transcriptomics identifies around 100 clusters representing the terminal leaves of this hierarchically branched organizationOverall, the landscape of cell types appears to be generated from a combination of specification through evolutionarily driven and developmentally regulated genetic mechanisms, and refinement of cellular identities through intercellular interactions within the network in which the cells are embedded. In this scenario, genetic mechanisms drive intrinsic or cell-autonomous determination of cell fate, as well as progressive temporal generation of cell types from common progenitor pools that explain global similarities and continuous features of cellular phenotypes reflecting developmental gradients. Network influences can drive further phenotypic refinement that may not be reflected in the adult genetic signature\u2014for example for axonal projection and synaptic connectivity that may reflect transient or stochastic developmental events, region or circuit-specific and/or activity or plasticity-dependent modification to form and reshape functionally specific circuits. Future studies focusing on these mechanisms and testing of the ensuing hypotheses will enable a deeper understanding of the nature of variability among related cell types in the mammalian brain.70.Evolutionary conservation is strong evidence of functional significance. The demonstrated conservation of cell types from mouse, marmoset, macaque and human suggests that these conserved types have important roles in cortical circuitry and function in mammals and even more distantly related species. We also find that similarity of cell types varies as a function of evolutionary distance, with substantial species differences that represent either adaptive specialization or genetic drift. For the most part, species specializations tend to appear at the finer branches of the hierarchical taxonomy. This result is consistent with a recent hypothesis in which cell types are defined by common evolutionary descent and evolve independently, such that new cell types are generally derived from existing genetic programs and appear as specializations at the finer levels of the taxonomic treeA surprising finding across all homologous cell types was the relatively high degree of divergence for genes with cell-type-specific expression in a given species. This observation provides a clear path to identify core conserved genes underlying the canonical identity and features of those cell types. Furthermore, it highlights the need to understand species adaptations superimposed on the conserved program, as many specific cellular phenotypes may vary across species including gene expression, epigenetic regulation, morphology, connectivity and physiological properties. As we illustrated in the Betz cells, there is clear homology across species in the L5 ET subclass, but variation in many measurable properties across species.Our findings have major implications for the consideration of model organisms to understand human brain function and disease. Despite major investments, animal models of neuropsychiatric disorders have often been characterized by \u2018loss of translation\u2019, fuelling heated debates about the utility of model organisms in the development of treatments for human diseases. Cell census information aligned across species will be highly valuable for making rational choices about the best models for each disease and therapeutic target. For example, the characterization of cell types and their properties shown in Fig. The approach we took to generate a cell census and atlas through a systematic dissection of cell types opens up numerous avenues for future work. The MOp census and atlas provides a foundational platform for the broad neuroscience community to accumulate and integrate cell-type information across species. Classification of cell types based on their molecular, spatial and connectional properties in the adult sets the stage for developmental studies to understand the molecular genetic programs underlying cell-type specification, maturation and circuit assembly. The molecular genetic information promises to deliver tools for genetic access to many brain cell types via transgenic and enhancer virus strategies. A combination of single-cell transcriptomics and functional measurements may further elucidate the roles of distinct cell types in circuit computation during behaviour, bridging the gap between molecular and functional definition of cell types. The systematic, multi-modal strategy described here can be extended to the whole brain, and major efforts are underway in the BICCN to generate a brain-wide cell census and atlas in the mouse with increasing coverage of human and non-human primates.Car3), the layer 5 neurons projecting to extratelencephalic targets (L5 ET), the CT-projecting neurons in layer 6 (L6 CT), the NP neurons found in layers 5 and 6, and the L6b neurons whose projection patterns remain largely unknown.In this manuscript we have adopted a nomenclature for major subclasses of cortical glutamatergic excitatory neurons, which have long-range projections both within and outside of the cortex, following a long tradition of naming conventions that often classify neurons based on their projection targets. This nomenclature is based on our de novo transcriptomic taxonomy Fig.  that orgWhile the IT, CT, NP and L6b neurons have been consistently labelled as such in the field, the L5 ET neurons have not been named consistently in the literature, largely owing to their large variety of projection targets and other phenotypic features that vary depending on cortical areas and species. Here we use the term L5 ET  to refer to this prominent and distinct subclass of neurons as a standard name that can be accurately used across cortical regions and across species, and we provide our rationale below.48. Accordingly, various names incorporating these features have been adopted to refer to L5 ET versus L5 IT cells, such as L5b versus L5a, thick-tufted versus thin-tufted and burst-firing versus regular-firing. The most common term used to refer to L5 ET cells residing in motor cortical areas has been PT, which refers to neurons projecting to the pyramidal tract. As accurately stated in Wikipedia, \u201cThe pyramidal tracts include both the corticobulbar tract and the corticospinal tract. These are aggregations of efferent nerve fibers from the upper motor neurons that travel from the cerebral cortex and terminate either in the brainstem (corticobulbar) or spinal cord  and are involved in the control of motor functions of the body.\u201dIt has long been appreciated that cortical layer 5 contains two distinct populations of neurons that can be distinguished, not only based on the presence or absence of projections to ET targets (ET and IT cells), but also based on their predominant soma locations, dendritic morphologies and intrinsic physiology73 and prominently highlighted in this manuscript, it is now clear that PT represents only a subset of L5 ET cells and is thus unable to accurately encompass the entire L5 ET subclass. This realization is informed by comparisons across species and cortical areas, and by single-cell transcriptomics and descriptions of the projections of single neurons, as well as studies linking transcriptional clusters to projection targets.Owing to the past wide use of the term PT, we do not take the decision to use L5 ET rather than PT lightly. However, in the face of multiple lines of evidence that have accumulated over the last several years38  PT refers to motor neurons that project into MY or spinal cord, but in many cortical areas  none of the L5 ET cells are motor neurons; and 2) even in the motor cortex many cells in the L5 ET subclass do not project to the pyramidal tract and instead project solely to the TH (or to TH and other non-PT targets). This is revealed by single-neuron reconstructions53 Figs. , 8, BARs even in 49. Given that the telencephalon is equivalent to the cerebrum, ET and subcerebral have the same meaning and the term L5-SCPN would be an accurate and equivalent alternative. But the \u2018L5\u2019 qualifier is crucial in either case to distinguish these cells from the L6 CT subclass. We favour the use of ET because SCPN has not been widely adopted and due to symmetry with the widely used \u2018IT\u2019 nomenclature. Alternatively, given their evidence that \u201cunlike pyramidal tract neurons in the motor cortex, these neurons in the auditory cortex do not project to the spinal cord\u201d, Chen et al64 used the term \u2018pyramidal tract-like\u2019 (PT- l). We also favour L5 ET over L5 PT-l which clings to an inaccurate and now outdated nomenclature.We recognize that another name that has been used to describe L5 ET cells is subcerebral projection neuron (SCPN)38. Code for generating Figs. http://data.nemoarchive.org/publication_release/Lein_2020_M1_study_analysis/Transcriptomics/flagship/. Analysis was performed in RStudio using R version 3.5.3, R packages: Seurat 3.1.1, ggplot2 3.2.1 and scrattch.hicat 0.0.22.To identify homologous cell types across species, human, marmoset and mouse 10x v3 snRNA-seq datasets were integrated using Seurat\u2019s SCTransform workflow. Each major cell class  was integrated separately across species. Expression matrices were reduced to 14,870 one-to-one orthologues across the three species . Nuclei were downsampled to have approximately equivalent numbers at the subclass level across species. Marker genes were identified for each species cluster using Seurat\u2019s FindAllMarkers function with test.use set to \u2018roc\u2019, >\u00a00.7 classification power. Markers were used as input to guide alignment and anchor-finding during integration steps. For full methods see ref. https://github.com/AllenInstitute/BICCN_M1_Evo). The integrated space (from the previously mentioned Seurat integration) was over-clustering into small sets of highly similar nuclei for each class (about 500 clusters per class). Clusters were aggregated into metacells, then hierarchical clustering was performed based on the metacell gene expression matrix using Ward\u2019s method. Hierarchical trees were then assessed for cluster size, species mixing and branch stability by subsampling the dataset 100 times with 95% of nuclei. Finally, we recursively searched every node of the tree, and if certain heuristic criteria were not sufficient for a node below the upper node, all nodes below the upper node were pruned and nuclei belonging to this subtree were merged into one homologous group. We identified 24 GABAergic, 13 glutamatergic and 8 non-neuronal cross-species consensus clusters that were highly mixed across species and robust. For full methods see ref. 38. A final dendrogram of consensus cell types was constructed by transforming the raw unique molecular identifier (UMI) counts to log2(counts per million (CPM)) normalized counts. Up to 50 marker genes per cross-species cluster were identified by using the scrattch.hicat (v0.0.22) (https://github.com/AllenInstitute/scrattch.hicat) display_cl and select_markers functions with the following parameters; q1.th\u2009=\u20090.4, q.diff.th\u2009=\u20090.5, de.score.th\u2009=\u200980. Median cross-species cluster log2 CPM expression of these genes were then used as input for scrattch.hicat\u2019s build_dend function. This analysis was bootstrapped 10,000 times with branch colour denoting confidence. Branch robustness was assessed by rebuilding the dendrogram 10,000 times with a random 80% subset of variable genes across clusters and calculating the proportion of iterations that clusters were present on the same branch. Consensus taxonomy agreement in Fig. To establish a robust cross-species cell type taxonomy, we applied a tree-based clustering method on integrated class-level datasets . For a given cross-species cluster, each sample was split by species and donor, then a Wald test was performed between each species pair. Genes with adjusted P-values\u2009<\u20090.05 and log2 fold-changes greater than 2 in either direction were counted and reported in Fig. Expression matrices were subsetted to include one-to-one orthologous genes across all three species. Spearman correlations shown in Fig. 75 into a target region in INTACT mice76, which turned on Cre-dependent GFP expression in the nuclei of MOp neurons projecting to the injected target region. Individual GFP-labelled nuclei of MOp projection neurons were then isolated using fluorescence-activated nucleus sorting (FANS)  of each single nucleus.We injected retrograde tracer rAAV2-retro-Cre45. Specifically, we quantified the ratio between the number of cells in expected on-target subclasses  versus in expected off-target subclasses , denoted as rp, and compared the ratio with the one expected from the unbiased data without enrichment for specific projections, denoted as ru. This provides an estimation of signal-to-noise ratio of each FANS experiment. For IT projections, we used IT subclasses as on-target and L6 CT + inhibitory as off-target, and for ET projections, we used L5 ET as on-target and IT + inhibitory as off-target. For the MOp neurons without enrichment of projections, the expected ratio between cells in IT subclasses and in L6 CT + inhibitory are ru\u00a0=\u00a02,652:1,775, whereas the expected ratio between cells in L5 ET subclass and in IT + inhibitory are ru\u00a0=\u00a0202:3,434. The fold enrichment in the text was computed by rp/ru for each FANS run separately and averaged across IT or ET targets respectively.The methods used to evaluate contamination level and potential reasons are described in detail in ref. We want to point out that, in addition to this computational method, other methods are available to evaluate and minimize potential contamination in Epi-retro-seq. In cases in which differences in expected results from on- versus off-target populations are unknown, other available methods would need to be used to eliminate cases in which injections might have directly labelled cells outside the intended target region, such as examination of labelling along the injection electrode track.37. The read counts were normalized by the total read counts per cell and log transformed. Top 5,000 highly variable genes were identified with Scanpy78 (v1.8.1) and z-score was scaled across all the cells. For Epi-retro-seq, the posterior methylation levels of 12,261 genes in the 848 L5 ET cells were computed45. Top 5,000 highly variable genes were identified with AllCools79 and z-score was scaled across all the cells. The 1,512 genes as the intersection between the two highly variable gene lists were used in Scanorama80 (v1.7.1) to integrate the z-scored expression matrix and minus z-scored methylation matrix with sigma equal to 100.For snRNA-seq, the 4,515 cells from 10x v3 B dataset labelled as L5 ET by SCF were selected45, or from the integrated clustering of multiple datasets37. The integrated clustering and embedding of the 11 datasets are then generated by projecting all datasets into the 10x v2 scRNA-seq dataset using SingleCellFusion79. Genome browser views of IT and ET cell types . MERFISH data were analysed using custom Python code, which is available at https://github.com/ZhuangLab/MERlin.To integrate IT cell types from different mouse datasets, we first take all cells that are labelled as IT, except for L6_IT_Car3, from the 11 datasets as listed in Fig. es Figs.  are take81 v2.2.7.1. using Python 3.6 with parameter: \u201c--nomodel --shift \u2212100 --ext 200 --qval 1e-2 \u2013B --SPMR\u201d. First, we extended peak summits by 250 bp on either side to a final width of 501 bp. Then, to account for differences in performance of MACS2 based on read depth and/or number of nuclei in individual clusters, we converted MACS2 peak scores ) to \u2018score per million\u201982. Next, a union peak set was obtained by applying an iterative overlap peak-merging procedure, which avoids daisy-chaining and still allows for use of fixed-width peaks. Finally, we filtered peaks by choosing a score per million cut-off of 5 as cCREs for downstream analysis.For peak calling in the snATAC-seq data, we extracted all the fragments for each cluster, and then performed peak calling on each aggregate profile using MACS283 with the following parameters: aggregation k\u2009=\u200950, window size\u2009=\u2009500 kb, distance constraint\u2009=\u2009250 kb. In order to find an optimal co-accessibility threshold, we generated a random shuffled cCRE-by-cell matrix as background and calculated co-accessible scores from this shuffled matrix. We fitted the distribution of co-accessibility scores from random shuffled background into a normal distribution model by using the R package fitdistrplus84. Next, we tested every co-accessible cCRE pair and set the cut-off at co-accessibility score with an empirically defined significance threshold of FDR\u00a0<\u00a00.01. The cCREs outside of \u00b11 kb of transcriptional start sites in GENCODE mm10 (v16) were considered distal. Next, we assigned co-accessibility pairs to three groups: proximal-to-proximal, distal-to-distal and distal-to-proximal. In this study, we focus only on distal-to-proximal pairs. We calculated the Pearson\u2019s correlation coefficient (PCC) between gene expression (scRNA SMART-seq) and cCRE accessibility across the joint clusters to examine the relationships between the distal cCREs and target genes as predicted by the co-accessibility pairs. To do so, we first aggregated all nuclei or cells from scRNA-seq and snATAC-seq for every joint cluster to calculate accessibility scores (log2 CPM) and relative expression levels (log2 transcripts per million). Then, PCC was calculated for every gene-cCRE pair within a 1-Mbp window centred on the transcriptional start sites for every gene. We also generated a set of background pairs by randomly selecting regions from different chromosomes and shuffling the cluster labels. Finally, we fit a normal distribution model on background and defined a cut-off at PCC score with an empirically defined significance threshold of FDR\u00a0<\u00a00.01, in order to select significant positively correlated cCRE-gene pairs.First, co-accessible cCREs are identified for all open regions in all neuron types (cell clusters with less than 100 nuclei from snATAC-seq are excluded) using Cicerocis-regulatory modules based on their relative accessibility across cell clusters. We adapted NMF (Python package: sklearn v.0.24.2) to decompose the cluster-by-cCRE matrix V  into a coefficient matrix H  and a basis matrix W (N\u00a0\u00d7\u00a0R), with a given rank R: V\u00a0\u2248\u00a0WH.We used nonnegative matrix factorization (NMF) to group cCREs into R . Several criteria have been proposed to decide whether a given rank R decomposes the occupancy profile matrix into meaningful clusters. Here we applied a measurement called sparseness85 to evaluate the clustering result. Median values were calculated from 100 times for NMF runs at each given rank with a random seed, which will ensure the measurements are stable. Next, we used the coefficient matrix to associate modules with distinct cell clusters. In the coefficient matrix, each row represents a module and each column represents a cell cluster. The values in the matrix indicate the weights of clusters in their corresponding module. The coefficient matrix was then scaled by column (cluster) from 0 to 1. Subsequently, we used a coefficient\u2009>\u20090.1 (~95th percentile of the whole matrix) as a threshold to associate a cluster with a module. Similarly, we associated each module with accessible elements using the basis matrix. For each element and each module, we derived a basis coefficient score, which represents the accessible signal contributed by all clusters in the defined module.The basis matrix defines module related accessible cCREs, and the coefficient matrix defines the cell cluster components and their weights in each module. The key issue to decompose the occupancy profile matrix was to find a reasonable value for the rank P-value\u2009<\u00a00.05, cluster average fold change\u2009>\u00a02). To perform the motif enrichment analysis, we used known motifs from the JASPAR 2020 database86 and the subclass specific hypo-CG-DMR identified in Yao et al.37. The AME software from the MEME suite (v5.1.1)87 was used to identify significant motif enrichment  using default parameters and the same background region set as described37. All genes in Extended Data Fig. All analyses for this section were at the subclass level. For RNA expression, we used the scSMART-seq dataset and compared each subclass with the rest of the population through a one-tailed Wilcoxon test and FDR correction to select significantly differentially expressed transcription factors  of Cold Spring Harbor Laboratory, University of California Berkeley and Allen Institute, in accordance with NIH guidelines. Mouse knockin driver lines are being deposited to the Jackson Laboratory for wide distribution.Tle4-2A-CreER, Fezf2-2A-CreER, PlexinD1-2A-CreER, PlexinD1-2A-Flp and Tbr2-2A-CreER were generated by inserting a 2A-CreER or 2A-Flp cassette in-frame before the STOP codon of the targeted gene. Targeting vectors were generated using a PCR-based cloning approach47. In brief, for each gene of interest, two partially overlapping BAC clones from the RPCI-23&24 library (made from C57BL/b mice) were chosen from the Mouse Genome Browser. 5\u2032 and 3\u2032 homology arms were PCR amplified  using the BAC DNA as template and cloned into a building vector to flank the 2A-CreERT2 or 2A-Flp expressing cassette as described27. These targeting vectors were purified, tested for integrity by enzyme restriction and PCR sequencing. Linearized targeting vectors were electroporated into a 129SVj/B6 hybrid ES cell line (v.6.5). ES cell clones were first screened by PCR and then confirmed by Southern blotting using appropriate probes. DIG-labelled Southern probes were generated by PCR, subcloned and tested on wild-type genomic DNA to verify that they give clear and expected results. Positive v6.5 ES cell clones were used for tetraploid complementation to obtain male heterozygous mice following standard procedures. The F0 males and subsequent generations were bred with reporter lines  and induced with tamoxifen at the appropriate ages to characterize the resulting genetically targeted recombination patterns. Drivers Tle4-2A-CreER, Fezf2-2A-CreER and PlexinD1-2A-CreER were additionally crossed with reporter Rosa26-CAG-LSL-Flp and Tbr2-2A-CreER;PlexinD1-2A-Flp with reporter dual-tTA, and induced with tamoxifen at the appropriate age to perform anterograde viral tracing, with Flp- or tTA-dependent AAV vector expressing EGFP (AAV8-CAG-fDIO-TVA-EGFP or AAV-TRE-3g-TVA-EGFP), to characterize the resulting axon projection patterns.Driver and reporter mouse lines were generated using a PCR-based cloning. Knockin mouse lines Npnt and Slco2a1, respectively, using ribonucleoprotein (RNP) complexes composed of SpCas9-NLS protein and in vitro transcribed sgRNA (Npnt: GATGATGTGAGCTTGAAAAG and Slco2a1: CAGTCTGCAGGAGAATGCCT). These RNP complexes were nucleofected into 106 v6.5 mouse embryonic stem cells  along with repair constructs in which P2A-FlpO or P2A-Cre was flanked with the following sequences homologous to the target site, thereby enabling homology-directed repair.To generate lines bearing in-frame genomic insertions of P2A-FlpO or P2A-Cre, we engineered double-strand breaks at the stop codons of Npnt-P2A-FlpO: TGGCCCTTGAGCTCTAGTGTTCCCACTTGCCATAGAAATCTGATCTTCGGTTTGGGGGAAGGGTTGCCTTACCATGCTCCATGAGTGAGCACTGGGAAAAGGGGCAGAGGAGGCCTGACCAGTGTATACGTTCTCTCCCTAGGTCATCTTCAAAGGTGAAAAAAGGCGTGGTCACACGGGGGAGATTGGATTGGATGATGTGAGCTTGAAGCGCGGAAGATGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCTCCTAAGAAGAAGAGGAAGGTGATGAGCCAGTTCGACATCCTGTGCAAGACCCCGCCGAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAAAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCGATCAAGAGGGCCACCTTCATGAGTTATAACACCATCATCAGCAACAGCCTGAGTTTTGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCATGGGAGTTCACGATTATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAAAGCAGCGAGGAGGCCGACAAGGGGAATAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAAGGCGAGAGCATCTGGGAGATTACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGATTTACCAAAACGAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCGAAGAGCTTCAAGCTCGTCCAGAACAAGTATCTGGGCGTGATCATTCAGTGCCTGGTCACGGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATTAAAAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGTAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATTATAAGCCAGGAGGTGCTGGACTACCTGAGCAGCTACATCAACAGGCGGATCTGAAAGAGGTCGCTGCTGAGAAGACCCCTGGCAGCTCCCGAGCTAGCAGTGAATTTGTCGCTCTCCCTCATTTCCCAATGCTTGCCCTCTTGTCTCCCTCTTATCAGGCCTAGGGCAGGAGTGGGTCAGGAGGAAGGTTGCTTGGTGACTCGGGTCTCGGTGGCCTGTTTTGGTGCAATCCCAGTGAACAGTGACACTCTCGAAGTACAGGAGCATCTGGAGACACCTCCGGGCCCTTCTGSlco2a1-P2A-Cre:TGCCCCTGGGCCTCACCATACCTGTCTCTTCCTGCCTCATAGGTACCTGGGCCTACAGGTAATCTACAAGGTCTTGGGCACACTGCTGCTCTTCTTCATCAGCTGGAGGGTGAAGAAGAACAGGGAATACAGTCTGCAGGAGAATGCTTCCGGATTGATTGGAAGCGGAGCTACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCTGGACCTATGGCTCCTAAGAAGAAGAGGAAGGTGATGAGCCAGTTCGACATCCTGTGCAAGACTCCTCCAAAGGTGCTGGTGCGGCAGTTCGTGGAGAGATTCGAGAGGCCCAGCGGCGAGAAGATCGCCAGCTGTGCCGCCGAGCTGACCTACCTGTGCTGGATGATCACCCACAACGGCACCGCCATCAAGAGGGCCACCTTCATGAGCTACAACACCATCATCAGCAACAGCCTGAGCTTCGACATCGTGAACAAGAGCCTGCAGTTCAAGTACAAGACCCAGAAGGCCACCATCCTGGAGGCCAGCCTGAAGAAGCTGATCCCCGCCTGGGAGTTCACCATCATCCCTTACAACGGCCAGAAGCACCAGAGCGACATCACCGACATCGTGTCCAGCCTGCAGCTGCAGTTCGAGAGCAGCGAGGAGGCCGACAAGGGCAACAGCCACAGCAAGAAGATGCTGAAGGCCCTGCTGTCCGAGGGCGAGAGCATCTGGGAGATCACCGAGAAGATCCTGAACAGCTTCGAGTACACCAGCAGGTTCACCAAGACCAAGACCCTGTACCAGTTCCTGTTCCTGGCCACATTCATCAACTGCGGCAGGTTCAGCGACATCAAGAACGTGGACCCCAAGAGCTTCAAGCTGGTGCAGAACAAGTACCTGGGCGTGATCATTCAGTGCCTGGTGACCGAGACCAAGACAAGCGTGTCCAGGCACATCTACTTTTTCAGCGCCAGAGGCAGGATCGACCCCCTGGTGTACCTGGACGAGTTCCTGAGGAACAGCGAGCCCGTGCTGAAGAGAGTGAACAGGACCGGCAACAGCAGCAGCAACAAGCAGGAGTACCAGCTGCTGAAGGACAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCATCTTCGCTATCAAGAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACCAGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTGGAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATCACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGACCCCATCAGCAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAGGAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGATACCCCGCCTGGAACGGCATCATCAGCCAGGAGGTGCTGGACTACCTGAGCAGCTACATCAACAGGCGGATCTGACCTTCAGCTGGGACTACTGCCCTGCCCCAGAGACTGGATATCCTACCCCTCCACACCTACCTATATTAACTAATGTTAGCATGCCTTCCTCCTCCTTCCNpnt-P2A-FlpO left homology arm; GATTGAGGTCAGGCCAGAAG and TCGACATCGTGAACAAGAGC for Npnt-P2A-FlpO right homology arm; CTGGTGAAAGGGGAACTCTTGCT and GATCCCTGAACATGTCCATCAGG for Slco2a1-P2A-Cre left homology arm; TACAGCATCCCTGACAAACACCA and TAGCACCGCAGGTGTAGAGAAGG for Slco2a1-P2A-Cre right homology arm.Transfected cells were cultured and resulting colonies directly screened by PCR for correct integration using the following genotyping primers: flanking primer ATGCATTGCTTCATGCCATA and internal recombinase primer CCTTCAGCAGCTGGTACTCC for The inserted transgenes were fully sequenced and candidate lines were analysed for normal karyotype. Lines passing quality control were aggregated with albino morulae and implanted into pseudopregnant females, producing germline-competent chimeric founders which in turn were crossed with the appropriate reporter lines on the C57/BL6 background.All experimental procedures using live animals were performed according to protocols approved by Institutional Animal Care and Use Committees (IACUC) of all participating institutions: Allen Institute for Brain Science, Baylor College of Medicine, Broad Institute of MIT and Harvard, Cold Spring Harbor Laboratory, Harvard University, Salk Institute for Biological Studies, University of California Berkeley, University of California San Diego and University of Southern California. Macaque experiments were performed on animals designated for euthanasia via the Washington National Primate Research Center\u2019s Tissue Distribution Program.Postmortem adult human brain tissue collection was performed in accordance with the provisions of the United States Uniform Anatomical Gift Act of 2006 described in the California Health and Safety Code section 7150 (effective 1 January 2008) and other applicable state and federal laws and regulations. The Western Institutional Review Board reviewed tissue collection processes and determined that they did not constitute human subjects research requiring institutional review board (IRB) review. Before commencing the human Patch-seq, the donor provided informed consent and experimental procedures were approved by the hospital institute review board.Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03950-0.Reporting SummarySupplementary Table 1Confusion matrices corresponding to Extended Data Figure 5.Supplementary Table 2Cell type nomenclature.Peer Review File"}
+{"text": "Deroplatys truncate, D. lobate, Amorphoscelis chinensis and Macromantis sp. belong to Deroplatyidae, Amorphoscelidae and Photinaidae family, respectively. Our results indicated that the ATP8 gene may be lost in D. truncate and D. lobata mt genome, and four tRNA genes have not been found in D. truncate, D. lobata and Macromantis sp. A dN/dS pair analysis was conducted and it was found that all genes have evolved under purifying selection. Furthermore, we tested the phylogenetic relationships between the eight families of the Mantodea, including 35 species of praying Mantis. Based on the complete mitochondrial genome data, it was also suggested as sister to Deroplatyidae + Mantidae, Metallyticus sp., the only representative of Metallyticidae, is sister to the remaining mantises. Our results support the taxonomic system of Schwarz and Roy and are consistent with previous studies.Praying mantises are distributed all over the world. Though some Mantodea mitogenomes have been reported, an evolutionary genomic and phylogenetic analysis study lacks the latest taxonomic system. In the present study, four new mitogenomes were sequenced and annotated.  The praying mantis comprises up to 2,300 species having diversified morphological and ecological characteristics. These insects colonize at wider range of habitats, including arid and tropical rainforests, temperate regions and engaged in multiple hunting tactics , 2. PrayThe mitochondrial genome (mitogenome), as a robust molecular marker , has recAmorphoscelis belongs to the subfamily Amorphoscelinae, which can be distinguished from all other mantis through some characters such as: small, dorsoventrally flattened body, and adapted to a bark-living lifestyle Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0NoReviewer #2:\u00a0No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0In the manuscript titled 'Complete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analyses' the author present the complete mitochondrial genome of four species of mantid. Overall the study is good and should be published but some revisions need to be addressed before publishing.1) The authors need to have a native English speaker review the document for grammar. It is not unintelligible but there are many spots where it is difficult to follow or does not sound scientific.2) Throughout the document, the species D. truncata and D. lobata are converted to truncate and lobate due to autcorrect from their word processor . It is annoying but an easy fix.3) I would like to see a table or tables that list the gene order and position for all ORFs for each species, this generally is a useful reference for other readers.Reviewer #2:\u00a0The authors sequence and annotate the mitogenomes of four mantises species and combine this new data with existing mantis data to estimate phylogenetic relationships in the group. They find a few missing genes in some of the four new mitogenomes, but tRNA secondary structure and codon usage was similar to other mantises. The phylogeny also tended to agree with current taxonomy.I think the study is definitely a worthwhile addition to insect mitogenomics. I particularly appreciate that you discuss your findings in the context of insect and mantis mitogenomics. However, I do have several issues that should be addressed, some more major then others:1. You need to re-work some of the structure in the Introduction. For example, the second paragraph introduced mitogenomes and then abruptly switches to introducing the taxonomy of mantises. I would separate these.2. Because the Methods comes last, I would include some brief methodological details in the Results and Discussion section.3. Please include more specific results. For example, you vaguely state that there is a high A+T% and list the most-used codons. Please provide specific values in the text.4. You begin discussing the utility of using cox1 as a barcoding gene (starting line 151), but this discussion appears abruptly. Why is this relevant? Is it related to your nucleotide diversity results? Please explain.5. I\u2019m a little confused by your sampling strategy. Did you sample the 35 mantis species mentioned in lines 189-190? Or were these taken from GenBank?6. I am not convinced you are actually missing the atp8 genes. In my experience, it can be difficult to annotate this gene, and you often have to look for it manually based on ORFs, etc. It also seems like there is a non-coding region on the 5\u2019 end of your atp6 genes when a \u201cmissing\u201d atp8 is reported. I would bet that is the atp8 gene. Also, were your missing genes confirmed with your PCR step? Finally, it looks like both atp6 and atp8 are missing from one species , but I didn\u2019t see any discussion of this. Please elaborate.7. Did you confirm that these assembled mitogenomes are in fact complete and circular? I recommend doing this, especially since you report missing genes.8. Please provide more methodological details. For example, how did you calculate Ka/KS? What were your PCR conditions? What GBlocks parameters did you use?9. There are several portions of the manuscript that are difficult to read. I recommend going through the text and again and edit for grammar, awkward phrasing, etc.Some minor comments:Line 25: There is no context for referencing the \u201cSchwarz and Roy (2019) taxonomic system.\u201d Perhaps just say \u201cthe latest taxonomy.\u201dLine 31: No need to say \u201crespectively.\u201d Same in line 82.Line 42: Be careful about using \u201cbugs\u201d when you mean \u201cinsects.\u201d Entomologists will strongly object to this usage\u2026Lines 74-78 should be in the IntroductionLine 100: Why do you rule out this second possibility? Please elaborate.Line 144: Please provide a brief overview of dN/dS ratio .Line 217: Confusing phrasing. Please re-write. It\u2019s not clear what \u201cminiscule to align means.\u201dFigure 1: It\u2019s not clear which mitogenomes go with each species. Please add names to the figure.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 24 May 20214/5/2021Dear Editor, Thank you very much for your email message regarding our manuscript. We appreciated much the valuable comments from the reviewers for revising and improving our manuscript. Generally, we have incorporated all suggestions and comments of yours into this revision. More specifically, our corrections are as follows here under.We hope you can now accept this revised version. Most sincerely,All authorsReviewer #1: In the manuscript titled 'Complete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analyses' the author present the complete mitochondrial genome of four species of mantid. Overall the study is good and should be published but some revisions need to be addressed before publishing.1) The authors need to have a native English speaker review the document for grammar. It is not unintelligible but there are many spots where it is difficult to follow or does not sound scientific.Reply: We thank this referee for his/her suggestions. We have polished the full text and further revised the corresponding parts. 2) Throughout the document, the species D. truncata and D. lobata are converted to truncate and lobate due to autcorrect from their word processor . It is annoying but an easy fix.Reply: Thank you. 3) I would like to see a table or tables that list the gene order and position for all ORFs for each species, this generally is a useful reference for other readers.Reply: We agree and have added the related information on the position of all ORFs. See Table S2. For the gene order, we refer to Figure 1 with the mitochondrial gene structure.Reviewer #2: The authors sequence and annotate the mitogenomes of four mantises species and combine this new data with existing mantis data to estimate phylogenetic relationships in the group. They find a few missing genes in some of the four new mitogenomes, but tRNA secondary structure and codon usage was similar to other mantises. The phylogeny also tended to agree with current taxonomy.I think the study is definitely a worthwhile addition to insect mitogenomics. I particularly appreciate that you discuss your findings in the context of insect and mantis mitogenomics. However, I do have several issues that should be addressed, some more major then others:1. You need to re-work some of the structure in the Introduction. For example, the second paragraph introduced mitogenomes and then abruptly switches to introducing the taxonomy of mantises. I would separate these.Reply: Agree. We have separated the two parts. Please check the line 53.2. Because the Methods comes last, I would include some brief methodological details in the Results and Discussion section.Reply: Thank you. We have added the brief methodological details in the Results and Discussion section. Please check lines 78-83. 3. Please include more specific results. For example, you vaguely state that there is a high A+T% and list the most-used codons. Please provide specific values in the text.Reply: Agree. We have added the whole genome, A+T-rich region and PCGs specific values of four mitochondrial genomes. Please check Table S2.4. You begin discussing the utility of using cox1 as a barcoding gene (starting line 151), but this discussion appears abruptly. Why is this relevant? Is it related to your nucleotide diversity results? Please explain.Reply: Thank you. The dN/dS pairwise analysis showed that cox1 is undergoing a robust purifying selection (0.028). That is why cox1 can be used as a molecular marker of barcoding entire Mantidae. We also reorganized the writing of this part. Please check lines 152-153.5. I\u2019m a little confused by your sampling strategy. Did you sample the 35 mantis species mentioned in lines 189-190? Or were these taken from GenBank?Reply: We have reorganized the writing of this part. Please check lines 189-193 and Table 1.6. I am not convinced you are actually missing the atp8 genes. In my experience, it can be difficult to annotate this gene, and you often have to look for it manually based on ORFs, etc. It also seems like there is a non-coding region on the 5\u2019 end of your atp6 genes when a \u201cmissing\u201d atp8 is reported. I would bet that is the atp8 gene. Also, were your missing genes confirmed with your PCR step? Finally, it looks like both atp6 and atp8 are missing from one species , but I didn\u2019t see any discussion of this. Please elaborate.Reply: Thank you for this advice. We know ATP8 which is difficult to annotate. We repeated to analysis the sequence for annotation of ATP8, and in addition we ran the PCR to amplify the corresponding part of about 1000bp to confirm the sequence. Unfortunately, we could not annotate the relevant homologous sequences. For the ATP6 of Macromantis sp. species, we have added this annotation name in Figure 1  and the Table S2. Please check Figure 1 and Table S2. 7. Did you confirm that these assembled mitogenomes are in fact complete and circular? I recommend doing this, especially since you report missing genes.Reply: We thank this referee for his/her comment. We received the assembled mitogenomes sequence and this was confirmed by PCR technology. The starting and the ending of sequence overlapped with 184bp, so we are sure that this is a complete and circular structure. We redesigned the primers for PCR and sequenced the PCR product to confirm the sequencing results. The primers are listed in Table S1. We also used ORF Finder and BLAST search engines against the GenBank database to identify protein-coding and rRNA genes. Please check the lines 206-208.8. Please provide more methodological details. For example, how did you calculate Ka/KS? What were your PCR conditions? What GBlocks parameters did you use?Reply: We agree and have added the methods of calculating Ka/KS. Please check lines 219-221. We also added the conditions of long PCR reactions. Please check lines 213-217. Also the GBlocks parameters were added in lines 237-239.9. There are several portions of the manuscript that are difficult to read. I recommend going through the text and again and edit for grammar, awkward phrasing, etc.Reply: Thanks for your suggestion. We polished the text and revised the corresponding parts. Some minor comments:Line 25: There is no context for referencing the \u201cSchwarz and Roy (2019) taxonomic system.\u201d Perhaps just say \u201cthe latest taxonomy.\u201dReply: Done; please check lines 26-27Line 31: No need to say \u201crespectively.\u201d Same in line 82.Reply: Done; please check lines 31, 84Line 42: Be careful about using \u201cbugs\u201d when you mean \u201cinsects.\u201d Entomologists will strongly object to this usage\u2026Reply: Done, please check line 42.Lines 74-78 should be in the IntroductionReply: Agree. We have moved this text to the Introduction. Please check line 64-68.Line 100: Why do you rule out this second possibility? Please elaborate.Reply: We apologize for this. Indeed the second possibility is also very likely. So, we have reorganized this text part. We have deleted our conclusion: \u201cWe concluded that a lack of the atp8 gene in D. truncate and D. lobate is most likely because of the former or the later reason.\u201d Please check lines 112-113. Thank you.Line 144: Please provide a brief overview of dN/dS ratio .Reply: Agree. Please check lines 151-152.Line 217: Confusing phrasing. Please re-write. It\u2019s not clear what \u201cminiscule to align means.\u201dReply: We thank this referee and have rephrased this part text, which means Nad4L is too short to align among the Mantodea species. ATP8 was not identified in D. truncate and D. lobate. So nad4L and atp8 were excluded in the phylogenetic analysis. Please check lines 238-241. Figure 1: It\u2019s not clear which mitogenomes go with each species. Please add names to the figure.Reply: Thanks and done.AttachmentR1-rebuttal letter-final.docxSubmitted filename: Click here for additional data file. 25 Jun 2021PONE-D-21-03889R1Complete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analysesPLOS ONEDear Dr. Smagghe,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Minor issues were raised which can be considered since they will improve the manuscript somewhat.plosone@plos.org. 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For instructions see:\u00a0We look forward to receiving your revised manuscript.Kind regards,Ben J Mans, PhDAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #2:\u00a0(No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #2:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2:\u00a0Thank you for your thorough responses and edits to your manuscript based on my comments. I think the manuscript has been improved. I do have a few comments that still should be addressed, based on my first round of comments:1. I still think the paragraphs in the Introduction are a little disconnected. Perhaps switch paragraph #2 (on the utility of mitogenomes for mantid phylogenetics) and paragraph #3 (on mantid taxonomy).2. I think you should add the AT% to the main text, not just in a supplement.3. I\u2019m still somewhat skeptical that the atp8 gene is actually missing, but your combination of NGS and PCR results are more convincing. It is difficult for me to fully evaluate this claim without access to the sequence data; but as is stated in the manuscript, atp8 and other genes are missing from other taxa. Is this the first case of missing mt genes in mantises?4. I recommend one more round of editing for writing clarity. For example, Lines 50-51 are a little confusing, and should probably be split up into two sentences.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0 27 Jun 202126/06/2021Dear Editor, Thank you very much for your email message regarding our manuscript entitled \u201cComplete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analyses\u201d. We appreciated the valuable comments from the reviewers and editor to revise and improving our manuscript. Generally, we have incorporated all suggestions and comments into this revision. More specifically, our adjustments are stated below in a point-by-point manner.We hope that this revised version can be accepted for publication. Most sincerely,All authorsReviewer #2: Thank you for your thorough responses and edits to your manuscript based on my comments. I think the manuscript has been improved. I do have a few comments that still should be addressed, based on my first round of comments:1. I still think the paragraphs in the Introduction are a little disconnected. Perhaps switch paragraph #2 (on the utility of mitogenomes for mantid phylogenetics) and paragraph #3 (on mantid taxonomy).Reply: We agree and have switched the paragraph #2 and paragraph #3. See lines 46-68.2. I think you should add the AT% to the main text, not just in a supplement.Reply: Indeed, we have added the AT% information to the main text; see in Table 2.3. I\u2019m still somewhat skeptical that the atp8 gene is actually missing, but your combination of NGS and PCR results are more convincing. It is difficult for me to fully evaluate this claim without access to the sequence data; but as is stated in the manuscript, atp8 and other genes are missing from other taxa. Is this the first case of missing mt genes in mantises?Reply: Thanks for your suggestion. Here we attach mitochondrial sequencing results for D. truncate species, and we really did not identify the atp8 gene. So, we imply that atp8 gene may have been lost. Yes, from our sequence of D. truncate and D. lobata, we can confirm the atp8 is lost. This is the first case of missing Mt gene in mantises. TAATTATCCCATGAAAGATTAGTATTAAACCAATAACTCTAAATTTATTTTAAATAATAGAGTAAAATGCCTGATTTAAAAGGAATATCATGATAGGATAAAATATGTAAATTAATTACTTTTACTAATATTATCATTACACATAACAGAGTTAAACTGTTTCCTAAAATATCAAAAATTTTTGTGCATCCTACACTAAAATATAGAAAAGTAAGCTAAATTTAAGCTAATGGGTTCATACCCCATATATAGAGTTTACACTCTCTTTTCTAGTGCCCAACAATTCGATAAAAATCCTCTTTTTAATAACTTTAATTAGAGGTGTGTTAATTTCATTATGTGCTAATTCATGAATAGGAGCATGAATAGGATTAGAAATTAACTTACTCTCCTTCATTCCTTTACTTTCTTCCAGAAAAAACTTGTTCTCAACTGAAGCTTCACTTAAATATTTTCTTATTCAAGCTATTTCATCATCTACATTTCTATTTCTAATTATGATAAAAATTAATATCCAAGAAATGTTTTATCTAATAAAATTTAATAATTGAAACACCTTAATTACAATTCCTATTCTAATGAAAATTGCATCTGCACCTTTTCATTGATGATTACCTTCCGTGATAGAAGGTTTATCTTGAATAAATTGTTTTATTATTCTGTCAATTCAAAAAGTAGCCCCATTAATATTCATTTCCTACTTAATCACTAATAACTTCTTTATTCAAATCATCATTACAATTTCTGCAATTATAGGAGCTATTGGAGGTCTCAATCAAATTTCTCTACGAAAAATTTTATCATTTTCATCTATCAACCATATTGGATGAATATTAACAACCATAATTATAGGTTCTAACTTTTGAATAACATACTTTATCATTTATACGGTAAATATTATCCCTATTATTTTTATGGTAAACAAAATAAACCTATCTTTTATTCCTCAGACCTTTAATTCCTTCAATAACAAAAAAATTATTAAATTCATTCTATTTATCTCCCTCCTTTCTTTAGGGGGCCTCCCTCCTTTTATTGGTTTTTTCCCAAAATGAATTATCATTCAAATTATAATCCAAAATTTAATAATTCTTACATCAATAACCCTAATTATATCCTCCCTTTTAACCCTTTATTATTATCTACGAATTATTTACACAACATTAATAATCACAAATTCAGAAATAACTTGAATAGCAATCTACTCAAATAACAACTTAGGAAAAAGAACATTTCTTTTCTTATTCATTCTTATTTTTGGTTTATCAATCTGTACACTTATCTTAACAATCTATTAAGATTTTAAGTTAACCAAACTAATATCCTTCAAAGTTATAAATAAAGTGATTCATCTTTAAGTCTTAGTATTACTTACACCTTTAGAATTGCATTCTAATATCATCTCCATGAATATAAAACTTTAGTAAAAGAGATAATAATCTCATTAATAAATTTACAATTTATTACCTAATATCAGCCATTTTACTTTTTTTTTTGCAACGATGATTATTCTCAACAAATCATAAGGATATTGGAACATTATATTTTATTTTTGGTGCATGGGCAAGTATATTAGGAACATCTTTAAGAATTTTAATTCGAACAGAGTTAAGACAACCCGGATCTTTAATTGGAGATGATCAAATTTATAATGTCATTGTAACAGCTCACGCTTTTATTATAATCTTCTTTATAGTTATACCCATTATAATTGGTGGATTTGGTAATTGATTAATTCCTTTAATACTTGGAGCTCCAGATATAGCCTTTCCTCGAATAAATAATATAAGATTTTGATTATTACCTCCTTCTATTCTATTTCTTATTATCGGAAGAGTTGTAGAAAGAGGAGCTGGTACAGGATGAACAGTCTATCCCCCTCTTTCAGCCAGAATTGCTCACGCCGGCCCCGCAATTGATTTAACCATTTTTTCCTTACACTTAGCTGGAATATCAAGAATTATAGGAGCTGTAAATTTTATCACAACTATAATTAACATAAAATCAATTCATATAAATCATACTCAAATTCCTTTATTTGTTTGATCTGTAGGAATTACAGCAATTCTTCTTCTTTTATCTCTCCCTGTACTTGCTGGGGCAATCACTATACTTCTAACTGATCGAAACTTAAATACATCCTTTTTCGACCCTGCTGGAGGGGGGGATCCTATTCTCTATCAACATCTATTTTGATTTTTTGGACATCCTGAAGTATATATTTTAATTTTACCAGGATTTGGTATAATTTCTCATGTCATTTCTCATGAAAGAGGAAAAAAAGAAGCTTTTGGAAATTATGGAATAATTTGAGCTATATCAGCCATTGGTTTTCTGGGGTTTATTGTATGAGCTCATCATATATTTACAGTAGGAATAGATGTAGATACACGAGCCTACTTTACAGGAGCTACTATAATTATTGCTGTTCCTACGGGTATTAAAATTTTTAGTTGACTCACAACTATATACGGAACAAAAATAATTTATAGTGTAGTATCTTTATGAGCATTAGGATTTATTTTTTTATTTACAGTTGGAGGTCTTACAGGAGTAATTTTAGCCAATTCAGCTATTGATATTATTCTTCATGACACTTATTATGTAGTAGCCCATTTTCACTATGTACTTTCAATAGGAGCCGTTTTTGCTATTATAGCAGGGTTCATTCATTGATATCCCTTATTCACTGGATTATCATTAAATCCTAATTGATTAAAAAGTCAATTTTTCACAATATTTGTAGGAGTTAATTTAACATTTTTTCCACAACATTTCTTAGGATTAGCTGGAATACCTCGACGCTATTCAGATTACCCTGACGCCTATAGATCATGAAACTTCTTATCATCTGTAGGAGCAATAATTTCTTTCGCTGCTGTTATCATATTCATTCTTATCTTATGAGAAAGAATTACCTTAAATCGATTTATATTATTTTCATCTCAAATAAATAGATCAATTGAATGAATTCATAATTTTCCCCCAGCTGAACACACCTACAATGAACTAACTCTAATTACAAATTAAAACCTAAATTGTTGATAATTTTCCTCATTTCTAATGTGGCAGAATAGTGCACTGGGTTTAAGCTCCAAAAATAAAGATAAACTTTTTTTAGAAGTTATTTTAATGGCTACAAATGCAAATTTAGGATTTCAAGATAGAGCCTCCCCCTTAATAGAACAACTCATGTATTTTCATGATCATTCCATATTTATTATTACTATAATTGTAATTACAGTAAGTTATATAATTATAGCTTTAATAGTAAATAAATTTTCTGATCGTCATGTTATAGATGGTCAATATTTAGAAATTTTTTGAACAGTTCTTCCAGCCATAGTTCTAGTCTTCATCGCTCTACCTTCTCTACGGATTTTATACCTAATTGATGAAAATACAAATCCAACATTAACCTTAAAAACAATTGGTCATCAATGGTATTGAAGTTACGAATATTCAGATTTTACCAATATTGAATTTAACTCTTACATAATTCCACAAAATGATTTAAATCTATTTAACATGCGTTCACTTGAAGTTGACAATCGAACATCATTACCCATAAATACCTTAACACGAATTTTAATCACCTCAGATGATGTTATTCATTCTTGAACAATTCCGAGAATTGGAGTAAAAGCTGATGCTACTCCGGGACGATTAAATCAAGCAACATTTTGATTTAATCGTCCCGGAGTATTTTATGGTCAATGTTCAGAAATTTGTGGAGCAAATCACAGATTTATACCTATTGTAATTGAAAGAACTTTGATTAATAATTTTCTTAGTTGAATCTTAAATTATATTGAATCACTAGATGACTGAAAATAAGTGATGGTCTCTTAAACCATATCATAGTAACATAATAACTACTTCTAGTGATTGACTAACAATTTATCAAGAAGTTAGTTAAAAAATAACATTAGTATGTCAAACTAAAGTCATTATCATTTAATACATCTTTATACCCCAAATAATACCCCTAAATTGGCTAATCCTATTTTACATTGTTTCTACTAGATTAATTTTTTTTAATGTAATAAATTTTTTTATATTTTCCCACAAAATCCCATTAACTTCAAATAAAATTTTACTAAAAACCCTAATTTGAAAATGATAACCAACCTATTTTCAATTTTTGATCCCTCTTCAAATTTTATAAACCTATCAATAAATTGACTTAGAATTTGAATCGGATTATTATTATTTCCTTCCTCGATATGGTTAATTTCATCACGAAATAAAACCCTTTGAAGTTTTATTTTAAGTAAACTTCATGAAGAATTTAAATTATTAATTGGTAAAAAAAAAATTAACAAAGGATCAACATTCATATTTATTTCAACATTTTTACTTATTATATATAATAATTGTATAGGATTATTTCCATATATTTTTACTGGGACAAGTCATATAGCTATAACTCTATCTTTTGCTTTACCTTTATGACTAAGATTTATACTCTTTGGTTGAATTAATAACTCTAATCACATATTTATTCATTTAGTTCCTCAAGGAACTCCAAATATATTAATACCTCTTATAGTTTGTATTGAAACAATTAGAAATTTAATTCGTCCCGGAACTTTAGCTATTCGACTCGCAGCAAATATAATTGCAGGACATTTATTAATAACCCTCCTAGGAAATTCTGGGAGAAACATTATAGATTCATTTTTACCCCTATTAATTTTAGTTCAAATTATACTTTTAACTCTAGAATCCGCAGTTGCTATTATCCAATCATATGTTTTTGCAGTATTAAGTACTTTATACTCTAGAGAAGTAAATTAATAATGATAATACACACTAATCACCCCTACCATTTAGTTACTTATAGTCCCTGACCTATTATAACTACTTTAAGAATCATAATCATAATATTAGGTTTTATTAAATTTTCTTATGAGTTTAGTGAAAAATTTATGCTATTAGGAACTTTAATTTTAATTTTAATTACTACTCAATGATGACGTGACGTTGTACGAGAAAGTACATATCAAGGATTACACACTAAAAAAGTAATCTTTGGACTACGATGAGGAATAATTTTATTTATTATTTCAGAAATTTTTTTCTTTGTATCTTTTTTTTGAACTTTTTATCATAGAAGCTTAACTCCTACTATTGAATTAGGGTCCTTTTGACCACCTCAAGGAATCTGACCCTTTAATGCTCTTCATGTTCCTCTTCTTAATACAACGGTACTTTTAGCATCAGGCATCACTATTACATGAAGTCACCATGGACTATTAATAAATAATTATAATCAAGCCACCCAAGGATTAATATTTACCATTATCCTTGGGATTTATTTTACCATCTTACAACTCTATGAATATTATGAAGCTCCGTTTACAATTGCGGATTCAGTTTTTGGGTCAATCTTTTTTATAGCAACTGGATTTCATGGACTTCATGTAATTATTGGAACTACATTCTTAGTTACATGCTTATTTCGAATAATTTATAAACATTTTACATCTATTCACCACTTTGGTTTCGAGGCAGCAGCCTGATATTGACATTTTGTGGACGTAGTATGATTATTCCTGTACATTTCTATTTATTGATGAGGGAGATAAATCCAATTTATTTAGTATAAAAGTACAATTGATTTCCAATCAAAAAGTCTATATTTAATTAGAATAAATAATTAAAATTTTAATTTTTATCTCATTCATTACTATATCAATTACCTTAACAATCATATTACTAACAAATTTCTTGTCAAAGAAAAAAATTGAAGACCGAGAAAAAAATTCACCTTTTGAATGTGGATTTGATCCGATTAGATCCTCGCGCCTTCCCTTTTCTTTACGTTTCTTTTTGATTGGAGTAATCTTTTTAATTTTTGATGTAGAAATCGCCTTTATCTTACCAATAATTATCATTCCTCTTACATCAAAAATAACATCTTGAATATCTACTAGAATTATATTCTTATTGATCTTAACAACTGGTTTATTTCATGAATGAAATCAAGGTTCTCTCGACTGAGCAACTTAACTTTATAAGGGTTATAGTTAAAAATAACATTTGACTTGCACTCAAAAAGTATTGAAATATCAATTTTCCTTATTATAAGTAAGAAGCAAATTCATTGTAATCAGTTTCGACCTGATAGTAAGATATTCATATCCTTATTTGTTGATTAATTGAAACCAAATAGAGGTATATCACTGTTAATGGTAAAATTGAAATTAATGCTTTCCAATTAAGAAAATGTGTAGATCGAATATAAGTTGCTAATTTATTATTCAAGTGGTTTAATCCCATTTACATTTTAATTTATATAGTTTAAATAAAACATTACATTTTCATTGTAAAAATAAATTTTTCAATTTTATTAATAGTAAGTACTCTATTTAAAGATAAATTAGTTATCTCAATAACAGCTTCAATGTTATACTCTCTATAAGATATTTAAATAAATACAAAATATTATTATAAGTAAAATACTAAAAAAAATCATTAAATAAGATTTTAAATCATTATATTGAAATCATTGATTAAAACATCTTAATTTTATCAAAATATAATATAAATTTTGTGCTCCAAAATATTCTCTTCAGCCTAAATCAATATATATCATTGAATTTAAACTAATAATTAAAGGTATTTTTCTTACTCCTTTAGTAAAAATTAAAGGTATATATCATATTGAAGCAAAAAAAATTGTCATTTTATAATACTTAAATGTTGAAAAATAATAATTTATTCTATACTTAAATATAATTCTTCCTAATCATAAACCTACAAATCTACCTAATAAAGGTATTATTTTCATTATTAATGTCATATAAATTATATAGGGAGTAAGAAAAATAATCCAATTTAATATCCCACCTCCAATAATTGCAAATAATATTAAACCTAATATTCCATATACTATTATTCAACTTTCATTTAATTTACATATTGATATTATATTAAAATCTCCTCATAATACATAATAGGATAAACGAAAAGAATAACTTACTGTTAAACCTGTTGAAAAAAAAAATAAAACATATATAAAGATATTTAAATTTCTTAAAGATAATATTTCTAAAATTATATCTTTTGAATAAAATCCTGCTAAAAAAGGTATACCACATAAAGCAAAATTTGATACTATAAAACAAGATGAAGTAAAAGGCATAAATATTACTAAATTTCCTATAAAACGAATATCCTGTAAATTTTTTATTCTATGGATTATTATTCCTGTACACATAAATAACAATGCCTTGAATAAAGCATGAGTTAATAAATGAAAAAAAGCCAAATCTGAAAAACCTAATGATAAAATTCTTATTATTAATCCTAATTGACTTAAAGTTGATAAAGCAACAATCTTTTTTAAATCATATTCAAAATTTGCTCCTAACCCTGATATAAACATTGTTAAAACAGATATTACTAGTAAAAACTTTAATAATCAATCCGGAAAAACCTTACAAAATCGGATTAATAAATAAACCCCAGCCGCAACCAAAGTTGATGAATGAACTAGGGCTGAGACAGGAGTTGGAGCAGCTATTGCTGCAGGCAATCAAGCTGAAAAGGGAATTTGTGCACTTTTTGTTATTCCTGCTAAAAGTACTAAAAATGAAATTAAATATATTTCAATTTCATTTAATGTACAATCTAAATAAAAAATATAATTTCATCTTCCAAAATTTAATATTCATGAAATTGCTATTAATAAAGCAACGTCCCCTACTCGATTAGATAAAGCTGTCAATATTCCAGCATTATAAGACTTAGTATTTTGATAATAAATTACAAGACAATAAGAAATTAAACCTAACCCATCTCAACCTAATAAAATTCTAATCAAATTTGGACTAATAATTAAAAACATTATAGACAATACAAATATTAATACTAAAAAAATAAACCGATTTAGTGATGAATCTCCAGTTATATAATCTTCTCTATATAAAATTACTAAGGATGAAATTAATAAAACAAAACTCATAAATATGAGAGACATTCAATCCAACAATATTGTTATAACAACTGAAGAAGTACTTAATCTAACAATTTCTCATTCAATAAAAATAATTAAATCATTTAAAATAAAAACCATTCTAGAAATAAATATTATTAAAGAAAATAATGCTAATAAAAAAAATCTAATAAAACATAATGATAAATAAACCATAACTTAAAATAACTATTCATTATATCCATGATACCACAAATCATAATTTTATGATAAACTATTTAAGTAAATTAACTTTTATTTAAAATCAAATTAAAACATAATCTCTCTTTAAAATTAATAAATTTAAGGGTAATCAATGAAGTATTATTAATAAATATTCACGTCTATACCCTCCAACTCTTCTATAAATGCCTGAATAATAAAATCCATGTTGACTATATGAATATAAATATAAAGTATAAGCAGCTCTAAAAAATGAAATTAATATAAGAAAAAATATAGATATTCATACCCAACTTACTATACTATTGAATAATCCAATTTCTCCTAATAAATTTAAAGTAGGGGGAGCAGCTATATTGCTAGATGAAAGTAAAAATCATCATAATGTTAAACTTGGCATCAAATTTAATAAACCCTTATTAATTAACAATCTTCGTCTTCCCAGACGCTCATAACTAATATTAGCTAAACAAAATAAACCAGATGAACATAAACCATGAGCAATTATTATTACAAAAGTTATATAAAACCCTCAAATATTTAATGTTATTAAGCCCCCAATAATTAAACCTATATGAACCACAGACGAATAGGCAATTAATGCTTTTACGTCTATTTGACGTAAACACATTAAACTAACTATAACCCCACCGATTAATCTAGTAGAAAATCAAAATACATTAACTAATATCCCAACTCTCTTCAAGAATTCATATACTCGTAATAAACCATAACCTCCTAACTTTAATAAAACTCCAGCTAAAATCATTGAACCTGAAACTGGAGCTTCAACATGAGCCTTAGGTAATCATAAATGCACTAAAAATATAGGCATCTTAATCAAAAAAGCTAAAATTATTCTAAAATATAAATATCAATTCAATAAATTATTTTTATAAACTAACGAAAAAACTAAATAGCCCATGTTGTTATAAAAATATATAATACCCATCAATAATGGTAATGAAGCTAAAAGAGTGTATAATAATAAATAAATACCAGCTTGCAATCGCTCAGGTTGATATCCTCACCCAAAAATTAAAAATAAAGTTGGAATCAAACTACCCTCAAAAAAAAAATAAAATGAAATGAAATTTATTCTACAAAAAGTACAAATTAATATCAATAACAATATTAAAATTATAAATATAAATAAATTATTATAAAATTTATATCGAATAACAGAATAACTAGCTAAGATTATTAAAACACAAATTCAAAATCTTAGCACAATAAGACCATATGATAAGTAATCATATCCAAATATATAACTCATTCTTATTCAATAAAAAAAATCCACTCCAATAAAAAATTTAAAAGATATAAAAAATAATAAATTCTGAATTAATAATCAATTTTTATTCATTAAACATAATGGAATCAAAAACATTAAACTTAAAATATATCTTAACATTGCAATAAGCTAAACGAATTAAAATAATCATTTCCATGAGTTCGAATTATAGAAACCAAAATTGATAATCCTAATACACCTTCACAAACTGCAAATGAAAGAAAAATTATAGTCATATATAACTCTCCTCTTATTATTAAATAAAAATACAAAATAATGAACAAAACTAAAACAATAAATTCTAAACTTAATAAAGTTACTAATAAATGTTTACGTCCAGAAGAAAAAACTCACAAACCACATAAAAATATAAAACAGAATATTATTAACATTTTTTAGTTTTAATAGTTTACTAAAAACACTGGTCTTGTAAACCAAAATTAAGAATAATTACTTTTAAAACTTCAAAGGAAAGAAACTCATCATTAATTCCCAAAATTAATATTTTATTATTAAACTACCCTTTGATATTTTATACTCAT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ATATAAACTTTCAAAAAAAAAATCCACTTTACCCAAAAAATAAGTATGCTTTTTTTAAAACGTTTTCCTCCCTAGAAGTAGGAATTAAGTGCCCAATTCAATTCGATTTAATGTTTTTTTTTAAACATTGGTCTACTAAAGTATATTTAGTATATAATAAATTTAATTATATATATGTCTTATCAAAGATTAATAAAAAAAATATTCTCAAACAGCGGTATACAAAAATATAAAGTAAGTAAGGTCCAACGCGGATTATCAATTAAATAATAGACTCCTCTAAATA4. I recommend one more round of editing for writing clarity. For example, Lines 50-51 are a little confusing, and should probably be split up into two sentences.Reply: Thanks for your suggestion. Lines 50-51 have been split up into two sentences. Please check lines 56-57.AttachmentR2-Reply letter-GS2.docxSubmitted filename: Click here for additional data file. 7 Jul 2021Complete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analysesPONE-D-21-03889R2Dear Dr. Smagghe,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ben J Mans, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 21 Oct 2021PONE-D-21-03889R2 Complete mitochondrial genomes of four species of praying mantises  with ribosomal second structure, evolutionary and phylogenetic analyses Dear Dr. Smagghe:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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+{"text": "Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, \u2018nuclear morphogenesis\u2019 may be a microscopic counterpart of the macroscopic process that shapes the animal body.An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in  Summary: The nuclear space becomes progressively heterogeneous during embryo development, forming distinct transcriptional sub-nuclear environments, paralleling the partitioning of the embryo body during morphogenesis. During embryogenesis, transcriptional regulation controls the expression of genes that pattern the animal body . This prDrosophila embryos at a late stage of development and that the developmental gene shavenbaby  resided in regions enriched for TFs when it is transcriptionally active , rhomboid (rho) and snail (sna) enhancers to measure their local chromatin environment during different stages of development and at different locations in the embryo. In summary, we found that the distributions of TFs and epigenetic marks became progressively heterogeneous as development proceeded. Thus, the nuclear space may undergo a transformation that parallels morphogenesis, where the animal body becomes increasingly heterogeneous and segmented. Additionally, even for the same gene, the nuclear environments at transcription sites depended on the location and timing of expression. Organizing the nucleus to form multiple reconfigurable regulatory environments in a single nucleus could permit genes to perform different tasks at different times and facilitate the complicated regulatory patterns during later stages of embryogenesis.However, the origins and spatial-temporal behaviors of these transcriptional microenvironments during development remain unclear. When does the nuclear microenvironment form? How and when do genes interact with these microenvironments? Here, we imaged the distributions of TFs and histone modifications in Hunchback (Hb) is a TF that is a gap gene in the early embryo . We obsey-axis) when the image has been shifted by a given distance compared with the original (x-axis) indicates the abundance of structures at that length scale . There was a monotonous increase in autocorrelation as the embryo ages, suggesting that Hb environments spanning several hundred nanometers became more prevalent as development progressed. Indeed, stimulated emission depletion (STED)  distributions across similar developmental stages with Airyscan and computed their autocorrelation . There was a similar upward trend, suggesting the formation of local TF environments in older embryos.To quantify the spatial distributions of Hb, we computed the spatial autocorrelation of Hb intensity G. The len (STED)  imaging n (STED) H, bottomn (STED) H, middlen (STED) H, top, tFig.\u00a0S1F,G) and Engrailed , which are active within a narrower time frame during development, showed high autocorrelation when they are first expressed (stage 10) and when they reach maximum expression (stage 14) in nuclei on the ectoderm. At stage 16, when both are no longer expressed, their distributions became sparse with low autocorrelation. This two-state behavior for factors only expressed at later stages of development is unlike Hb and Kr, which showed a progressive increase in autocorrelation with intermediate states. This suggests that the general distributive properties of TFs may conform to the underlying nuclear architecture at the time of their expression.The TFs Ultrabithorax  is expressed  and imaged embryos at stages 5 and 10, in nuclei within 20\u2005\u00b5m of the ectoderm to preserve optical resolution. We stained for H3K4me1 . However, using RNA fluorescence in situ hybridization (FISH) to detect transcription sites degraded the signal from histone modifications . To improve quantitative image analysis, we detected transcription sites without the denaturing step in RNA FISH to preserve the signal from histone modifications (Drosophila melanogaster line expressing a reporter mRNA containing MS2 stem loops driven by 18\u2005kb of the cis-regulatory region of hb (hbBAC)   with a l (hbBAC) . This sp embryos . The mRNhbBAC transcription sites between stages 5 and 10 showed changes that are distinct from the global trend in To quantify the spatial distribution of histone marks around transcription sites, we computed the average radial intensity distribution centered on the transcription site F-J acco. We obseFig.\u00a0S3A). We generated transgenic lines with MS2 reporter mRNA driven by enhancers from two genes active during this process: snail (sna) and rhomboid (rho). The constructs are named sna A2.2 W0.10 and rhoNEE, respectively. We observed that both were expressed in patterns corresponding to their endogenous genes : in an anterior-posterior band along the ventral side for sna A2.2 W0.10 . rhoNEE, on the other hand, showed no change in H3K4me1  and an increase in H3K4me3 only between early and mid cellularization . Thus, the regulatory environments around genes can change over relatively short time scales in a manner that depends on their cis-regulatory regions.To test whether we can resolve finer temporal changes in local histone environments, we focused on the 1 h cellularization process at end of the rapid nuclear division cycles at stage 5 , which wus genes , 2007  has a sharp ventral edge resulting from Snail repression and trails off gradually in the dorsal direction due to a decreasing gradient of the activator Dorsal  but differential engagement from enhancers, depending on whether the repressor Snail is present. Together, these results imply that histone environments around genes depend on the properties and states of cis-regulatory elements. These differences could refine expression patterns over time, as is the case for many developmental genes  of hunchback (hb) on a bacterial artificial chromosome (BAC) driving a yellow reporter mRNAs containing 24 MS2 stem loops generated by hb. The reporter constructs for rhoNEE and sna A2.2 W0.10 were derived from the plasmid pIB-hbP2-P2P-lacZ-\u03b1Tub3 UTR (hbP2 enhancer between the restriction sites HindIII and AscI replaced with the rhoNEE or the sna A2.2 W0.10 sequence. The hbP2 promoter was retained. The plasmids were synthesized by Genscript. The constructs were injected into Bloomington Drosophila Stock Center line 27388 and integrated using RMCE by GenetiVision. Virgin flies from a transgenic D. melanogaster line containing MS2 coat protein (MCP) fused to EGFP driven by the nanos promoter  staining. The MCP-EGFP line does not contain a nuclear localization signal (NLS) to minimize MCP-EGFP forming aggregates in the nucleus in the absence of MS2 mRNAs.All Tub3 UTR  with theter from  were crorhoNEE, CTTGGGCAGGATGGAAAAATGGGAAAACATGCGGTGGGAAAAACACACATCGCGAAACATTTGGCGCAACTTGCGGAAGACAAGTGCGGCTGCAACAAAAAGTCGCGAAACGAAACTCTGGGAAGCGGAAAAAGGACACCTTGCTGTGCGGCGGGAAGCGCAAGTGGCGGGCGGAATTTCCTGATTCGCGATGCCATGAGGCACTCGCATATGTTGAGCACATGTTTTGGGGGAAATTCCCGGGCGACGGGCCAGGAATCAACGTCCTGTCCTGCGTGGGAAAAGCCCACGTCCTACCCACGCCCACTCGGTTACCTGAATTCGAGCT; and sna A2.2 W0.10, GTCGACCTAGTTCTGTTTTGTGACTCGGATTTACTATTTCGCATGGCTCCTCTTCGAACAATGTCAGTCGAGCTCTGTAGATCCCTGTGTTCCCTCTTCATTGTCAACTTGAACAAATGAGCCAGGGAACAAGGTGCAAAAATGGGACGGTCCTATTCTCAGCAAAAATTGACAAGAACAACAACAATGTCTATGGAAAATCGAACTTCATCCCAGCACCTGCAGAAATCCCGAGCGAGTCGGGGAAAAAGTATTTAACCCCCGAAAGGGTTTTCCCCAAAATAATGAAGTAATGAATGAAGCGGAAAACACTGGCCGCCAATCTACCTAATACTAATGAGCGGGCCAACCCGACCAGGAATTTTTGCAAGTCAGGTACTTCAACGGATATATGGGTTCGACAAGTGCGGATTTTCCCGCGACATCAATGAGGACTTGGCCGGGTTATCCGCGGTGCTCATCGGGCAATTCCGCGGCCGAGGACTTCATCGTAGTGATCATTAGGTAGATATGTGCATGGATGTGACATGGCGATCATTGCGCGGAATAACACACGTAATAACCGAGATATCCGGGATGACCCACCAGGTAGGATGTGAGGACATATAGAAAACCCCCAGCCAGTTTTTCCACTCGTCGTGGCTTGTTTTGCTTGAGTTTCGCTGACTGCGTAATTGGATAAGATGGGAAATTACTTTAAATCCTTCGCTGATCCACATCCGGACATTCGTCGAAGGAAAATCCATTGCAGGGAAATACGAAATGGAAATGCGGCTGGGTTATTGGCTCGACATTTCCCATCTTCCCTCACGCCATTGGTTGCAGGATCGCGGGGAATTGGAATTCCGCGCTGGAATTTTTTGTCACCTCTTGGGTTTATCAAAACTTTTGGGTTTGCTATGGATTTTTTCCAATTTTACCACCGCGCCTGGTTTTTTTTTTTTGACGACGCGGAAAATCGGACTTGGCTATGCGGGCTTGTCTGTTTTTCCGGGTACAAAGTCTGCATGTCAGCCTCCATGCGGGAGTGGGAGTTGGGAAAGTTTCCCATCGATAGTTGGAGGGGTGGCTTGAAAGTCTGGAGGTGCTAGCTGGGAAAGTTGTGTGTGCGCGATGAGGCAAGGAGTCAAAGATCAGGGGAGTTGGAAAGCGAGAATTGTGGGAATCGTCCAGGACTCAGCTGGATGCTGAGGGGCAGTATGATTTTTTTTACGTTATCAATCGAATTGATTTTAAGACAGCAGAACTTCACATACTAATAAGATGACCATGGGATTAGTTAAAATGTGTAACTCGTATTCGAATCGTCATTCTTTCACGGACCAATCGTGGGAACAGGAGATCTCTTCGATCCAAGCTCACAGGAGACTTGACACTCTTCGTCTATTCCTTGTCAAGTTTTTAATGACATCTCCTATGCCCTGAGCTATGTTTTCCTAGCTCTCATCGATCGCTGCCAATGAGCCACTGGAGATGATCCATAAGTCAGCGTAGAGTGCACCCCAGAGTTGACACTTGGTGTCTCGGAATTCGGCTCATTATCAGTGCTATTTTTGGAACACCTCTCTGCGAAGGTGTCATTTTTGTCAGTGCGTATCGCTCAGGTTCAACTCCCCACCAAAAACCGAATTTAGAGCATCGGCAGATGTACTTGAAGCACTCAATCTAAGTGAGGAAACCACCCCATGAACGAAGAGTACTAGGAGTCCTATTTGACTCGTGCTTAAAAATAGAAAATTACTTAGGGTGATCCATAGGTAGGGAGGCGATATTGTAACTTGCATTTCGGACCCGGACCTGCACGAGTTATTACGGGTGGGTTGTGAGCGTATCGGGAAATTGGAGAGCCACCAGATCTGTCATAACTTATACGGGGGATCCTTATTCCTGGGAGGGTGCGCCTGCGTCTGCTCTTCCGAGAGAGAGGTGGGAAATGGAGGAAGAGAGAGAGAGAGAGAGTGAGAGAGCAGGTAGAGGGAAGTGAGGGAAATACGCAATAAGGGTATGGGAAAAGTGCTGTTGTTGTTGCTAGGTAGCGACGCACACGTGCGAGTGTTTTTCTGTTTTGAAGAAGAACCACCACCAAATGG.The enhancer sequences were as follows: 1118w flies (for imaging TFs) and from crosses of the hbBAC, rhoNEE or sna A2.2 W0.10 reporter lines with the MCP-GFP line (for imaging transcription sites and histone modifications) were collected, fixed and IF stained using previously described protocols ;The secondary antibodies were as follows: Alexa488 donkey anti-mouse ; Alexa488 donkey anti-rabbit ; Alexa555 donkey anti-rabbit ; Alexa594 donkey anti-mouse ; and STAR RED goat anti-rabbit .in situ hybridization (FISH) and histone modifications using IF, we followed a previously described FISH protocol . One additional secondary antibody was used: Alexa488 donkey anti-sheep .To stain for transcription sites using RNA fluorescence protocol  and the x-y  and z direction  of stacks used for quantification. The laser power and gain were adjusted to maximize the signal to noise ratio within the dynamic range of the PMT or Airyscan detector for each sample preparation condition (i.e. different antibodies). Within a given sample preparation condition, laser power and gain were kept the same across embryos. The acquired Airyscan stacks were processed with Zen 2.3 SP1  in 3D mode to obtain super-resolved images.Confocal and Airyscan  imaging Drosophila embryos was carried out in ProLong Diamond without DAPI . Fixed embryos at the appropriate stage and orientation were imaged on a STEDYCON  in 2D STED mode on an Olympus BX53 microscope. Excitation lasers with wavelengths of 594 and 640\u2005nm were used as appropriate for the specific fluorescent dyes. The wavelength of the STED laser was 775\u2005nm. All samples were imaged using an Olympus UPlanSApo 100\u00d7 Oil/1.4 objective. The resolutions used for the x-y and z directions were 25\u2005nm and 250\u2005nm, respectively. The laser power and gain were adjusted to maximize the signal-to-noise ratio within the dynamic range of the APD detector on the STEDYCON for each sample preparation condition. Within a given sample preparation condition, laser power and gain were kept the same across embryos.Mounting of fixed r = 0. Radially averaged auto-correlation functions were computed using an ImageJ macro . Airyscan images were used for the autocorrelation of Hb and Kr for their wider field of view compared with STED, in order to include a larger number of nuclei. Intensity distributions and autocorrelation functions were plotted using Matlab (MathWorks).Image processing was carried out using Fiji  with the"}
+{"text": "Aurkb is an essential gene, we conclude that this analog-sensitive allele is either catalytically inactive or not fully catalytically active in mouse.The mammalian genome encodes three Aurora protein kinase homologs (AURKA/B/C) which regulate chromosome segregation in nearly every cell type. AURKC expression is largely limited to meiotic cells. Because of the similarity in sequences between AURKB and AURKC, determining their separate functions during meiosis is challenging. We designed a chemical genetics approach to investigate AURKB function. Using Crispr/Cas9 genome editing in mouse, we replaced an ATP binding pocket amino acid to permit binding of cell-permeable ATP analogs. We also introduced a second site supressor mutation to tolerate the pocket enlargement. Heterozygous mice were fertile, but never produced homozygous analog-sensitive mice. Because  Because AURKC shares high sequence homology with AURKB, standard approaches such as RNAi knockdown  and small molecule inhibition  to understand their roles do not provide the specificity required to assess non-overlapping functions. Furthermore, these kinases can compensate for one another in oocytes from mouse knockout strains , making phenotype assessments less clear. Therefore, alternative strategies to provide specificity are needed.The Aurora protein kinases (AURK) are essential regulators of chromosome segregation in mitotic and meiotic cell divisions (Carmena and Earnshaw 2003). Unlike mitotically dividing cells which express two AURKs (AURKA and AURKB), mammalian oocytes, which undergo meiosis, express three AURK homologs: AURKA/B/C . Although most protein kinases tolerate the ATP pocket enlargement, approximately 30% of protein kinases do not . A previous report demonstrates that human AURKB ATP-pocket enlargement (Leucine 154 to Alanine) abolishes its catalytic activity. Excitingly, this activity can be rescued by mutation of a second site suppressor in the C-terminus of the kinase (Histidine 250 to Tyrosine). When HeLa and U2OS cells were transiently transfected with the analog sensitive human AURKB allele, the kinase was active and cell division was not altered . Based on these data, we were keen to adopt this approach to study the potential unique functions of AURKB. We used Crispr/Cas9 genome editing to introduce the ATP-binding pocket point mutation (L159 in mouse) and second site suppressor mutation (H255 in mouse) into the mouse genome . After confirmation by restriction digestion  and Sanger sequencing , we obtained 4 founders  that were heterozygous for the analog-sensitive allele on the same chromosome. To expand the colony, we set up breeding cages using the heterozygous founders. Heterozygous animals were fertile and gave birth to pups. We also crossed heterozygous pups to increase chances of obtaining homozygous animals. After genotyping 80 pups that were born to 8 heterozygous mating pairs, we did not obtain any homozygous mice; wild-type and heterozygotes were born at altered Mendelian ratios . Because Aurkb is an essential gene , these data indicate that this analog-sensitive allele of Aurkb is either catalytically inactive or not fully catalytically active in mouse and that it cannot be used to determine specific AURKB functions in mouse oocytes.Chemical genetics is a strategy that documents superior specificity in inhibiting protein kinases. This approach involves enlarging the kinase ATP-binding pocket by introduction of an amino acid substitution. This enlargement allows specific acceptance of cell-permeable, bulky ATP analog inhibitors that cannot bind other kinases in the cell (Garske C57BL/6J mice: Jackson LaboratoryCas9 IDTL-A RNA guide (PAM sequence in parentheses) from Sigma: ACTACTTCTACGACCAGCAG (AGG)L-A donor oligo (mutations in lower case):CAGGCACTAGATCCCACACCAGGTAGGCCCTGACCGTGGCAGTCCGCTGCTCATCGAAGGTCCGACTCTTCTGCAGTTCCTTGTAGAGTTCCCCGCGAGGGGCGTATTCCgcGATTAAGTAGATtCTCTGCTGGTCGTAGAAGTAGTTGTAGAGTTGAAGGATGTTGGGATGTCTGGH-Y RNA guide (PAM sequence in parentheses) from Sigma: TGCCCCCAGAGATGATTGAG (GGG)H-Y donor oligo (mutations in lower case):TCCGACGATACGTCTCACTGTGGCTAGGGCTCTCGAAGGGTGGGTTCCCCACCATCAGTTCATAGCAGAGCACCCCGATGCACCATAGATCTACCATTTCATTATaCATGCGgCCCTCAATCATCTCTGGGGGCAGATAGTCCAGCGTGCCGCACATGGTCTTCCTCCTGGTGGGATGAGGenotyping Primers:L159A substitutionL159WT F: 5\u2019- CCAGCAGAGGATCTACTTAATCCT -3\u2019L159AS F: 5\u2019- CCAGCAGAGGATCTACTTAATCGC -3\u2019WT R: 5\u2019- ATGATctgtgagggtccacacaag -3\u2019H255Y substitutionH255WT F: 5\u2019- GAGATGATTGAGGGGCGCATGCAT -3\u2019H255Y F: 5\u2019- GAGATGATTGAGGGGCGCATGTAT -3\u2019WT R: 5\u2019- CTGGCTCCTACAGTACACAAAGG -3\u2019"}
+{"text": "STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic\u2014intestinal and retinal\u2014disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.Biallelic The online version contains supplementary material available at 10.1007/s00439-021-02284-1. STX3, OMIM 600876) by us and by others  . All affected individuals had homozygous loss-of-function For all clinical data presented including the molecular genetic studies appropriate informed consent was obtained from subjects or their parents in accordance with the guidelines of the respective institutes at which the subjects were seen and approved by the institutional review boards and ethics committees as required. Human cadaver tissue was donated to the Willed Body Program at the McGovern Medical School at the University of Texas Health Science Center in Houston. Use of cadaver specimens is deemed exempt by the Institutional Review Board.Animal procedures conformed to National Institutes of Health guidelines and were approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston. Mice were kept under standard housing conditions with unlimited access to food and water and with a 12\u00a0h light/dark cycle. Genotyping was performed by PCR using DNA isolated from tail snips as described  with DNA extracted from peripheral blood leukocytes in P3, P4 and P5 as described . Variant designations are based on NCBI transcript reference NM_004177.5, using\u2009+\u20091 as the A of the ATG translation initiation codon. The DNA samples from all patients\u2019 parents, and from a number of additional family members were tested for the segregation and zygosity of STX3 variants identified in index patients .Novel pathogenic re Table . STX3 muSTX3 loss-of-function variants; STX3 variant identification by WES was reported for P1 and P2   or Alexa Fluor 488  where used for labeling. All antibodies were diluted with blocking solution . Specificity of immunolabeling was confirmed by processing a second set of sections in the absence of the primary antibodies, or by substituting normal rabbit serum for polyclonal antibodies, as appropriate.Images were captured with a ZEISS LSM 800 confocal microscope  or with a Nikon A1R Confocal Laser Microscope  at a thickness of 0.3\u20130.5\u00a0\u00b5m. Confocal images were processed using the manufacturer\u2019s software Zen 2.5 (Blue edition) or Nikon Imaging Software Element (Version 4.20) and with Adobe Photoshop.STX3 transcripts was analyzed by identifying matching reads from recent Illumina TruSeq\u00ae RNA-seq projects obtained with human retina samples  . Probes of 120\u00a0bp length covering 60\u00a0bp upstream and downstream of the respective splice sites were analyzed. Default BLAST settings were used with the exception of the Expect threshold, which was set to 1000. Reads were identified as matching if they had at least 90\u00a0bp identity with the probe, corresponding to an E value of\u2009<\u200910\u201330. Reads per kilobase of transcript, per Million mapped reads (RPKM) where calculated for each probe.The expression of the different Sequences of probes used:HS STX3 8_9BAGCAGCATCAAGGAGCTTCACGACATGTTTATGGACATCGCCATGCTGGTGGAGAATCAGGGATCCATGATTGACCGTATTGAGAACAACATGGACCAGTCAGTGGGCTTTGTGGAGCGG:HS STX3 9B_10BGTGGAGCGGGCCGTGGCAGATACCAAAAAGGCTGTCAAGTATCAGAGTGAAGCCCGGAGGAAGAAGATCATGATCATGATCTGCTGTATTATCCTTGCGATCATCTTAGCTTCCACCATT:HS STX3 8_9AAGCAGCATCAAGGAGCTTCACGACATGTTTATGGACATCGCCATGCTGGTGGAGAATCAGGGTGAGATGTTAGATAACATAGAGTTGAATGTCATGCACACAGTGGACCACGTGGAGAAG:HS STX3 9A-10AAATAATCAATGCTAAAATGCCCAGCAACACAACTACTAGCACAATGATAATTATCAATTTCTTCCGGGCCTGACTCTGGTATTTCACAGCTTTTTTCGTTTCATCTCGTGCCTTCTCCAC:HS STX3 9A-11AGTGGAGAAGGCACGAGATGAAACGAAAAAAGCTGTGAAATACCAGAGTCAGGCCCGGAAGAAACTGATTTCACTCCAGACTGGTGTGGCCACCCTTGTCTTCAGATGAGAATGGAGTCTGHuman Tissue cDNA samples were purchased from Takara Bio USA, Inc. . Standard PCR reactions were performed with the OneTaq Hot Start Quick kit (New England Biolabs) using these primers:A: HS Syntaxin 3AB Sense exon 1:GGCTTCAGGATGAAGGACCGB: HS Syntaxin 3B Sense Exon 7:GCAACCCGGCCATCTTCACTTCTGC: HS Syntaxin 3A Antisense exon 10A:AGCCCAACGGAAAGTCCAATD: HS Syntaxin 3B Antisense Exon 11B:CCTGTCCCTGTCCTCCGCCCAATGAPDH1: ATGACATCAAGAAGGTGGTG,GAPDH2: CATACCAGGAAATGAGCTTGConditions: 35 cycles for each reaction, annealing temperatures STX3: 55\u00a0\u00b0C; GAPDH: 50\u00a0\u00b0C. PCR reactions were analyzed using agarose gel-electrophoresis and correctness of the amplified fragments was confirmed by direct sequencing.STX3 expression in the murine and non-mammalian retina and roles for Stx3 in protein trafficking in photoreceptors  carried the same pathogenic variant  that is located in exon 9A and thus spares STX3B.Given the high level of Severe visual impairment was seen and a diagnosis of EOSRD rendered in individuals P2-P9, as evidenced by the inability to respond to or track visual stimuli, locate or reach for objects. Half the subjects also exhibited nystagmus. Five subjects underwent fundoscopic examination, revealing pallor of the optic disks Fig.\u00a0 and fundSTX3A and STX3B, we asked whether both splice forms were present in the human retina. A GeneBank search identified a large number of ESTs and several full-length cDNA clones corresponding to STX3A. However, while no clones corresponding to STX3B were identified, analysis of the human STX3 gene sequence showed the presence of putative STX3B-specific exons 9B, 10B and 11B  and inner plexiform layer (IPL) respectively, labeled for the major structural protein of the synaptic ribbon, ribeye, exhibited strong STX3 immunolabeling Fig.\u00a0a\u2013d, consStx3 was achieved with a mouse line that expresses an optimized iCre under the control of the rhodopsin promoter , as well as homozygous Stx3 floxed control mice  and heterozygous Cre expressing control mice .To probe for an essential role of Stx3 in vision, we generated a cell-specific Stx3 mouse knockout line, as complete inactivation of the Stx3 gene in mice produces an embryonic lethal phenotype . Double-labeling for Stx3 and ribeye/CtBP2 and for Stx3 and cone opsin revealed that the remaining Stx3 signal was in cone photoreceptors  and in the number of neuronal somata in the ONL was observed Fig.\u00a0a, c indiOPL Fig.\u00a0a. In somOPL Fig.\u00a0a, arrowsAt 8 and 12\u00a0weeks of age, an increasing cell loss and an increase in the ectopic expression of rhodopsin were observed. Cone photoreceptor loss and the ectopic expression of opsin in cones was also observed Fig.\u00a0b, c. TheSTX3 loss-of-function variants that affect both STX3A and STX3B transcript isoforms display visual impairment consistent with an EOSRD. We show that STX3B rather than STX3A is highly expressed in the human retina, where the protein is found in the inner and outer segments of rod and cone photoreceptors, and in both plexiform layers, where the synaptic endings of photoreceptors and bipolar cells reside. Inactivation of Stx3 in murine rod photoreceptors resulted in their rapid degeneration as shown here and in a very recent study . All the other data supporting the findings of this study are available within the article and from the corresponding authors upon request.The sequence of the Supplementary file1 Supplementary Fig.\u00a01. Simplified family trees, segregation and Sanger chromatograms relating to novel STX3 variants (TIFF 3415 KB)Below is the link to the electronic supplementary material."}
+{"text": "Amputoearinus is given. Gnathopleurajosequesadai Sharkey, sp. nov. is reported as a hyperparasitoid of fly larvae, the first such record for the genus. The following new species are diagnosed primarily using COI barcode data; Sharkey is the authority for all: Agathidinae: Aerophilusdavidwagneri, Aerophilusfundacionbandorum, Aerophilusnicklaphami, Lytopylusdavidstopaki, Lytopylusdavidschindeli; Alysiinae: Gnathopleurajosequesadai; Braconinae: Braconandreamezae, Braconfranklinpaniaguai, Braconrafagutierrezi, Braconguillermoblancoi, Braconoscarmasisi, Braconpauldimaurai, Braconshebadimaurae, Saciremakarendimaurae; Cheloninae: Chelonusminorzunigai; Homolobinae: Homolobusstevestroudi; Macrocentrinae: Macrocentrusmichaelstroudi; Orgilinae: Stantoniagilbertfuentesi; Rhysipolinae: Rhysipolisstevearonsoni; Rogadinae: Aleiodeskaydodgeae, Aleiodeskerrydresslerae, Aleiodesjosesolanoi, Aleiodesjuniorporrasi, Aleiodesrocioecheverri, Aleiodesronaldzunigai, Choreborogasjesseausubeli, Triraphisdoncombi, and Yeliconesmayrabonillae.Twenty-nine species are treated, most of which have host caterpillar and food plant records, and all but one are new to science. The first host record for the agathidine genus  Agathidinae, Braconinae, Cheloninae, Macrocentrinae, Orgilinae, Proteropinae, Rhysipolinae, and Rogadinae. Of these 28 species, 23 are reared from host caterpillars and their food plants are documented. This is an addendum to COI DNA barcode sequences. The purpose of this research is to diagnose and name 28 new species of Costa Rican braconids. We deal with braconid subfamilies: Alabagrus (Braconidae) and the Mesochorus (Ichneumonidae). We emphasize that in these two publications the keys and descriptions required enormous time, effort, and expense; they are little used; and when they are used, the results are usually erroneous. This makes them useless or even detrimental to the taxonomy and understanding of these groups.We briefly describe the fate of large monographs that treat small portions of hyper-diverse Neotropical ichneumonoids by exemplifying the Alabagrus revision has 32 citations and the Mesochorus revision has 39. The majority of the citations are geographical surveys that simply copy the distributional records that are in these papers. For example, Ichneumonidae occurring in Peru and included a number of species cited as being present in Peru by According to a search in Google Scholar (May 2021), the Alabagrus employed the key of Alabagrus albispina, A. imitatus, A. juchuy, A. kagaba, A. latisoma, A. latreillei, A. maya, A. mojos, A. nahuatl, A. nigrilitus, A. pachamama, A. paruyana, A. parvifaciatus, A. semialbus , A. tricarainatus, A. tripartitus, and A.warrau. Furthermore, five of the species reported by Alabagrus cocto, A. englishi, A.pecki, A.roibasi, and A. yaruro .Only four publications dealing with Neotropical . yaruro . There ian fauna . In thisAlabagrus, only one person other than Sharkey has used the key to arrive at a determination for Neotropical species, and in that instance most of the identifications are probably incorrect. It took Sharkey more seven years to produce the 1988 revision, and it is worse than useless because it is full of misleading information on species limits and species distributions, owing to misidentifications. Some might argue for an integrative approach, such as the revision of Alabagrus by COI barcode is the only reliable source for identification (barring much more expensive and complex multi-gene information)?In summary, in the 30-plus years since the publication of the morphology-based revision of Mesochorus , we estimate that there are approximately 688 species in Costa Rica. These species are almost exclusively from the Area de Conservaci\u00f3n Guanacaste, from rearings that have been conducted exclusively in the provinces of Guanacaste and Alajuela   . To helptierrezi .Macrocentrusgeoffbarnardi . The specimens on C1 cannot be separated from those on C2 on morphological grounds. However, the entity (C1 + C2) can be separated on morphological grounds from all of the other specimens in BIN BOLD:ACK7466. Finally, no other specimens in the BIN are parasitoids of species of Neurophyseta. Therefore, we considered the entire cluster (C1 + C2) as one species. If further examination or data suggest that it is two, then one more will be also described. Similar arguments were used to delimit the other nine species in the BIN . The NJ tree produced by BOLD indicates slightly different relationships but, as with the Bayesian tree, the three morphologically indistinguishable species are not sister species nor are they nearest neighbors by any definition of that concept. We may have made different decisions if these lineages shared hosts or formed a monophyletic clade and were represented by very few sequences.Figure As with those of COI barcodes and submitting them to BOLD. Instructions on how to do this are included below.Identification of specimens to the subfamily level can be achieved using the key by Antaeotricha Janzen233 is identified to the genus Antaeotricha by classical morphology-based criteria and to Janzen233 by barcode and ecological information. However, no formal scientific species name is available until a barcode-match is obtained with an existing holotype or until it is described as new, or interim matched morphologically with a described species by a taxonomic specialist, which may take decades. Equally, Antaeotricharadicalis EPR03 is also an interim name based on what the species looks like, however, it is not a scientific name. It temporarily retains the information that this species is recognized by similarity with its look-alike, A.radicalis, before barcoding and associating it with other ecological data. Finally, a name such as gelJanzen01 Janzen407 signifies a caterpillar in the family Gelechiidae for which even a generic name is not obtainable at present.Some host species treated here are awaiting full identification and are given interim names. For example, c oxidase subunit I (COI) gene using standard insect primers LepF1 (5'-ATTCAACCAATCATAAAGATATTGG-3') and LepR1 (5'-TAAACTTCTGGATGTCCAAAAAATCA-3') (Molecular work was carried out at the CBG using their standard protocols. A leg of each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol . ExtractATCA-3') . If initBarcode sequences presented in the species descriptions herein are a consensus of the barcode sequences of all included individuals, meaning base pairs that differ between conspecific specimens are replaced by IUPAC ambiguity codes.http://janzen.sas.upenn.edu/caterpillars/database.lasso). Specimen voucher codes beginning with BIOUG are from the BOLD database (http://www.boldsystems.org), and most of the specimens obtained from ACG Malaise traps have this prefix. The DHJPAR and their associated SRNP codes can also be found on the BOLD database. The abundant collateral information obtainable from these two databases complements the species treatments. See Voucher codes are presented for all holotype specimens  treated herein. All host caterpillars are individually vouchered to their individual records (yy-SRNP-xxxxx). Codes beginning with DHJPARxxxxxxx are for the parasite (or hyperparasite) specimens reared from the caterpillar; therefore, each wasp carries two voucher codes, one for the rearing (host) record and one for the wasp itself. The SRNP voucher codes are from the Janzen and Hallwachs\u2019 database (http://www.boldsystems.org/index.php/IDS_OpenIdEngine). 2. Paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library . 3. The search results page shows the top hits based on % similarity starting with the closest matches. This page also provides additional information to help verify the identity of a match, such as links to the BIN where specimen data (including images) can be found, a distribution map, and a tree-based identification tool. 4. Use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.The BOLD database can be used to identify specimens using the following steps: 1. Navigate to the identification tab of the BOLD Systems database  feeding on Desmopsisschippii (Annonaceae). Host caterpillar voucher code13-SRNP-972Hosts are all the same as that of the holotype: DHJPAR0054734, DHJPAR0055235, DHJPAR0051139, DHJPAR0051915, DHJPAR0055516, DHJPAR0054741, DHJPAR0055237, DHJPAR0054728, DHJPAR0055233.Aerophilusdavidwagneri is named in honor of David Wagner of the University of Connecticut, Storrs, Connecticut, USA, for his recent work as an environmental activist for a healthier global climate and wild biodiversity.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.EBD717AC-52CC-5638-BAA9-AA8747A70927http://zoobank.org/A73266E4-1332-4185-816A-4AD9EEDACEC4Figure BOLD:ACN0950; nearest neighbor: AerophiluscalcaratusBOLD:AAU4711; distance to nearest neighbor is 5.81%. Consensus barcode AATTTTATATTTTATTTTTGGAATTTGATCTGGTATTTTAGGATTATCAATAAGAATCATTATTCGTATAGAATTAAGATTAGGGGGTAATTTAATTGGTAATGATCAAATTTATAATAGAATTGTTCTGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCGATTATAATTGGAGGATTTGGAAATTGATTAGTTCCTTTAATGTTAGGAGGTCCAGATATGGCCTTTCCACGRATAAATAATATAAGATTTTGATTATTAATTCCTTCATTAACTTTATTAATTTTAAGATCAATATTAAATGTTGGTGTAGGTACGGGATGAACTGTYTATCCTCCCTTATCATTAAATATAAGTCATAGAGGAATATCTGTAGATTTAGCAATTTTTTCTTTACATAYTGCTGGAATTTCTTCTATTATAGGAGCAATAAATTTTATTACTACAATTATTAATATATGRATAATAAATGTAAAAATTGATAAAATACCTTTATTGGTATGATCTATTTTTATTCTGCTATTTTATTATTATTATCTTTACCAGTATTAGCTGGGGCTATTCTATATTATTAACTGATCGAAATTTAAATCTAGATTTTTTGATCCTTCTGGAGGAGGAGATCCAATTTTATATCAACACTTATTTBIN: Aerophilusfundacionbandorum keys to A.macadamiae in Aerophilusfundacionbandorum differs in many ways. One of the most obvious is its wide, sharply angled, median propodeal areola  feeding on Schnellaguianensis (Fabaceae), caterpillar voucher code13-SRNP-71694.Same host species as that of holotype DHJPAR0054547.Aerophilusfundacionbandorum is named in honor of the BAND Foundation of the USA, in recognition of its decades of support for growth and development of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste and most recently for adding 85 more hectares to ACG of original forest, Bosque Transici\u00f3n, lying on the nearly extinct fusion of dry forest with rain forest (http://www.gdfcf.org/content/introducing-bosque-transici\u00f3n).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7B32CE61-1022-5440-8680-A2BCE450C6AEhttp://zoobank.org/5C52FDA0-10E1-4152-9EE5-4D7BFE64516EFigure BOLD:ACT7814; nearest neighbor: AerophiluscolleenhitchcockaeBOLD:ACA4890; distance to nearest neighbor is 5.16%. Consensus barcode: AATTTTATATTTTATTTTTGGAATTTGATCTGGAATTTTAGGATTATCAATAAGAATAATTATTCGTATAGAATTAAGATTAAGGGGCAATTTAATTGGAAATGATCAAATTTATAATAGAGTTGTT-CTGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCAATTATGATTGGGGGTTTTGGTAATTGATTAATTCCTTTAATATTAGGAGGTCCAGATATAGCATTTCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTATTTTTATTAATTTTAAGTTCAATATTAAATATTGGAGTAGGTACAGGATGAACTGTTTATCCTCCTTTATCATTAAATATAAGACACAGAGGAATATCTGTAGATTTAGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCTATTATAGGGGCAATAAATTTTATTACTACAATTATTAATATATGAATAATAAACGTAAAAATTGATAAAATACCTTTATTAGTATGATCCATTTTTATT-CTGCTATTTTATTATTATTATCTTTACCAGTATTGGCTGGAGCTATT-CTATATTATTAACAGATCGAAATTTAAAT-CTAGATTCTTTGATCCTTCAGGGGGAGGAGATCCTATTTTATATCAACATTTATTTBIN: A.jessicadimauroae in A.nicklaphami differs in many ways. Two of the most obvious are the wide sharply angled median propodeal areola and the sharp lateral longitudinal carinae on the first metasomal median tergite. In A.jessicadimauroae the areola is gradually narrowed anteriorly and the carinae are not sharp.This species keys to Aerophiluscolleenhitchcockae, by having the hind coxa and femur entirely brown  feeding on Ficuscitrifolia (Moraceae), caterpillar voucher code: 14-SRNP-41346.Aerophilusnicklaphami is named in honor of Nick Lapham of the BAND Foundation of the USA, in recognition of his decades of support for growth and development of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste, Costa Rica, and most recently adding 85 more hectares to ACG of original forest, Bosque Transici\u00f3n, lying on the nearly extinct fusion of dry forest with rain forest (http://www.gdfcf.org/content/introducing-bosque-transici\u00f3n).Taxon classificationAnimaliaHymenopteraBraconidae?Lindsay & Sharkey, 2006D52030DC-E128-5B95-8D89-CA6C069F82EDFigure There is no BIN for this specimen because the barcode is too short to merit a BIN code. The short barcode follows:ATATTTATTTAATTTTTGGAATTTGATCAGG-ATTTTAGGATTATCAATAAGAATAATTATTCGTATAGAATTAAGAATGGGGGGAAATTTTATTGGTAATGATCAAATTTATAATAGAATTGTT-CTGCTCATGCATTTATTATAATTTTTTTTAAAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGGGGCCCAGAAAAAGCTTTCCCCCGAATAAATAATATAAAATTTTGATThis specimen was identified based on morphological criteria from the key in Reared specimen: ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Puente Mena, 280 m, 11.04562 -85.45742; host caterpillar collection date: 11/07/2007, parasitoid eclosion: 27/07/2007; depository CNC, voucher code: DHJPAR0028287.Reared specimens host data: Dysodiaspissicornis (Thyrididae) a leaf-roller feeding on Heisteriaconcinna (Olacaceae), caterpillar voucher code: 07-SRNP-22487.This is the first host record for this wasp genus.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.9AA5EF7E-2B85-5723-B06A-C334F012820Bhttp://zoobank.org/B88988CF-D2D5-4B57-ACC5-B3D5A8E7A2DFFigure BOLD:ACJ2185; nearest neighbor: LytopylusdavidschindeliBOLD:ACB1289; distance to nearest neighbor is 2.56%. Consensus barcode: AATTTTATATTTTATATTTGGTATTTGATCAGGAATTTTAGGTTTATCATTAAGATTAATTATTCGAATAGAATTAAGAATTGGTGGAAATTTAATTGGAAATGATCAAATTTATAATAGAATTGTTACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGTAATTGATTAATTCCTTTATTATTAGGAGGTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGATCTTTAATTAATATTGGTGTAGGTACAGGATGAACAGTTTATCCTCCATTATCTTTAAATATAAGTCATAGTGGTATATCTGTAGATATAGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGAGCTATAAATTTTATTACTACTATTATAAATATATGAATTTTAAATTTAAAATTTGATAAAATACCTTTATTAGTTTGATCAATTTTAATTACAGCAATTTTATTATTATTATCATTACCAGTTTTAGCTGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACAAGATTTTTTGATCCTTCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTBIN: L.youngcheae in L.youngcheae and entirely orange in L.davidstopaki. This species can be morphologically distinguished from its nearest neighbor, Lytopylusdavidschindeli, by having its mesosoma and coxae entirely tan  feeding on Calatolacostaricensis (Metteniusaceae), caterpillar voucher code:13-SRNP-30082.elachJanzen01 Janzen527 , in recognition of his decades of editorial understanding and accepting the strange research emerging from the biodiversity inventory of Costa Rica\u2019s \u00c1rea de Conservaci\u00f3n Guanacaste.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.8C468F73-0A3F-557C-849D-396D447833BFhttp://zoobank.org/5006423F-2393-4963-9EE1-8423DC2CE954Figure BOLD:ACB1289; nearest neighbor: LytopylusdavidstopakiBOLD:ACJ2185; distance to nearest neighbor is 2.56%. Consensus barcode AATTTTATATTTTATATTTGGAATTTGATCAGGAATTTTAGGATTATCATTAAGATTAATTATTCGAATAGAATTAAGAATTGGAGGAAATTTAATTGGTAATGATCAAATTTATAACAGAATTGTAACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGGTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTATTATTATTAATTTTAAGGTCTTTAATTAATATTGGAGTAGGAACAGGATGAACAGTTTATCCTCCTTTATCTTTAAATATAAGTCATAGTGGTATATCTGTAGATATGGCAATTTTTTCTTTACATATTGCTGGAATTTCTTCAATTATAGGAGCTATAAATTTTATTACTACTATTATAAATATATGAATTTTAAATTTAAAATTTGATAAAATACCTTTATTAATTTGATCAATTTTAATTACAGCAATTTTATTATTATTATCATTACCAGTTTTAGCTGGTGCTATTACTATATTATTAACTGATCGAAATTTAAATACAAGATTTTTTGATCCATCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTBIN: L.angelagonzalezae in Lytopylusdavidschindeli is almost completely smooth with the central areola barely indicated. This species can be morphologically distinguished from its nearest neighbor, Lytopylusdavidstopaki, by having the mesosoma and coxae entirely black or dark brown  feeding on Prunusannularis (Rosaceae), caterpillar voucher code:12-SRNP-35088.elachJanzen01 Janzen185  which is a primary parasitoid of Pachyliaficus (Sphingidae) feeding on Macluratinctoria (Moraceae). Including the holotype, five specimens were reared from the fly puparia parasitizing the caterpillar, voucher code 08-SRNP-13289.The host flies were identified from their surviving sibs, one of which is DHJPAR0027924 of BIN BOLD:ACE9310.Hyperparasitoid of the fly Reared from the same caterpillar as the holotype DHJPAR0028038, DHJPAR0028039, DHJPAR0028040, DHJPAR0028041.Gnathopleurajosequesadai is named in honor of Jos\u00e9 Ram\u00f3n Quesada Mora, the manager of the 2020\u201321 BioAlfa Malaise traps for the Hacienda Baru Wildlife Refuge, Costa Rica.Gnathopleura confirmed to be a hyperparasitoid.This is the first species of Coleoptera and Lepidoptera, but use many other insect orders as well. A key to the Braconinae genera of the New World is in Braconines are mostly primarily idiobiont parasitoids of Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.9281613C-E428-552C-94D0-B39D9FD09F6Chttp://zoobank.org/C8FDFE7C-5289-4EF6-A9F0-BC1C493AEBBDFigures BOLD:AAJ8891; nearest neighbor: BraconfranklinpaniaguaiBOLD:ACK6897; distance to nearest neighbor is 5.64%. Consensus barcode: TATATTATATTTTATACTTGGTATTTGATCTGGTATAATTGGTTTATCAATAAGTTTAATTATTCGGTTAGAATTAAGAATACCAGGAAGTTTATTAAGTAATGATCAAATTTATAATAGAATAGTTACAGCACATGCTTTTGTAATAATTTTTTTTATAGTTATACCAGTGATATTAGGAGGGTTTGGTAATTGATTAATCCCTTTAATATTGGGATCTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCATTAATTTTATTATTATTAAGAAGAATATTAAATGTAGGAGTGGGTACTGGTTGAACTATTTATCCTCCATTATCTTCTAACTTAGGGCATAGAGGTGTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTATTTCATCTATTATAGGTTCAATTAATTTTATTTCTACAATTTTAAATATACATTTATTAATATTAAAATTAGATCAATTAACTTTATTTATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGAGGAGGGGATCCAATTTTATTTCAACATTTATTTBIN: B.franklinpaniaguai  feeding on Chrysochlamysglauca (Clusiaceae). The species is a gregarious parasitoid; the holotype is one of 56 specimens that emerged from the host, caterpillar voucher code: 07-SRNP-30348.07-SRNP-30348 Ten specimens, reared from the same caterpillar as the holotype, were mounted and designated as paratypes (DHJPAR0066400 to DHJPAR0066409), depository CNC.Braconandreamezae is named in honor of Ministra Andrea Meza Murillo of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of her taking on this difficult ministerial task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.3CC0B67E-7C11-5917-9AE6-2D3E0B4B56D3http://zoobank.org/45DC951E-E7E0-4C69-9518-E2628FAA99DEFigure BOLD:ACK6897; nearest neighbor: BraconalejandromasisiBOLD:AAA5367; distance to nearest neighbor is 4.49%. Consensus barcode: ATATTATATTTTTTATTTGGAATTTGAGCTGGAATAATTGGTTTATCAATAAGATTAATTATTCGTTTAGAATTAGGTATACCAGGTAGTTTATTAGGTAATGATCAAATTTATAATAGTATAGTTACAGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCAGTAATATTAGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCTTCATTAATTTTATTATTATTAAGAAGAATATTAAATGTTGGTGTAGGGACAGGTTGAACTATTTATCCTCCTTTATCATCTAGGTTAGGTCATGGAGGTATATCTGTTGATTTAATTATTTTTTCTTTACATTTAGCTGGTATTTCATCTATTATAGGATCTATTAATTTTATTACTACAATTTTAAATATGCATTTATTAATATTAAAGTTAGATCAATTAACTTTATTTATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTATTAGCAGGAGCTATTACTATATTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGAGGTGGGGATCCAATTTTATTTCAACATTTATTTBIN: B.alejandromasisi  feeding on Crotonbillbergianus (Euphorbiaceae), caterpillar voucher code: 04-SRNP-56695.Eight specimens reared from the same host specimen as the holotype were mounted and designated as paratypes (DHJPAR0066410 to DHJPAR0066417), depository CNC.Braconfranklinpaniaguai is named in honor of Vice-Minister Franklin Paniagua Alfaro of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of his taking on this difficult task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.4A5F25E2-2180-5096-8BC8-2B5996EE17B3http://zoobank.org/0093066D-6B8F-4674-9D40-9538CD2ECE5CFigure BOLD:ACB1290; nearest neighbor: Bracon sp.BOLD:AAV0639; distance to nearest neighbor is 2.24%. Consensus barcode: GATTTTATATTTTTTATTTGGTATATGAGCAGGTATAGTTGGTTTATCAATAAGGTTAATTATTCGATTAGAATTAGGTATACCTGGGAGTTTACTAGGTAATGATCAAATTTATAATAGTATAGTGACTGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTGTAATATTAGGAGGATTTGGTAATTGATTAATTCCTTTAATATTAGGAGCTCCAGATATAGCTTTTCCTCGTTTAAATAATATAAGATTTTGGTTATTATTTCCTTCTTTAATTTTATTATTATTAAGAAGAATTTTAAATGTTGGTGTAGGTACTGGGTGAACAATATACCCACCTTTGTCATCTAGATTAGGTCATAGAGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGGGTTTCTTCTATTTTAGGTTCAGTTAATTTTATTACAACAATTTTAAATATACATTTATTAATATTAAAATTAGATCAATTAACTTTATTAATTTGATCAATTTTTATTACAACTATTTTATTATTATTATCTTTACCTGTTTTAGCTGGTGCTATTACTATATTATTAACAGATCGAAATTTAAATACTTCTTTTTTTGATTTTTCTGGTGGAGGGGATCCTATTTTATTTCAACATTTATTTBIN: This species can be morphologically distinguished from its nearest neighbor by having all femora dark brown and the metasoma dark brown dorsally starting at the third tergum Fig.  comparedHolotype ?: Costa Rica: Alajuela, Area de Conservaci\u00f3n Guanacaste, Sector Rincon Rain Forest, Palomo, 96 m, 10.962 -85.28; host caterpillar collection date: 05/iii/2012, parasitoid eclosion: 18/iii/2012; depository CNC, holotype voucher code: DHJPAR0049049.Cosmorrhynchaalbistrigulana (Tortricidae) feeding on Dialiumguianense (Fabaceae). This is one of the only two species of Bracon reared by us that is solitary; the ten species treated by Braconrafagutierrezi is named in honor of SINAC Director Rafa Guti\u00e9rrez Rojas of the Ministerio de Recursos Naturales y Energ\u00eda de Costa Rica (MINAE) in recognition of his taking on this difficult task mid-government.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.CE2B1B46-C058-572B-A15B-75CF854B1A96http://zoobank.org/2E651361-5D12-419B-8A55-A2504505C00EFigure BOLD:AAT8852; nearest neighbor: Bracon sp. BOLD:AED7502; distance to nearest neighbor is 8.17%. Consensus barcode: TATTTTATATTTTTTATTTGGTATATGATCAGGAATAATTGGTTTATCAATAAGTTTAATTATTCGATTAGAATTAGGGATACCTGGAAGATTATTAGGTAATGATCAAATTTATAATAGAATAGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCAATTATATTAGGAGGATTTGGGAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGTTTAAATAATATAAGATTTTGATTATTAATTCCTTCATTAACTTTATTATTATTAAGAAGAATTTTAAATGTAGGTGTAGGAACTGGGTGAACAATGTATCCTCCATTATCATCTAATTTAGGTCATAGAGGTTTATCTGTAGATTTAGCTATTTTTTCTTTACATTTAGCTGGAATTTCTTCTATTATAGGTTCAATAAATTTTATTACTACTATTTTAAATATACATTTAAAAATATTAAAACTTGATCAATTAACGTTATTAATTTGATCTATTTTTATTACTACAATTTTGTTATTATTATCTTTACCTGTTTTAGCTGGGGCAATTACTATACTATTAACTGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGGGGAGGAGACCCAATTTTATTCCAACATTTATTT.BIN: There is only one low-quality image on BOLD for the nearest neighbor, but the p-distance makes it doubtful that it is conspecific.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Mundo Nuevo, Mamones, 365 m, 10.771 -85.429; host caterpillar collection date: 01/viii/2010, parasitoid eclosion: 12/viii/2010; depository CNC, holotype voucher code: DHJPAR0040470.Dysodiasica (Thyrididae) feeding on Pipermarginatum (Piperaceae). This is one of the only two species of Bracon reared by us that is solitary; the ten species treated by Braconguillermoblancoi is named in honor of Guillermo Blanco, the BioAlfa Malaise traps manager for Parque Nacional Isla del Coco, ACMIC (\u00c1rea de Conservaci\u00f3n Marino Isla del Coco), Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.CDA27D08-BB75-5E8B-9802-9C1D254958FEhttp://zoobank.org/48249926-80DE-4AB4-98E2-D204D3A3CFB7Figures BOLD:AAY4686; nearest neighbor: Bracon sp. BOLD:AEF4783; distance to nearest neighbor is 6.09%. Consensus barcode: AGTTTTGTATTTTTTATTTGGTATATGAGCTGGTATAGTTGGTTTATCAATAAGTTTAATTATTCGTTTAGAGTTAGGTATACCTGGAAGTTTATTAGGTAATGATCAAATTTATAATAGAATAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCTGTTATAATTGGAGGATTTGGTAATTGATTAATTCCTTTAATATTAGGAGCTCCTGATATAGCTTTTCCTCGAATAAATAATATGAGATTTTGGTTATTAGTTCCTTCATTAACTTTATTATTATTAAGTAGAATTTTAAATGTAGGGGTAGGTACAGGTTGGACAATATATCCACCTTTATCTTCAAGTTTAGGTCATAGAGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTGTTTCTTCAATTATAGGGGCAATAAATTTTATTACTACTATTTTAAATATGCATTTATTAATATTAAAATTAGATCAGTTAACTTTATTAGTTTGATCAATTTTTATTACTACTATTTTATTATTATTATCTTTACCTGTTTTAGCAGGAGCAATTACAATATTATTAACTGATCGAAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGTGGAGATCCTATTTTATTTCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having the hind femur dark brown Fig.  comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Mundo Nuevo, Punta Plancha, 420 m, 10.742 -85.427; host caterpillar collection date: 17/x/2010, parasitoid eclosion: 28/x/2010; depository CNC, holotype voucher code: DHJPAR0041854.Anadasmus Janzen25 (Depressariidae) feeding on Mespilodaphneveraguensis (Lauraceae); four specimens emerged from the host, caterpillar voucher code: 10-SRNP-56886.Gregarious parasitoid of Two males, same data as holotype  depository CNC.Braconoscarmasisi is named in honor of Oscar Masis, the BioAlfa Malaise traps manager for Parque Nacional Los Quetzales, ACOPAC , Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.6C04CB63-B7C7-59F2-AAC1-E1415E3F7692http://zoobank.org/723581AA-45CD-4F42-9AB1-CED38EBEA383Figure BOLD:AEF4305; nearest neighbor: Bracon sp. BOLD:ACG3693; distance to nearest neighbor is 9.62%. Consensus barcode: TGTTTTATATTTTTTATTTGGTATATGAGCTGGGATACTAGGTCTATCAATAAGATTAATTATCCGACTAGAGCTCGGAATACCGGGAAGTTTACTTGGTAATGACCAAATTTACAATAGAATAGTAACAGCTCATGCTTTTGTAATAATTTTTTTTATAGTTATACCTGTAATAGTAGGAGGATTTGGAAATTGACTATTACCTTTAATATTAGGAGCCCCTGATATAGCATTTCCTCGTTTAAATAATATAAGATTTTGATTACTTATTCCTTCCCTAACTTTATTATTAATAAGAAGAATTTTAAATGTAGGAGTAGGGACTGGATGAACAGTTTATCCTCCTTTATCCTCTTCACTAGGTCATAGAGGGTTATCAGTTGATTTGGCTATTTTTTCTTTACATATTGCAGGAATTTCCTCAATTTTGGGGGCTATTAACTTTATTTCAACTATTTTAAATATACATTTATTAATTTTAAAACTAGATCAACTAACATTATTAATTTGATCAATTTTTATTACAGCTATTTTATTATTATTATCTTTACCAGTATTAGCAGGAGCTATCACAATATTATTAACCGATCGAAATTTAAATACATCTTTTTTTGATTTTTCAGGAGGGGGTGACCCAATTTTATTTCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having large portions of the mesoscutum and mesepimeron brown Fig.  comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Cacao, Sendero Cima, 1460 m, 10.933 -85.457, 23/iii/2009, Malaise trap, depository CNC, holotype voucher code: DHJPAR0051516.Braconpauldimaurai is named in honor of Paul Dimaura of Boston, Massachusetts for his decades of support of the University of Pennsylvania in general and D. H. Janzen\u2019s position as a professor of conservation biology specifically.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.D39CF64B-0CA1-5A5C-ABC6-CE4C0016B05Chttp://zoobank.org/E87C71B3-2C89-4EB5-AFE9-47FD75A1C533Figure BOLD:ADW8464; nearest neighbor: Bracon sp. BOLD:AEB6448; distance to nearest neighbor is 4.81%. Consensus barcode: TTTATATTTTTTATTTGGTATATGAGCAGGAATATTAGGATTATCACTAAGTTTAATTATTCGTTTAGAATTAGGGATACCTGGAAGATTATTAGGTAATGATCAAATTTATAATAGTATAGTTACAGCTCATGCATTTGTTATAATTTTTTTTATAGTTATACCAGTAATATTAGGAGGATTTGGTAATTGATTATTACCTTTAATATTAGGGGCTCCTGATATAGCTTTCCCACGAATAAATAATATAAGATTTTGGTTAATTATCCCTTCTTTAATTTTATTATTAATAAGAAGAATTTTAAATGTAGGAGTAGGAACAGGTTGAACAGTTTATCCTCCTTTATCATCTTCATTAGGGCATAGTGGGTTATCTGTTGATTTAGCTATTTTTTCTTTACATATTGCAGGAATTTCTTCAATTATAGGTTCAATTAATTTTATTACTACTATTTTTAATATACATTTATTTAAATTAAAATTAGATCAATTAACATTATTAATTTGATCTATTTTTATCACAACTATTTTACTTTTATTATCTTTACCAGTTTTAGCGGGGGGAATTACTATATTATTAACAGATCGTAATTTAAATACATCATTTTTTGATTTTTCTGGAGGGGGAGATCCTGTTTTATTTCAACATTTATT.BIN: No images of the unnamed nearest neighbor are available.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pailas Dos, PL12-1, 828 m, 10.7642 -85.335, 14/v/2015, Malaise trap, depository CNC, holotype voucher code: BIOUG44786-F08, GenBank accession MW627534.Braconshebadimaurae is named in honor of Sheba Dimaura of Boston, Massachusetts for her decades of support of the University of Pennsylvania in general and D. H. Janzen\u2019s position as a Professor of Conservation Biology.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.1A4A7A9B-09BA-58BA-87AA-4A05BC58362Fhttp://zoobank.org/5BE4D1E3-9F52-444D-8F49-AC0130EEE9FFFigure BOLD:ADY0104; nearest neighbor: Sacirema sp. BOLD:AEH2057; distance to nearest neighbor is 2.24%. Consensus barcode: TTTATATTTTTTATTTGGGATATGATCTGGTATATTAGGTTTATCAATAAGTTTAATTATTCGATTAGAACTTGGAATACCATCAAGTTTATTAACAAATGATCAAATTTATAATAGAATAGTAACTGCCCATGCATTTGTCATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTAATTCCTTTAATATTAAGAGCTCCAGATATAGCTTTCCCTCGTATAAATAATATAAGTTTTTGATTACTAATTCCTTCTTTAATAATATTAATTTTAAGAAGAATTATTAATACAGGTGTAGGTACTGGTTGAACAGTTTACCCTCCTTTATCTTCTTCTATAGGACATAGAGGAATTTCAGTTGATTTAGCAATTTTTTCTTTACATTTAGCTGGAGCTTCCTCAATTATAGGGTCTATTAATTTTATTTCAACTATTATTAATATACGACTTTATTTAATAAAAATAGATCAATTAACATTATTAATTTGATCTATTTTTATTACTACAATTTTATTATTATTATCATTACCAGTTCTAGCTGGGGCAATCACAATATTATTAACAGATCGAAATTTAAATACTACTTTTTTTGATTTTTCAGGAGGTGGGGATCCAATTTTATTCCAACACTTAT.BIN: Sacirema  feeding on Hameliapatens (Rubiaceae), caterpillar voucher code:19-SRNP-35166.19-SRNP-35166 Same host data as holotype, DHJPAR0064500, DHJPAR0064502, depository CNC.Chelonusminorzunigai is named in honor of Minor Z\u00fa\u00f1iga Siles, the BioAlfa Malaise traps manager for Estaci\u00f3n Esquinas, Parque Nacional Tortuguero, ACTO (\u00c1rea de Conservaci\u00f3n Tortuguero), Costa Rica.Homolobinae are endoparasitoids of lepidopteran larvae. A key to the genera of the New World is included in Members of Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.4D94064C-D823-5FDA-B2A0-49F2323CD7DFhttp://zoobank.org/D21DFA7D-C461-4F83-9521-79A1E9982771Figures BOLD:AAA7060. The nearest neighbor: Homolobus sp. BOLD:ACM2462 is separated by a p-distance of only 1.12%. Consensus barcode: TATTTTATATTTTATATTTGGAATTTGAGCTGGAATTTTAGGTATATCAATAAGAATTATTATTCGAATAGAATTAAGAATACCAGGTAATTTAATTGGTAACGATCAAATTTATAATAGTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGAGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGATGTGTTGATATAGCTTTTCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCATCATTAATTTTATTAATTTTAAGAAGAATTTTAAATGTTGGTGTTGGTACTGGATGAACTGTTTATCCTCCTTTATCTTTAAATATTGGTCATGGAGGTTTATCTGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGAATTTCTTCAATTATAGGAGCTATTAATTTTATTACTACTATTTTAAATATACGATCTAATTTAATTACAATAGATAAAATTTCTTTATTAAGTTGATCAATTTTAATTACTGTAATTTTATTATTATTATCTTTACCAGTTTTAGCTGGGGCTATTACAATATTATTAACTGATCGTAATTTAAATACATCTTTTTTTGATCCATCTGGTGGAGGGGATCCAATTTTATATCAACATTTATTT.BIN: Homolobusinfumator in H.stevestroudi and the size of the basal tooth of the hind tarsal claws, which are longer in H.stevestroudi. The convincing difference can be found by looking at the NJ tree produced from the BOLD website; H.stevestroudi is found in its own BIN, far removed from any other species of Homolobus and particularly distant from specimens identified as H.infumator from Norway. The type locality of H.infumator is England. All nine specimens in the unnamed nearest neighbor are from Canada. They might represent the same species as the Costa Rican specimens, but more sampling will need to be done between Canada and Costa Rica to confirm or refute. There are no obvious morphological differences based on the BOLD images of the Canadian specimens.The specimen keys to Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Cacao, Sendero Toma Agua, 1140 m, 10.928 -85.467; host caterpillar collection date: 23/iv/2009, parasitoid eclosion: 18/v/2009; depository CNC, holotype voucher code: DHJPAR0035530, GenBank accession: MW627552.Pherotesiaminuisca (Geometridae) feeding on Zygiapalmana (Fabaceae), caterpillar voucher code: 09-SRNP-35488.Homolobusstevestroudi is named in honor of Steve Stroud as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Bar\u00fa Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste.Members of all genera are koinobiont endoparasitoids of caterpillars from a wide range of families. Most are solitary, but several gregarious species are known. A key to the genera of the New World is in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.C02F5F5C-22FE-545C-8AC9-DDF6868514D7http://zoobank.org/6E04139A-E5D6-4F29-BC63-DBD88D4EBADAFigure BOLD:ACK7466. Consensus barcode: TGTTTTATATTTTTTATTAGGTATTTGATGTGGATTAGTAGGTTTATCTTTAAGGTTACTTATTCGGTTAGAGTTGAGAAATTTAGGAAGATTATTAGGTAATGATCAAATTTATAATGTAGTTGTTACTATACATGCTTTTATTATAATTTTTTTTATGGTGATACCTATTATAATTGGAGGTTTTGGAAATTGATTAATTCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTGTTATTAATATTAATAAGAGGATTTATTATAGTTGGTAGTGGAACTGGGTGAACTATATATCCTCCTTTAAGTTCTTTAATTGGGCATAGAAGGTTTTCTGTTGATATAGTAATTTTTTCTTTACATTTAGCAGGGGTTTCTTCAATTATAGGAGCTATTAATTTTATTACAACAATTTTTAATATAAAATTAATTT---TAAAATTAGATCAGATTATATTATTTGTATGATCTGTATTAATTACTGCTTTTTTATTATTACTTTCTTTACCTGTTTTGGCAGGAGGAATTACTATATTATTAACAGATCGTAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGGGGAGATCCTGTTTTATTTCAACACTTATTT.This is the eighth species to be described in BIN Macrocentrusmichaelstroudi will key to M.gustavogutierrezi. Macrocentrusmichaelstroudi differs in its pale basal flagellomeres, contrasting with the melanic basal flagellomeres of M.gustavogutierrezi. The host caterpillar also differs from those of M.gustavogutierrezi. It belongs to the group of species with vein M+Cu of the forewing distinctly widened apically.In the morphological key to the species in this BIN , MacroceHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Sendero Rotulo, 510 m, 11.0135 -85.4241; host caterpillar collection date: 22/i/2016, parasitoid eclosion: 01/iii/2016; depository CNC, holotype voucher code: DHJPAR0058830, GenBank accession: MW627584.Phaedropsis leialisDHJ03 (Crambidae) feeding on Gouanialupuloides (Rhamnaceae), caterpillar voucher code: 16-SRNP-30230.Macrocentrusmichaelstroudi is named in honor of Michael Stroud Bonilla as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Baru Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste.Members of all genera are koinobiont endoparasitoids of caterpillars. A key to the genera of the New World can be found in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.AC50A86E-2624-5E92-9CCD-172E1C60F06Bhttp://zoobank.org/6058EB69-E85F-4CFB-8771-92DD917FA191Figure BOLD:AEC3983; nearest neighbor: StantoniamiriamzunzaeBOLD:ACB1896; distance to nearest neighbor is 4.47%. Consensus barcode: TATATTGTATTTTTTGTTTGGTATATGGTCTGGGGTGTTAGGTTTATCACTAAGTTTAATTATTCGTATAGAATTAGGTCAAATTGGTTCATTTATTGGAAATGATCAAATTTATAATAGTATTGTTACTTCTCATGCTTTTATTATAATTTTTTTTATAGTTATGCCTATTATAATTGGGGGGTTTGGAAATTGATTGATTCCTTTAATATTAGGAAGTGTTGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTAATATTATTAATTTTAAGAGGATTTATAAATATTGGTGTAGGTACAGGATGAACAGTTTACCCTCCTTTATCATTAAATGTTAGTCATATAGGAATTTCTGTAGATATAGCTATTTTTTCATTACATTTGGCTGGTATTTCTTCAATTATAGGTGCTATTAATTTTATTGTTACTATTATAAATATACGAAATTATGGGGTATTAATAGATAAAATTAGATTATTATCATGATCAATTTTAATTACAGCTATTTTATTATTGTTATCTTTACCTGTGTTAGCTGGTGCTATTACAATATTGTTAACTGACCGTAATTTAAATACATCCTTTTTTGATCCTGCTGGAGGAGGGGATCCTATTTTATATCAACATTTATTTBIN: S.miriamzunzae  feeding on Vismiabaccifera (Hypericaceae), caterpillar voucher code:19-SRNP-46256. Erebidae is a new host-family record for Stantonia.Stantoniagilbertfuentesi is named in honor of Gilbert Fuentes of the Organizaci\u00f3n de Estudios Tropicales of Costa Rica in recognition of his decades of intensive management of the OET library of tropical publications.Members of the subfamily are thought to be solitary, koinobiont ectoparasitoids of caterpillars. A diagnosis for the subfamily is included in Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.B58E733C-3E93-5BB7-918B-A9768C49D776http://zoobank.org/B5D5C71B-6810-4120-976F-D30A453FF262Figure BOLD:ADA0151; nearest neighbor: Rhysipolis sp. BOLD:ADL9389; distance to nearest neighbor is 2.91%. Consensus barcode: TGTATTATATTTTTTATTTGGAATTTGATCTGGAATAGTAGGTTTGTCTATGAGTTTAATTATTCGTTTAGAGTTAGGTATACCCGGTAGTTTGTTATTTAATGATCAGATTTATAATACGATAGTTACAGCTCATGCTTTTATTATAATTTTTTTTATAGTGATACCTGTAATGATTGGGGGGTTTGGTAATTGGTTAGTTCCATTAATGTTGGGGGCTCCTGATATAGCTTTTCCTCGTATGAATAATATAAGATTTTGATTATTAATTCCTTCTTTAATTTTATTATTTTTGAGGGGATTAGTAAATGTTGGGGTAGGTACTGGATGAACAGTTTATCCTCCTTTATCTTCTTCTATAGGTCATAGAGGTATTTCTGTTGATTTGGCTATTTTTTCTTTACATTTAGCTGGTATTTCTTCTATTATAGGAGCTATTAATTTTATTTCAACAATTTTTAATATGTGTTTATATTCAATTAATATAGATCAAATTAGTTTATTTATTTGATCTATTTTGATTACTGCTTTTTTATTATTGTTGTCTTTACCTGTTTTGGCAGGGGCTATTACTATATTGTTAACGGATCGAAATTTAAATACTTCATTTTTTGATTTTTCTGGBIN: This species can be morphologically distinguished from its nearest neighbor by having its mesoscutum entirely black Fig.  comparedHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector San Cristobal, Estaci\u00f3n San Gerardo, 575 m, 10.8801 -85.389, 04/viii/2014, Malaise trap, depository CNC, holotype voucher code: BIOUG28483-E12, GenBank accession MW627555.BIOUG27682-G07.Rhysipolisstevearonsoni is named in honor of Steve Aronson of San Jose, Costa Rica, in recognition of decades of concern and involvement with the betterment of Costa Rica\u2019s positive relationship with its wild environment, and specifically with providing broadband internet to \u00c1rea de Conservaci\u00f3n Guanacaste as the first Costa Rican \u00c1rea de Conservaci\u00f3n to be so facilitated.Members of all genera are koinobiont endoparasitoids of caterpillars from a wide range of families. A key to the genera of the New World is in Sharkey (2021a).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.41DDCF67-4214-5C16-A53A-95B1E9F9DDE6http://zoobank.org/B8C158F4-07CA-4776-91B7-D7507870695FFigure BOLD:AEB2985; nearest neighbor: Aleiodes sp. BOLD:AAV6245; distance to nearest neighbor is 10.74%. Consensus barcode: AGTATTGTATTTTTTATTTGGTATATGAGCTGGAATAGTTGGTTTATCTATAAGATTAATTATTCGGTTAGAATTAAGAGTTGGGGGGAGAGTCTTAAAAAATGATCAGATTTATAAYGGTATAGTAACTTTGCATGCTTTTGTAATAATTTTTTTTATAGTTATACCTATTATATTAGGAGGGTTTGGAAATTGGTTAGTTCCTTTAATATTGAGAGCTCCAGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTATTAATTCCATCTTTTTTTTTATTATTGATTAGAGGTGTTATTAATTCAGGRGTAGGAACAGGTTGAACAATATATCCTCCTCTTTCTTTATTAATTGGTCATGATGGAATTTCTGTAGATATATCAATTTTTTCTTTACATTTAGCAGGAGCTTCTTCCATTATAGGTTCAATTAATTTTATTTCTACTATTTTTAATATAAAATTAAAAGATTTAAAATTAGATCAAGTTTCTTTATTTGTTTGATCTATTTTAATTACAACAATTTTATTATTGTTATCTTTACCTGTTTTAGCGGGGGCAATTACTATATTATTGACTGATCGAAACTTAAATACAAGATTTTTTGATTTTGCTGGAGGAGGGGATCCAATTTTATTTCAACATTTGTTT.BIN: This species can be morphologically distinguished from its nearest neighbor by having the base of the stigma brown Fig. , contrasHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Brasilia, Gallinazo, 360 m, 11.0183 -85.372; host caterpillar collection date: 10/vii/2019, parasitoid eclosion: 23/vii/2019; depository CNC, holotype voucher code: DHJPAR0064529, GenBank accession: MW627545.Isogona Poole07 (Erebidae) feeding on Celtisiguanaea (Ulmaceae), caterpillar voucher code:19-SRNP-65351.DHJPAR0065341.Aleiodeskaydodgeae is named in honor of Kay Dodge of Costa Rica\u2019s Nicoya Peninsula today, original and decades long facilitator of ACG support from Peter Wege (RIP) of the Wege Foundation of Grand Rapids, Michigan, USA.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.3A4BC25A-6E30-5F9F-A290-6CBA3F6752E7http://zoobank.org/273165E1-C8BD-43E8-8EED-75004A28DD99Figure BOLD:AEF3944; nearest neighbor: Aleiodes sp. BOLD:AAG1309; distance to nearest neighbor is 8.29%. Consensus barcode: AGTATTATATTTTTTATTTGGAATATGAGCAGGAATAATTGGGATATCAATAAGTTTAATAATCCGATTAGAATTAAGAACAAATGGAAGAATCTTAAAAAATGATCAAATTTATAATGGTATGGTAACTTTACATGCCTTTATTATAATTTTTTTTATAGTAATACCAATTATAATTGGAGGATTTGGAAATTGATTAATTCCTTTAATATTAGGAGCTCCTGACATAGCTTTCCCACGTATAAATAATATAAGATTTTGATTACTAATACCTTCTTTAATACTTTTATTACTTAGAGGAATAATTAATACCGGGGTAGGAACAGGATGAACTATATATCCCCCTTTATCATCACTAATTGGACATAATGGAATTTCAGTAGATATATCTATTTTTTCTTTACACCTTGCAGGGGCTTCTTCAATTATAGGAGCAATCAACTTCATTTCAACTATTTTTAATATAAATATTATAACAATTAAAATAGATCAAATTATACTATTAATTTGATCTATTTTAATTACTACAATCCTTTTATTATTATCTTTACCAGTATTAGCAGGAGCAATTACTATATTACTAACAGATCGAAATTTAAATACAAGATTTTTTGACTTTTCAGGGGGAGGAGACCCTATTTTATTCCAACATCTTTTT.BIN: This species can be morphologically distinguished from its nearest neighbor by its pale stigma Fig. , which iHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Bullas, 440 m, 10.98670 -85.38503; host caterpillar collection date: 25/i/2019, parasitoid eclosion: 13/ii/2019; depository CNC, holotype voucher code: DHJPAR0063995, GenBank accession: MW627548.Anomisgentilis (Erebidae) feeding on Peltaeaovata , caterpillar voucher code:19-SRNP-70250.DHJPAR0063970.Aleiodeskerrydresslerae is named in honor of Kerry Dressler for her life career of deep and intense work and interest in the taxonomy of orchids, and of supporting Bob Dressler\u2019s enthusiasm for the same.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7B3CF334-BA1C-592A-952E-AC5502B0E972http://zoobank.org/2A3DC697-674E-4EB5-AE11-A15680375504Figure BOLD:AEB1913; nearest neighbor: Aleiodes sp. BOLD:AAM5681; distance to nearest neighbor is 2.24%. Consensus barcode: AATTTTATATTTTTTATTTGGTTTATGAGCAGGAATAATTGGGATATCAATAAGATTAATTATTCGATTAGAATTAAGAACTAGAGGTAGAATTTTAAAAAATGATCAAATTTATAACGGCATAGTAACTTTACATGCATTTATTATAATTTTTTTTATAGTAATACCCATTATAATTGGAGGGTTTGGAAATTGATTAATYCCTCTAATATTAGGAGCCCCTGATATAGCATTTCCTCGAATAAATAATATAAGATTTTGATTACTAATTCCATCATTAATATTTTTATTAATTAGAGGAATTATTAATACAGGTGTAGGAACAGGATGAACAATATATCCTCCATTATCTTCATTAATTGGACATAATAGAATTTCAGTTGATATATCAATTTTTTCTTTACATATAGCAGGTGCTTCATCAATTATAGGAGCTATTAATTTTATTTCAACAATTTTCAATATAAACTTAATAAAAATCAAAATAGAYCAAATTATATTATTAGTTTGATCAGTTTTAATTACAGCCATTTTATTATTACTTTCATTACCTGTTTTAGCAGGAGCAATCACTATATTRTTAACCGACCGTAACTTAAATACAAGTTTTTTTGATTTTTCAGGAGGAGGAGATCCTATTTTATTCCAACATTTATTT.BIN: The nearest neighbor is a sole specimen from Canada. There is no image available on BOLD but due to the distribution, conspecificity is doubtful.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Meteorologico, 590 m, 11.002 -85.4617; host caterpillar collection date: 25/vi/2019, parasitoid eclosion: 15/vii/2019; depository CNC, holotype voucher code: DHJPAR0064517, GenBank accession: MW627567.Herbitamedama (Geometridae) feeding on Dendropanaxarboreus , caterpillar voucher code:19-SRNP-20457.Prenestascyllalis feeding on Forsteroniaspicata (Apocynaceae), depository CNC.DHJPAR0064001 (DHJPAR0062033 not barcoded), host data Aleiodesjosesolanoi is named in honor of Jose Andr\u00e9s Solano, the BioAlfa Malaise traps manager for Estaci\u00f3n El Ceibo, Parque Nacional Braulio Carrillo, ACC, Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.7F53D704-D338-583F-AF41-E21CF4F5BA18http://zoobank.org/474FE466-BEF5-4451-8623-252D716F6B6FFigure BOLD:AAV7490; nearest neighbor: Aleiodes sp. BOLD:AAV6239, from French Guiana; distance to nearest neighbor is 8.65%. Consensus barcode. AATTTTATATTTTTTATTTGGTTTATGGTCAGGAATAATTGGCATGTCAATAAGATTAATTATTCGATTAGAATTAAGAACGAGAGGTAGAATTTTAAAAAATGACCAAATTTATAATGGCATAGTAACTTTACATGCATTTATTATAATTTTTTTTATAGTAATACCAATTATAATTGGTGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGAGCCCCTGATATAGCATTTCCTCGTATAAATAATATAAGATTTTGATTATTAATCCCATCACTAATATTTTTATTGATTAGAGGTATTATTAATACAGGAGTAGGGACAGGATGAACTATATATCCTCCCCTATCTTCCTTAATTGGCCATAATAGAATATCAGTTGATATATCAATTTTTTCTCTCCATATAGCTGGAGCCTCATCAATCATAGGAGCAATTAATTTCATCTCAACAATTTTTAACATAAATCTAATAAAAATTAAAATAGACCAAATTATACTATTAGTATGGTCAGTTTTAATTACAGCTATTTTATTACTACTTTCATTACCTGTTTTAGCAGGAGCAATTACAATATTATTAACTGACCGTAATTTAAATACAAGATTTTTTGATTTTTCAGGAGGAGGGGACCCCATTTTATTCCAACATTTATTT.BIN: This species can be morphologically distinguished from its nearest neighbor by its uniformly colored hind legs Fig. , compareHolotype ?: Costa Rica: Alajuela, Guanacaste Area de Conservaci\u00f3n, Sector Rincon Rain Forest, Sendero Aura, 432 m, 10.9654 -85.3239; host caterpillar collection date: 04/vii/2019, parasitoid eclosion: 31/vii/2019; depository CNC, holotype voucher code: DHJPAR0064521, GenBank accession: MW627570.Geometridae) feeding on Serjaniaschiedeana (Sapindaceae), caterpillar voucher code:19-SRNP-27158.geoJanzen01 Janzen7158 .Aleiodesjuniorporrasi is named in honor of Junior Porras Quir\u00f3s, the BioAlfa Malaise traps manager for Estaci\u00f3n Altamira, Parque Nacional Chirripo, ACLAP, Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.55F61DB3-C558-584F-8E23-B05A51A872EBhttp://zoobank.org/E8FBEA44-9DE3-4E93-81F8-0CBAD405DB42Figure BOLD:AAM5673; nearest neighbor: Aleiodes sp. BOLD:AAH8820; distance to nearest neighbor is 4.03%. Consensus barcode: GTTTTATATTTTTTATTTGGGATATGAGCTGGTATATTAGGRTTATCTATAAGGTTAGTTATYCGTTTAGAATTAAGAAYTGTTGGRAGAGTTTTAAAAAATGATCAAATTTATAATGGKATGGTTACATTACATGCTTTTGTAATAATYTTTTTTATAGTTATACCTATTATAATTGGTGGGTTTGGAAATTGATTAATTCCTTTAATATTAGGGGCTCCTGATATAGCATTYCCTCGGATAAATAATATGAGATTTTGRTTATTAATTCCTTCATTTTTTTTATTATTAATTAGAGGTGTTATTAATTCAGGGGTAGGTACAGGTTGAACAATATACCCTCCCCTTTCTTTATTAATTGGTCATAATGGTTTATCAGTGGATATATCTATTTTTTCTTTACATTTAGCTGGRGCTTCTTCTATTATAGGATCAATTAATTTTATTTCAACTATTTTTAATATAAATTTATTTTATATTAAATTAGATCAGATTTCTTTATTAGTATGGTCAGTATTAATCACTACTATTTTATTATTATTATCTTTACCTGTTTTRGCAGGGGCTATTACTATATTATTGACTGATCGTAATTTAAATACAAGATTTTTTGATTTTTCGGGGGGAGGGGATCCAATTTTATTTCAACATTTA.BIN: There is no image on BOLD, but the p-distance makes it doubtful that it is conspecific.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector San Cristobal, Estaci\u00f3n San Gerardo, 575 m, 10.88 -85.389, 18/xi/2013, Malaise trap, depository CNC, holotype voucher code: BIOUG20202-G10, GenBank accession: MW627543.Other material. BMNHE897799, from Belize deposited in the Natural History Museum (London), based on barcode, not viewed and lacking image on BOLD.Aleiodesrocioecheverri is named in honor of Rocio Echeverri of San Jose and Liberia, Costa Rica, in recognition of her lifetime of concern and involvement with the betterment of Costa Rica\u2019s positive relationship with its wild environment.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.01BDD77E-547F-5861-BF97-A6940D4496B8http://zoobank.org/A73F3D24-4F8D-42C3-94F3-EC56B5219C77Figure BOLD:AAH8707; nearest neighbor: Aleiodesnr.speciosusBOLD:AAM4342; distance to nearest neighbor is 2.4%. Consensus barcode: AATTTTATAYTTCATATTTGGAATATGAGCAGGTATAATTGGAATATCAATAAGATTAATTATTCGAATAGAATTAAGAACAAGAGGAAGAATTTTAAAAAATGACCAAATTTATAATGGTATAGTAACTTTGCATGCTTTTATTATAATTTTTTTTATAGTTATACCARTTATAATTGGGGGATTCGGAAATTGATTAATCCCCTTAATATTAGGGGCCCCTGATATAGCATTCCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCATCTTTATTATTTTTACTAATAAGAGGTATTATTAATACAGGAGTTGGGACAGGATGAACTATATACCCTCCTTTATCRTCTTTAATTGGACATAATAGAATTTCARTTGATATGTCAATTTTTTCTTTACATTTAGCAGGAGCTTCTTCTATTATAGGRGCAATTAATTTTATTTCAACAATTTTTAATATAAATTTAATAAAAATTAAATTAGACCAAATTTCATTGTTAATTTGATCAATTTTAATTACTACTATTTTATTATTATTATCTTTACCTGTACTAGCAGGAGCAATCACCATATTATTAACTGATCGTAACTTAAACACAAGATTTTTTGATTTTTCTGGAGGAGGAGAYCCAATTTTATTTCAACATTTATTT.BIN: No images are available on BOLD for the three specimens in the nearest neighbor, all from Ecuador.Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Del Oro, Sendero Puertas, 400 m, 11.01087 -85.48816; host caterpillar collection date: 28/xii/2018, parasitoid eclosion: 07/i/2019; depository CNC, holotype voucher code: DHJPAR0064524, GenBank accession: MW627585.Geometridae) feeding on algae, caterpillar voucher code:18-SRNP-21223.geoJanzen01 19-SRNP-20029 , Costa Rica.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.236166A3-241E-5A99-9034-2FAAC25B4297http://zoobank.org/20C776D0-74D9-4B3D-B0E0-EE4E117B28CBFigure BOLD:AAM5951; nearest neighbor: Choreborogas sp. BOLD:ACG8400; distance to nearest neighbor is 2.71%. Consensus barcode:BIN: AGTATTGTATTTTTTTTTTGGTATATGATCAGGTATATTGGGYTTATCAATAAGGTTAATTATTCGGTTTGAATTAGGGGTTCCTGGATCATTTTTAGGTAATGATCAGATTTATAATAGAATTGTTACGGCYCATGCCTTGGTTATAATTTTTTTTATGGTTATACCTGTAATAATTGGGGGATTTGGTAATTGATTAATTCCTTTAATATTAGGRGCACCTGATATAGCTTTYCCTCGAATAAATAATATAAGATTTTGGTTATTAATTCCTTCTATTTTGTTATTGTTAGTTAGATCTTTAGTTAATGTTGGGGYAGGTACAGGATGAACAATTTATCCTCCTTTATCTTCRTTAATAGGTCATGGSGGGATTTCAGTTGATTTAGCTATTTTTTCTTTACATTTAGCTGGTGCATCATCAATTATAGGTGCAATTAATTTTATTTCTACAATTTTTAATATAAATTTATTTTCAATGAAAATAGATCAAATTATATTATTAGTTTGATCTGTATTAATCACTGCTTTTTTATTATTATTATCATTRCCTGTTTTGGCGGGGGCAATTACTATATTATTATTTGATCGTAAYATTAATAGAACTTTTTTTGATTTTTCAGGGGGAGGGGATCCTATTTTATTYCAGCATTTATTThis species can be morphologically distinguished from its nearest neighbor by its swollen hind basitarsus Fig. , which iHolotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pailas Dos, PL12-6, 853 m, 10.7637 -85.3331, 04/xii/2014, Malaise trap, depository CNC, holotype voucher code: BIOUG46391-F12, GenBank accession: MW627542.Malaise trapped, BIOUG46544-F12, BIOUG49790-H06, BIOUG07453-F05, BIOUG28810-A07, BIOUG29020-B09.Other material: BMNHE897774 from Belize is in the same BIN and likely conspecific. There is no image on BOLD and the specimen was not examined.Choreborogasjesseausubeli is named in honor of Jesse Ausubel of Rockerfeller University, New York, USA, for his very strong support of the germination and early development of DNA barcoding as an identification tool.Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.BC1796D0-995C-5151-89B9-479740A8C5D5http://zoobank.org/8228960E-CF6E-4BF9-8C86-696F7EA67FE5Figures BOLD:AAH8815; nearest neighbor: Triraphis sp. BOLD:AAG5003 from Guyana. Distance to nearest neighbor is 6.28%. Consensus barcode:TGTTTTATATTTTTTATTTGGAATTTGAGCTGGTATAGTCGGGCTGTCTATAAGGTTAATTATTCGGTTAGAATTAAGTATACCAGGGAGATTATTGGGGAATGAYCAGATTTATAATGGTATAGTTACCGCTCATGCTTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGTGGTTTTGGAAATTGATTAATTCCATTAATGTTGGGGGCYCCTGATATGGCTTTCCCTCGTATAAATAATATGAGGTTTTGGTTATTAATTCCYTCATTGACGTTATTAATTTTAAGGGCTGTAGTTAACGTTGGAGTAGGTACTGGGTGAACTTTATATCCYCCCTTATCTTCTTTAGTTGGTCATGGGGGTATATCTGTAGATATAGCTATTTTTTCTTTACATTTAGCTGGTGCCTCTTCTATTATAGGAGTTGTTAATTTTATTTCTACTATTTTTAATATAAAATTAATATCAATTAATTTAGATCAAATTAATTTATTTGTTTGATCAGTATTAATTACGGCTGTTTTATTATTATTATCTTTACCAGTATTAGCTGGTGCAATTACTATATTATTGACAGATCGTAATTTAAATACAACATTTTTTGATTTTTCTGGGGGGGGYGATCCTATTTTATTCCAACATTTATTTBIN: COI distance and geographic distribution suggest that they are not the same species.No image of the nearest neighbor is available on BOLD, but the Holotype ?: Costa Rica: Guanacaste, Area de Conservaci\u00f3n Guanacaste, Sector Pitilla, Sendero Naciente, 700 m, 10.98705 -85.42816; host caterpillar collection date: 09/ii/2010, parasitoid eclosion: 13/ii/2010; depository CNC, holotype voucher code: DHJPAR0038023. GenBank accession: HQ548697.Holotype host data.Eucleamesoamericana (Limacodidae) feeding on Thelypterisnicaraguensis (Thelypteridaceae), caterpillar voucher code: 10-SRNP-30444.BCLDQ0860.Triraphisdoncombi is named in honor of Dr. Don Comb (RIP), founder of the New England Biolabs and New England Biolabs Foundation, in recognition of his serious and ongoing support for the management and biodiversity conservation of \u00c1rea de Conservaci\u00f3n Guanacaste in northeastern Costa Rica (http://www.acguanacaste.ac.cr), through the Guanacaste Dry Forest Conservation Fund (http://www.acguanacaste.ac.cr).Taxon classificationAnimaliaHymenopteraBraconidae?Sharkeysp. nov.92179AC2-B0F2-536F-A9EC-A5CE15FADB3Fhttp://zoobank.org/8BE983A2-E1D9-4FB8-AC3C-6E2FB4B5F8AAFigures BOLD:AAT9450. Its nearest neighbor on BOLD is identified by D. Quicke as YeliconesartitusBOLD:AAM6954; distance to nearest neighbor is 9.2%. Consensus barcode: TGTTTTATATTTTTTATTAGGTATTTGATGTGGATTAGTAGGTTTATCTTTAAGGTTACTTATTCGGTTAGAGTTGAGAAATTTAGGAAGATTATTAGGTAATGATCAAATTTATAATGTAGTTGTTACTATACATGCTTTTATTATAATTTTTTTTATGGTGATACCTATTATAATTGGAGGTTTTGGAAATTGATTAATTCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGTATAAATAATATAAGATTTTGATTATTAATTCCTTCTTTGTTATTAATATTAATAAGAGGATTTATTATAGTTGGTAGTGGAACTGGGTGAACTATATATCCTCCTTTAAGTTCTTTAATTGGGCATAGAAGGTTTTCTGTTGATATAGTAATTTTTTCTTTACATTTAGCAGGGGTTTCTTCAATTATAGGAGCTATTAATTTTATTACAACAATTTTTAATATAAAATTAATTT---TAAAATTAGATCAGATTATATTATTTGTATGATCTGTATTAATTACTGCTTTTTTATTATTACTTTCTTTACCTGTTTTGGCAGGAGGAATTACTATATTATTAACAGATCGTAATTTAAATACTTCTTTTTTTGATTTTTCAGGAGGGGGAGATCCTGTTTTATTTCAACACTTATTT.BIN: Y.vilawanae in the key of Yeliconesvilawanae has the apical 0.2 of hind tarsus and apical 0.8 of hind basitarsus brown. Yeliconesmayrabonillae has the basal four hind tarsomeres brown and the apical tarsomere yellow. This species can be morphologically distinguished from its nearest neighbor, Yeliconesartitus, by the color of the hind femur being entirely testaceous .This species keys to ous Fig.   feeding on Vochysiaguatemalensis (Vochysiaceae), caterpillar voucher code:10-SRNP-42391.epipajanzen01 Janzen882 (Yeliconesmayrabonillae is named in honor of Mayra Bonilla as the primary supporter of the BioAlfa Malaise trapping at the Hacienda Baru Wildlife Refuge, Savegre, ACOPAC, Costa Rica, as well as decades of support for the Area de Conservaci\u00f3n Guanacaste."}
+{"text": "Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-\u03b3 production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases. Genein vivo . In nonoin vivo . Of notein vivo , 10, indChronic inflammation is the hallmark of autoimmune diseases, characterized by massive infiltration and activation of immune cells , 12. NotThe amino acid sequences of recombinant proteins used in this study were indicated as below. All constructs carried a C-terminal histidine tag or IgG1 Fc fragment for purification and were cloned into pMT-puro vectors for expression in HEK293 cells. Stably transfected cells were selected with 6 \u03bcg/ml puromycin. The resulting proteins were purified using Ni-affinity or protein A agarose affinity chromatography column  followed by further purification by SEC using a Superdex 200 (S200) column with 10 mM Tris and 150 mM NaCl, pH 7.5 (1\u00d7 TBS). The purity of recombinant proteins was determined by SDS-PAGE .Protein sequences used in this study:Murine PD-L1-WT pHLIP :MDAMKRGLCCVLLLCGAVFVSNSHHHHHHHHFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLek?>LNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTGGGGSGGGGSGGGGSAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTMurine PD-L1-mFc (denoted to PD-L1-Fc):MDAMKRGLCCVLLLCGAVFVSNSFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTDDDDKAVPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKMurine PD-L1-mutated WT pHLIP (denoted to PD-L1-pHLIP(m)):MDAMKRGLCCVLLLCGAVFVSNSHHHHHHHHFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGML1LNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTGGGGSGGGGSGGGGSACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTEBOV-secreted glycoprotein (sGP)-WT pHLIP (denoted to control protein):MDAMKRGLCCVLLLCGAVFVSNSHHHHHHIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYASGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKGGGGSGGGGSGGGGSACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTA total of 96-well plated were coated overnight with the indicated antigens at 2 or 5 \u03bcg/ml, washed three times with PBS-T, and blocked with 4% nonfat dried milk in PBS for 1 h at room temperature. Twofold serial dilution of biotin-labeled anti-mPD-L1 antibody  from 6 \u03bcg/ml was measured. For mPD-1, which was biotinylated according to manufacturer\u2019s instructions , it was added at 25, 50, and 100 \u03bcg/ml and incubated for 1 h at room temperature. The wells were washed and avidin-conjugated HRP  in 0.5% BSA was added and incubated for 1 h at room temperature. A TMB substrate kit  was used for detection at 450 nm. IFN-\u03b3 levels in the supernatants were determined by sandwich ELSA assays .2 at 37\u00b0C. HEK293T cells were transiently transfected with mPD-1 and mPD-L1 expression plasmids using a PEI transfection protocol , respectively. 48 h later, the expression of PD-L1 and PD-1 on the surface of cells was detected by flow cytometry.Cell lines  were purchased from ATCC and cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) or RPMI 1640 medium  supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified atmosphere of 5% CORecombinant proteins were labeled by fluorescence dye (PE or AlexaFlour488) with Conjugation Kit, according to manufacturer\u2019s protocol . The HEK293T or HCT116 cell lines were placed in a laser confocal dish  and cultured overnight. AlexaFlour488-conjugated protein was added at 10 \u00b5g/ml and cultured at 37\u00b0C for 1 h. Cells were washed twice with PBS with corresponding pH, and then the fluorescence on the surface of cell membrane was observed under confocal laser scanning microscope .5 cells/well) with stimulation of anti-CD3 and CD28 antibody cocktail  for 72 h. BrdU cell proliferation assays were performed according to the manufacturer\u2019s protocol . In addition, lymphocytes were treated as described above with soluble proteins indicated in PBS with corresponding pH or containing lactic acid (10 or 20 mM). In some settings, neutralizing mAbs to murine PD-1 or PD-L1  were added.The splenocytes of BALB/c mice  were isolated by gradient centrifugation. Human PBMC were isolated from whole blood of healthy donors with signed written informed consents. The indicated proteins at titrated concentrations were coated in 96-well plates  at 4\u00b0C overnight. Lymphocytes were added (5 \u00d7 10The cells were incubated with PE-labeled proteins as indicated at 0.001\u201310 \u00b5g/ml for 0.5\u20134 h in PBS with corresponding pH or containing lactic acid (10 or 20 mM). In some settings, cells were incubated with the indicated proteins at 10 \u00b5g/ml for 1 h in PBS (pH 7.4 and 6.3). PE-labeled mPD-1 was added and incubated for another 30 min. Cells were washed twice with PBS with corresponding pH, and then detected by flow cytometry.For BrdU incorporation, mouse spleen lymphocytes were treated as described above. Six hours before the end of stimulation, BrdU solution  with the final concentration of 10 \u00b5M was added. Cells were washed and incubated with APC-antimouse CD4/CD8 antibody  at 4\u00b0C for 30 min. After washing, fixing, and permeabilizing , cells were incubated with PE-antimouse BrdU antibody  in the dark at 4\u00b0C for another 30 min. The fluorescence intensity was measured by FACS Calibur II .Extracted total mRNA with TRIzol reagent, Single-strand cDNA was made from 1\u00b5g total RNA by reverse transcription (RT) using a TransScript II First-Strand cDNA Synthesis SuperMix . qPCR was conducted using SYBR Green qPCR mixture  through 50 cycles in a IQ5 Real-Time PCR Detection Systems . The sequences of the primers used for qPCR were as follows: the forward primer of mouse IFN-\u03b3 is 5\u2019-ACAGCAAGGCGAAAAAGGATG-3\u2019 and the reverse primer is 5\u2019-TGGTGGACCACTCGGATGA-3\u2019. The forward primer of mouse GAPDH is 5\u2019-CATCAAGAAGGTGGTGAAGC-3\u2019 and the reverse primer is 5\u2019-CCTGTTGCTGTAGCCGTATT-3\u2019. Data were analyzed using the delta-delta Ct method.t-tests were used to compare experimental and control groups. Statistical significance is defined as p < 0.05.Data are expressed as the means \u00b1 SD according to at least 3 independent experiments. Two-tailed Student\u2019s 3 as a coupling linker and the fusion protein generated in the eukaryotic expression system. As well, PD-L1 (having His tags) and PD-L1-Fc protein were also expressed using the same methods. The identities of these proteins were validated by SDS-PAGE in vivo, pH was titrated by lactic acid (10 or 20mM). Similar effects were observed  were incubated with fluorescence-labeled PD-L1-pHLIP for 1 h in pH 7.4 or 6.3 solutions and then detected by flow cytometry. No fluorescence was found in neutral aqueous solutions. In contrast, high magnitudes of fluorescence were visible in pH 6.3 buffer , indeed, potently inhibited the expansion of CD4+ and CD8+ T cells simultaneously . To determine whether PD-L1/PD-1 ligation was responsible for the inhibitory function of PD-L1-pHLIP, neutralizing antibodies to PD-L1 and PD-1 were added. Blocking PD-L1/PD-1 interaction using anti-PD-L1 or PD-1 antibody almost entirely abrogated the inhibitory ability of PD-L1-pHLIP, including suppressing lymphocyte proliferation and IFN-\u03b3 secretion . Overall, these results indicate that plate-bound PD-L1-pHLIP actively inhibits T lymphocyte expansion and cytokine production via binding to PD-1 receptor on the surface of T cells.Thereafter, we evaluated the function of plated-bound proteins  to inhibit proliferation of lymphocytes at the intervals of 24\u201372 h following treatment with anti-CD3/CD28 mAbs. At 24 h poststimulation, none of them  displayed inhibitory function. All of them, however, actively suppressed lymphocyte expansion at 48 and 72 h following TCR stimulation, with over 90% of inhibition rates  at the soluble state in pH6.3 or 7.4 buffer respectively. As expected, these proteins at the soluble state did not suppress lymphocyte proliferation and IFN-\u03b3 secretion in neutral aqueous solution . In contrast, PD-L1 and PD-L1-Fc had no inhibitory effects on lymphocyte activation under the same condition . We further evaluated the function of these proteins in the solution containing 10mM lactic acid. Similarly, PD-L1-pHLIP displayed significantly inhibitory function on lymphocyte proliferation and IFN-\u03b3 production, instead of PD-L1 and PD-L1-Fc . Taken together, these data suggest that PD-L1-pHLIP actively suppresses T-cell activation under acidic condition.To test our hypothesis that under acidic conditions, PD-L1-pHLIP inserts and spans cellular membrane through conformational changes from unstructured coil to \u03b1 helix, thereby playing an immune-suppressive role in T-cell activation in vivo in the future. The reasons for choosing PD-L1 as a cargo are following: (a) PD-L1/PD-1 interaction is definitely critical to maintain T-cell tolerance and immune homeostasis. Long-term administration of PD-1 antibodies led to significant increase of risks for autoimmune diseases , which was due to that PD-1 mAb treatment abrogated endogenous signaling axis of PD-L1/PD-1, thereby pathogenically activated autoreactive T cells and ultimately caused the onset of autoimmune diseases  , 31. Howdiseases \u201335; (b) diseases ; (c) PD-diseases , 38; (d)diseases , we specdiseases , 41; (f)in vitro  and an inserted \u03b1-helical population (state III) (A recent study demonstrated that in NOD mice hematopoietic stem progenitor cells (HSPCs) were deficient in PD-L1 and transfusion of genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited autoimmune response and reverted diabetes . Howeverate III) . In loweThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics Committee of the Chinese PLA General Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Ethics Committee of the Academy of Beijing Institute of Pharmacology and Toxicology.YiS and LH performed the experiments and prepared the manuscript. PY, MZ, and XW were involved in optimization of the experimental protocols. HX, CQ, JW, LL, JF, and YZ provided methodological support. YW, YaS, and GC conceived and guided the study. All authors contributed to the article and approved the submitted version.This work is supported by the National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of  Computational approaches have recently made significant progress in the protein engineering field evolving from a tool for helping experimentalists to prioritize or short-list mutations for testing to being capable of making fully reliable predictions. However, not all the fields of protein modeling are evolving at a similar pace. That is why evaluating the capabilities of computational tools on different tasks is important to provide other scientists with up-to-date information on the state of the field. Here we tested the performance of Rosetta (one of the leading macromolecule modeling tools) in improving small molecule-binding proteins. We successfully redesigned a fluorogen binding protein DiB1 \u2013a protein that binds a non-fluorescent molecule and enforces its fluorescence in the obtained complex\u2013for improved brightness and better performance in super-resolution imaging. Our results suggest that such tasks can be already achieved without laborious library screenings. However, the flexibility of the proteins might still be underestimated during standard modeling protocols and should be closely evaluated. The biological role of many natural fluorogens is often directly connected to their ability to absorb light. For example, chlorophyll is used in photosystems of cyanobacteria, algae, and plants; flavins are essential parts of DNA photolyases [Fluorogenic molecules are compounds whose ability to fluoresce can be modulated, for example, by a chemical modification, change in the environment, or electronic structure . A numbetolyases  and cryptolyases ; rhodopstolyases .A number of fluorescent probes has been created by mutating natural fluorogen-binding proteins to promote their fluorescence. For example, starting from bacterial light-oxygen-voltage\u2013sensing domains, such an effort yielded flavin-binding fluorescent proteins ; differeIn addition to natural fluorophores, fluorogenic compounds can be synthesized. The existing examples include but are not limited by BODIPY dyes, rhodamines, cyanines, and coumarins . The utiIn silico shortlisting with the further screening of the lipocalin Blc mutants launched DiBs FAPs tags family binding green [de novo ligand-binding \u03b2-barrel protein design using Rosetta followed by two additional rounds of modeling-directed mutagenesis of proteins selected by in vitro screening of a limited number of hits from the previous step yielded two DFHBI-binding proteins designated mFAP1 and mFAP2 [Several FAPs have been designed so far to use unnatural fluorogenic molecules. Screening of libraries of antibodies resulted in the discovery of binders for derivatives of thiazole orange, malachite green , and cyang green  and red ng green  BODIPY dng green . Finallynd mFAP2 .in silico modelling of more than 100 000 mutants followed by a rigid body docking of a library of green fluorescent protein (GFP) chromophore-like ligands. Nineteen mutants and ten fluorogens were shortlisted to be evaluated experimentally [in silico might be already possible.All FAP modeling projects described above involved screening of libraries of proteins to select the one with the desired properties, which is time and resource consuming. For example, the first-generation lipocalin Blc-based FAPs (DiBs) were obtained after mentally . HoweverHere we tested the performance of one of the leading macromolecules modeling suites, Rosetta , on the Kd [DiB1 protein was selected as a starting point for the protein design project. Among first generation Blc-based FAPs this mutant showed the lowest Kd  indicatiKd . For thiKd . The besFig) was performed with gradually decreasing ligand sampling freedom until the solution converged on a single binding pose . First, 5 000 protein-ligand complexes were generated using a coarse protein binding pocket sampling strategy with 5 \u00c5 maximum ligand translation allowed per step and up to 360\u00b0 rotation. The top 500 models by total energy were further sorted based on their protein-ligand interface score and the 50 best were selected for a subsequent docking round. During the next step ligand translation and rotation were restricted to 1 \u00c5 and 45\u00b0, respectively. 100 structures were generated for each of 50 starting models resulting again in a total of 5 000 output structures. 50 best structures were selected as previously and the last round of docking was performed with 0.2 \u00c5 maximum translation and 5\u00b0 maximum rotation for fine-tuning of the ligand placement. The best scoring docking pose was further used for the ligand binding pocket design. In this model the ligand was located within an interaction distance from amino acids at positions 141 and 74, mutations in which were shown to influence the properties of the ligand:protein complex in our previous study [Docking of the M739 chromophore Fig was ing pose Fig. Firus study . While tus study . TherefoFig). The protocol was previously shown to perform better on apolar small molecules whose binding is dominated by van der Waals interactions [In fluorescent proteins, their ability to fluoresce and the brightness of the fluorescence highly depend on the capacity of the surrounding amino acids to stabilize the chromophore in a planar conformation . In the case of M739, the structure is already conformationally locked in the favorable planar conformation. Contrary, its fluorescence quantum yield (QY) was shown to vary dramatically depending on the solvent with an almost tenfold increase in dioxane compared to water . Thus, hractions .From the starting complex structure, we generated 5 000 designs. Amino acids with their C\u03b1 atoms within 6 \u00c5 of any ligand atom or with their C\u03b1 atoms within 8 \u00c5 of any ligand atom and with their C\u03b1-C\u03b2 vector pointing toward the ligand were allowed to be designed to any amino acid except cysteine. The residues with their C\u03b1 atoms within 10 \u00c5 of any ligand atom or with their C\u03b1 atoms within 12 \u00c5 of any ligand atom and their C\u03b1-C\u03b2 vector pointing toward the ligand were allowed to repack. The 2 000 best scoring designs were then sorted based on their ligand-protein interface score and the best 50 were selected for detailed analysis.Fig 1A and 1B). Mutations at positions 74, 108, 109, 116, 139, and 141 were relatively rare with the majority of the generated sequences retaining the native amino acid at those positions. In contrast, amino acids at positions 53, 76, 89, 90, and 107 were mutated in almost all 50 selected models. Four of these positions showed strong convergence toward one specific mutation: Phe at position 76, Tyr at position 89, Val at position 90, and Ala at position 107. Amino acid 53 was mutated to either alanine or glutamine with nearly equal frequency in the models.A total of 16 amino acid positions were found to be mutated at least in one of the selected designed models  contained only mutations at these five positions. Four residues  were converted to the most favorable amino acids for these positions. Alanine, the second most frequent amino acid in position 53 among 50 best-scored poses, was found in position 53 in that sequence. After additional manual examination, this sequence (designated DiB-RM) was selected for testing.Of note, one of the top-scoring models (in vitro (Table 1). In comparison with DiB1, the DiB-RM:M739 complex, with almost identical fluorescence spectra, demonstrated an increase in both quantum yield (QY) and extinction coefficient (\u025b) resulting in approximately 25% increased brightness. These changes were accompanied by some loss in the apparent binding affinity of the protein to the ligand.We first compared the photophysical properties of DiB-RM in complex with M739 chromophore with the parental protein DiB1 in cellulo . In concordance with the results obtained in vitro, DiB-RM demonstrated increased brightness compared to DiB1: ~ 1.38:1 versus ~ 1.29:1 observed in vitro.We next characterized the DiB-RM protein using transiently transfected living cells. We first generated a fusion protein of DiB-RM with histone H2B and blue fluorescent protein TagBFP, which was compared with a previously created H2B-TagBFP-DiB1 construct . Using TFig 2F and 2G).We next created DiB-RM fusions with various structural proteins  and imaged transiently transfected mammalian cells using confocal fluorescence microscopy. The target intracellular structures were brightly fluorescent immediately upon the addition of the fluorogen and did not show any signs of mislocalization or aggregation  and tested the behavior of the fusion protein composed of the newly obtained N fragment (designated DiB-RM-splitN1-109) with a fluorescent protein mNeonGreen in transiently transfected HEK cells. Interestingly, we did not observe aggregation  as was seen previously for DiB2-split N fragment in a similar experiment. Inspired by this observation we also checked free DiB-RM-split C fragment distribution in cells. There is only one amino acid difference between DiB2 and DiB-RM proteins downstream to the split point (L141N) and it did not affect the performance of the DiB-RM-split C fragment: HEK cells transiently transfected with the DiB-RM-splitC110-177-TagBFP construct showed uniform labeling in the blue fluorescent channel . We then tested the self-assembly capability of the DiB-RM N and C fragments obtained via split between amino acid residues 109 and 110. Transient cotransfection of H2B-DiB-RM-splitN1-109 and TagBFP-DiB-RM-splitC110-177 constructs in living cells revealed successful attraction of TagBFP-DiB-RM-splitC110-177 fusion protein to the nuclei despite lacking a nuclear localization signal  and staining nuclei in green upon the chromophore addition  as a result of efficient self-assembly of split-DiB-RM.We previously created a self-assembling split system from the first generation of DiB proteins . Despiteregation Fig as w channel Fig. We in vitro (Table 1) using purified protein. As was previously observed for other self-assembling DiB-splits [We also characterized the DiB-RM-split:M739 complex B-splits , splittiS3 Movies). All three tags provided reconstructions of vimentin fibers with better resolution than widefield fluorescence microscopy . DiB1 showed an initial exponential decrease in the number of localizations that later turned into a linear decrease down to ~20% of the initial localization count. The DiB-RM signal decreased only linearly throughout the experiment. That allows for the accumulation of a much higher number of localizations during the same time period using DiB-RM. DiB-RM-split tag performed similarly to DiB1 . DiB-RM also demonstrated higher single-molecule brightness  than DiB-RM-split and DiB1 . Both DiB1 and DiB-RM-split provided lower localization precision  compared to DiB-RM .The main power of DiB FAPs lies in single-molecule localization microscopy of living cells ,14. We tapo form. The asymmetric unit contains two copies of the protein with the canonical lipocalin fold, a \u03b2-barrel with an \u03b1-helix . The two copies align well (0.77 \u00c5 rmsd across 158 C\u03b1 atoms) with the main difference found in the E/F loop and at the N terminus of the protein .To further characterize DiB-RM we obtained a number of crystal structures. We first successfully crystalized full-length DiB-RM in Fig 4C).Interestingly, during structure refinement, we discovered positive difference density map features in the binding pocket of the molecules. Based on the shape of the density we speculated that they belong to the dodecyl chains of n-Dodecyl-\u03b2-D-maltoside (DDM) present in the crystallization buffer . Unfortunately, we still observed the same long carbon chain-like density in the binding pocket of DiB-RM in these crystals. It might be explained by the higher affinity of DiB-RM to DDM than to M739 or by the requirement of the presence of DDM in the binding pocket of the protein for crystal formation in these conditions.We also obtained colored crystals in similar conditions using soaking or co-crystallization of DiB-RM with M739 Fig. UnfTo test the former hypothesis, we assessed the binding affinity of DDM to DiB-RM using tryptophan fluorescence quenching assay. Surprisingly, we have not detected any spectral changes upon the addition of up to 50x molar access of the DDM to the protein solution in normal buffer conditions. While this experiment cannot fully reproduce the processes and their dynamics that are happening during crystallization, the requirement of the presence of DDM for crystal formation in these conditions seems to be a more likely explanation.apo DiB-RM crystals in DDM-free conditions. This crystal also contains two protein molecules in the asymmetric unit . However, the relative orientation of the molecules differs: we observed approximately 50 degrees rotation . This observation along with the data from \u2019Protein interfaces, surfaces and assemblies\u2019 service PISA [Fig 4E).We later obtained other ice PISA  suggestsFig 4F). Unsurprisingly, the crystals remained clear after the addition of the ligand in the drops.In comparison to the first structure, the main difference was observed in the conformation of the E/F loop of the protein. In the new structure, this loop is bent inwards and almost fully closes the entrance to the ligand binding pocket of the protein . Despite the backbone cleavage, the lipocalin fold is well preserved as it was previously observed for other DiB-derived split proteins [Fig 4I).We also obtained DiB-RM-split proteins  Fig). The carbon chain-like density in the binding pocket was absent, which further supports our hypothesis that the previously observed density in full-length DiB-RM crystals indeed belongs to DDM and not to some other molecule co-purified with the protein. However, we were not able to locate any density for the M739 ligand either.Multiple colored crystals from DiB-RM-split:M739 mixture were obtained in 1.8 M lithium sulfate and 6% 2-propanol buffer in presence of different additives Fig. Thein vitro (except for the dissociation constant) and as a tag for protein-PAINT [Table 2) of the DiB-RM:M739 complex compared to the parental DiB1:M739 through the better protein:ligand interface packing, in the absence of the ligand these bulky amino acids might partially hinder the entrance of the ligand in the binding pocket.Using a fixed backbone Rosetta Design protocol, we predicted a set of mutations to improve the first generation lipocalin Blc-based FAP DiB1. The resulting protein, designated DiB-RM, performs better than its parental variant both in-PAINT  with hig1-109 fragment, and the split protein successfully assembled in living cells without the need of including the additional overlapping \u03b2 strand in the C fragment. Regardless of lower brightness both in vitro and in super-resolution microscopy set up as well as less stable fluorescent signal compared to full-length DiB-RM tag, we speculate that the DiB-RM-split should be a scaffold of choice for further DiB-split system optimizations.Despite no intentional optimization for it, the DiB-RM-based split protein (DiB-RM-split) created analogously to the previously tested DiB2-split system  also behapo Blc structures [Fig 5A) and served as an entry point for the vaccenic acid that was previously co-crystallized with this lipocalin [Fig 5B), the chromophore was found much deeper and angled . This became possible due to the rotation of the lipocalin E/F loop. While high flexibility of this loop was previously shown [Fig 5C).Recently, the structure of DiB1 in complex with the chromophore M739 has been solved . To our ructures ,25 Despite our inaccurate initial docking of the chromophore, it is obvious that the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the DiB tag. To further investigate this phenomenon, we ran a number of experiments.Figs S7A) confirming that steric clashes with the backbone most likely caused our wrong initial binding mode prediction.First, to test the possibility that using a starting position of the E/F loop incompatible with the binding mode observed in the crystal structure was responsible for the failure of Rosetta docking, we reran the docking using either the DiB1 crystal structure minimized in the absence of ligand, or the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids). In both cases, all 50 top-scored structures had a crystal-like M739 placement . This observation together with our inability to obtain the DiB-RM:M739 co-crystal structure might indicate that we have stabilized an alternative binding site for the ligand by the introduced mutations but have not destroyed the other one. As a result, the ligand in DiB-RM might have multiple possible binding positions. This can also explain the larger apparent dissociation constant of this new complex.We then performed docking using the DiB1-based DiB-RM model generated the same way as the DiB1 model has been prepared before from the Table 2).To further investigate the role of the five introduced mutations we created and analyzed five reverse single mutant variants of DiB-RM , it is not surprising that this phenylalanine has been mutated by Rosetta. This substitution might be also important for the protein\u2019s performance in the crowded cell environment.All -exposed Fig, it Kd accompanies by the bathochromic shift of the fluorescence spectra and a decrease in both QY and \u025b. The side chain of the amino acid in position 76 packs against the ligand . These spectral changes upon introduction of the polar residue next to the ligand align well with the effects of the polarity of the environment on the chromophore M739 properties observed in the free ligand model [Reintroduction of asparagine at position 76 is the only mutation that results in tighter binding to the chromophore. That might be explained by the ability of bulkier phenylalanine to sample side chain conformations that are not compatible with the ligand\u2019s entrance to the binding pocket or its correct placement there. However, the lower e ligand Fig. Thend model .Fig). The observed changes might support the presence of an alternative ligand binding mode in DiB-RM where Y89 packs against the ligand. Alternatively, the introduction of tyrosine at position 89 can induce more complex rearrangements in the structure of the protein. For example, through interactions with the aromatic amino acids-rich flexible E/F loop.Reversing of the other introduced aromatic residue, DiB-RM-Y89S, results in an almost four-fold decrease in binding affinity as well as moderate spectral shifts and decrease in brightness similar to ones observed in DiB-RM-F76N mutant. The amino acid in position 89 is not in direct contact with the ligand, assuming DiB-RM interacts with the ligand similar to what is seen in the DiB1:M739 co-crystal structure Fig. TheFig). The most likely explanation of Rosetta\u2019s favoring of the E90V mutation is the better \u03b2-sheet propensity of valine compared to glutamate [Kd upon reversion of this mutation might indeed indicate its effect on the overall stability of the protein. Even less pronounced in vitro changes caused by A107S substitution located in a relatively flexible region of the protein (at the base of the E/F loop) make it difficult to propose its role, if any.The remaining two variants, DiB-RM-V90E and DiB-RM-A107S, carry substitutions at the positions that are pointing outside of the ligand-binding cavity Fig. Thelutamate . The obsOverall, it appears that the majority of the mutations proposed by Rosetta were beneficial for the DiB-RM performance with the polarity of the residues in the proximity of the ligand influencing the photophysical properties of the FAP the most. Hence, refining of the protein:ligand interface using a conventional RosettaLigand design protocol seems to be a possible option for optimization of FAPs with rigid, apolar ligands. The decrease in the ligand binding affinity might be further avoided in the future by employing a multistate design protocol  with simapo form in different conditions further confirmed the already known high flexibility of the E/F lipocalin loop that was not properly addressed during our computational redesign. Rigidifying of this loop through shortening or designed interactions can provide better stabilization of the ligand in the binding pocket. This in turn can allow for expanding the compatible ligand libraries towards more flexible, conformationally unlocked chromophores [Here we explored the power and limitations of Rosetta for the redesign of a protein-ligand complex. Our work resulted in the creation of an improved FAP-based fluorescent tag, however, potentially through stabilization of an alternative ligand binding site. Future optimization of DiB-RM might be focused on disabling one of the two suggested binding sites of the ligand. Crystallization analysis of DiB-RM in the mophores ,30. SuchFive mutations suggested by computational modeling were introduced into the previously described DiB1-pBAD bacterial expression vector  by self-The DiB-RM-split vectors for bacterial protein expression were created using plasmids pMRBad-Z-CspGFP (Addgene plasmid #40730), pET11a-Z-NspGFP (Addgene plasmid #40729) , and theThe H2B-TagBFP-DiB-RM plasmid was constructed by self-assembling cloning  using DiAll other plasmids for mammalian expression were assembled using Golden Gate cloning following MoClo standard \u201336. EachThe resulted constructs\u2019 amino acid sequences are provided below. The linker sequence is underlined.>vimentin-DiB1DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRFERGLEKVTATYSLRDDGGLNVINKGYNPDRGMWQQSEGKAYFTGAPTRAALKVSFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RMDPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>vimentin-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVYATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNLAEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKLHEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKFADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFAVEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRISLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLEG>DiB-RM-split110-177-TagBFPGDPPVATMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN*MGPFYGGYNVIALDREYRHALVCGPDRDYLWINSRTPTISDEVKQEMLAVATREGFDVSKFIWVQQPGS>mNeonGreen-DiB-RM-split1-109DPPVATMASSPTPPRGVTVVNNFDCKRYLGTWYEIARFDHRAERGLEKVTATYSLRDDGGLNVIFKGYNPDRGMWQQYVGKAYFTGAPTRAALKVAFFS*MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYKThe correctness of all obtained constructs was confirmed by sequencing.E. coli strain. Cells were grown in LB media or M9 minimal media supplemented with 100 \u03bcg/mL ampicillin (full-length DiB proteins) or 100 \u03bcg/mL ampicillin and 50 \u03bcg/mL kanamycin (split protein) at 37\u00b0C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10 \u03bcM IPTG (split protein) at 0.8 OD. Cells were harvested after 3 hours of expression in LB or after overnight expression in minimal media at 37\u00b0C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at -80\u00b0C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts.All lipocalin proteins were expressed in XJb(DE3) Autolysis (Zymo Research) E. coli strain. Cells were grown in LB media supplemented with 100 \u03bcg/mL ampicillin at 37\u00b0C. Expression was induced by the addition of 500 \u03bcM IPTG at 0.8 OD. Cells were harvested after overnight expression at 18\u00b0C. Before purification cells were resuspended in PBS buffer, pH 7.4, and sonicated on ice. The lysates were centrifuged to obtain cell-free extracts.Fluorescent protein Venus was expressed in BL21(DE3) The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg column  pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.Protein concentrations were estimated using the Bradford dye-binding method-based  colorimeKd). For each protein, the measurements were performed using at least two independent protein purifications and at least three technical replicates for each protein sample.Titrations were performed and analyzed as previously described  using FlHoriba Jobin Yvon Fluoromax-3 fluorometer was used to detect full fluorescence excitation and fluorescence emission spectra for excitation/emission maxima evaluation.apo form and in the presence of ~0.5\u20133.0 \u03bcM of the M739 chromophore. FAP concentrations for experiments were chosen individually for each protein based on the previously calculated Kd values to ensure that at least 95% of the added chromophore is bound to the protein . Spectra of the corresponding apo FAP solutions were subtracted from the absorption spectra of the protein-fluorogen complexes. The absorption at 510 nm values from these corrected spectra was also plotted against the area under the corresponding fluorescence emission curves and the linear approximation of the correlation has been calculated. The QYs were then calculated as a ratio of the slopes of the protein of interest and standard curves multiplied by standard\u2019s QY. For each protein, the measurements were performed using protein aliquots from at least two independent protein purifications. Reported is a mean value.Fluorescence quantum yield (QY) was measured relative to the fluorescent protein Venus . First, FAP\u03b5 is the FAP-fluorogen complex extinction coefficient, FAPA\u2013the FAP-fluorogen complex absorption at maximum, AM739\u2013free chromophore absorption at the FAP-fluorogen complex absorption maximum, cM739\u2013total added chromophore concentration, and \u03b1\u2013a fraction of added chromophore that is bound to the protein calculated based on the previously determined FAP-fluorogen complex Kds. Reported is a mean value \u00b1 s.d. of measurements obtained for at least two independently expressed and purified protein samples using at least five data points per sample.Absorption spectra collected for QY calculations were also used for excitation coefficients calculations. For each of the FAP-fluorogen complexes, the complex absorption maximum has been determined. Free M739 chromophore spectra were used to define free chromophore contribution to absorption at the given wavelength and to calculated chromophore concentrations. The FAP-fluorogen complexes\u2019 extinction coefficients were calculated using the following equation:apo DiB-RM  was crystalized at 21\u00b0C in 2 M ammonium sulphate, 2.5% 2-propanol supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1\u20133 days.Full-length apo DiB-RM  were also obtained at 21\u00b0C in 1.5 M lithium sulfate, 0.1 M tris hydrochloride pH 7.0, and 5% 2-propanol, supplemented with 200 mM cesium chloride using hanging drop vapor diffusion technique. For this 2 \u03bcL of protein solution were mixed with 1.2 \u03bcL of crystallization buffer  and 0.8 \u03bcL of 1 M cesium chloride from Hampton Research Additive Screen. Crystals grew within 2\u20133 weeks.Crystals of different morphology of full-length apo crystals were obtained at 21\u00b0C in 1.6 M ammonium sulfate, 0.1 M MES, pH 4.5 supplemented with 5% w/v n-Dodecyl-b-D-maltoside according to the Hampton Research Additive Screen protocol using hanging drop vapor diffusion technique. Crystals grew within 1 week.DiB-RM-split  All crystals were flash-frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.Table. The models have been deposited into the Protein Data Bank . Structure figures were prepared using PyMol .Diffraction data were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G or 21-ID-F at the Advanced Photon Source, Argonne National Laboratory. The diffraction data were processed using the xia2 software suite . The cry2. For transient transfections, FuGENE HD reagent (Promega) was used. Immediately before imaging DMEM was replaced with HHBS media (Hanks Buffer (PanEco) supplemented with 20 mM HEPES (Sigma)).HEK293 and HeLa Kyoto cells were grown in Dulbecco\u2019s modification of Eagle\u2019s medium (DMEM) (PanEco) supplied with 50 U/ml penicillin and 50 \u03bcg/ml streptomycin (PanEco), 2 mM L-glutamine (PanEco), and 10% fetal bovine serum  at 37\u00b0C and 5% COWidefield fluorescence microscopy was performed with the Leica DMI6000B inverted microscope equipped with HC PL Apo 100x NA 1.40 oil lens and HC PL Apo 40x NA 0.85 lens, CoolLED pE-300 light source, Zyla 5.5 sCMOS camera (Andor), using GFP and BFP filter sets.Confocal imaging was performed using an inverted Leica confocal microscope DMIRE2 TCS SP2  equipped with HCX PL APO lbd.BL 63.0x NA 1.40 OIL objective, excitation by 488 nm laser line (100 \u03bcW).-2 of 488 nm laser light, 33 ms frame exposure time, for 10,000 frames.Single-molecule localization super-resolution imaging of living cells was performed with Nanoimager S  microscope at 37\u00b0C. The microscope was equipped with Olympus UPlanSApo 100x NA 1.40 oil immersion objective. Imaging experiments performed with 1.1 kW cmAll Rosetta runs were performed with weekly release 2015.12.57698.The A36C and L141N mutations were manually introduced into the crystal structure (PDB ID 1QWD). The structure was further minimized using Rosetta Relax application  with the-flip_HNQ-no_optH false-relax:constrain_relax_to_start_coords-relax:ramp_constraints false-nstruct 50-ex1-ex2-use_input_scThe M739 chromophore geometry was optimized by the density functional method RB3LYP, using the 6\u2013311+G** basis set, a restricted hybrid HF-DFT SCF calculation was performed using Pulay DIIS + Geometric Direct Minimization to get a set of ideal bond lengths and angles. The conformers library for the ligand was further generated using BCL::Conf conformer generator .Docking of the M739 chromophore was performed in three steps. First, 5 000 structures were generated using 5 \u00c5 maximum ligand translation allowed per step and up to 360\u00b0 rotation. 50 structures were selected for the next docking round during which ligand translation and rotation were restricted to 1 \u00c5 and 45\u00b0 correspondingly. 100 structures were generated for each of the 50 starting models. Analogously 50 best structures from the second docking step were selected and the last round of docking was performed with 0.2 \u00c5 maximum translation and 5\u00b0 maximum rotation.The following RosettaScripts protocol has been used with the appropriate changes introduced to the Transform Mover during the second and the third docking rounds:<ROSETTASCRIPTS><SCOREFXNS>\u00a0\u00a0\u00a0\u00a0<ligand_soft_rep weights = ligand_soft_rep>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_elec weight = 0.42/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_bb_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = rama weight = 0.2/>\u00a0\u00a0\u00a0\u00a0</ligand_soft_rep>\u00a0\u00a0\u00a0\u00a0<hard_rep weights = ligandprime>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_intra_rep weight = 0.004/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_elec weight = 0.42/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_bb_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = rama weight = 0.2/>\u00a0\u00a0\u00a0\u00a0</hard_rep></SCOREFXNS><SCORINGGRIDS ligand_chain = \"X\" width = \"16\">\u00a0\u00a0\u00a0\u00a0<vdw grid_type = \"ClassicGrid\" weight = \"1.0\"/></SCORINGGRIDS><LIGAND_AREAS>\u00a0\u00a0\u00a0\u00a0<docking_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false minimize_ligand = 10/>\u00a0\u00a0\u00a0\u00a0<final_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false/>\u00a0\u00a0\u00a0\u00a0<final_backbone chain = X cutoff = 7.0 add_nbr_radius = false all_atom_mode = true Calpha_restraints = 0.3/></LIGAND_AREAS><INTERFACE_BUILDERS>\u00a0\u00a0\u00a0\u00a0<side_chain_for_docking ligand_areas = docking_sidechain/>\u00a0\u00a0\u00a0\u00a0<side_chain_for_final ligand_areas = final_sidechain/>\u00a0\u00a0\u00a0\u00a0<backbone ligand_areas = final_backbone extension_window = 3/></INTERFACE_BUILDERS><MOVEMAP_BUILDERS>\u00a0\u00a0\u00a0\u00a0<docking sc_interface = side_chain_for_docking minimize_water = true/>\u00a0\u00a0\u00a0\u00a0<final sc_interface = side_chain_for_final bb_interface = backbone minimize_water = true/></MOVEMAP_BUILDERS><MOVERS>\u00a0\u00a0\u00a0\u00a0<Transform name = \"transform\" chain = \"X\" box_size = \"8.0\" move_distance = \"5.0\" angle = \"360\" cycles = \"5000\" temperature = \"5\" initial_perturb = \"1.0\"/>\u00a0\u00a0\u00a0\u00a0<HighResDocker name = high_res_docker cycles = 1 repack_every_Nth = 1 scorefxn = ligand_soft_rep movemap_builder = docking/>\u00a0\u00a0\u00a0\u00a0<FinalMinimizer name = final scorefxn = hard_rep movemap_builder = final/>\u00a0\u00a0\u00a0\u00a0<InterfaceScoreCalculator name = add_scores chains = X scorefxn = hard_rep/></MOVERS><PROTOCOLS>\u00a0\u00a0\u00a0\u00a0<Add mover_name = transform/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = high_res_docker/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = final/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = add_scores/></PROTOCOLS></ROSETTASCRIPTS>The following RosettaScripts protocol has been used to for DiB1 redesign:<ROSETTASCRIPTS><SCOREFXNS>\u00a0\u00a0\u00a0\u00a0<ligand_soft_rep weights = ligand_soft_rep>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_elec weight = 0.42/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_bb_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = rama weight = 0.2/>\u00a0\u00a0\u00a0\u00a0</ligand_soft_rep>\u00a0\u00a0\u00a0\u00a0<hard_rep weights = ligandprime>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_intra_rep weight = 0.004/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = fa_elec weight = 0.42/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_bb_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = hbond_sc weight = 1.3/>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<Reweight scoretype = rama weight = 0.2/>\u00a0\u00a0\u00a0\u00a0</hard_rep></SCOREFXNS><SCORINGGRIDS ligand_chain = \"X\" width = \"16\">\u00a0\u00a0\u00a0\u00a0<vdw grid_type = \"ClassicGrid\" weight = \"1.0\"/></SCORINGGRIDS><TASKOPERATIONS>\u00a0\u00a0\u00a0\u00a0<DetectProteinLigandInterface name = design_interface cut1 = 6.0 cut2 = 8.0 cut3 = 10.0 cut4 = 12.0 design = 1/></TASKOPERATIONS><LIGAND_AREAS>\u00a0\u00a0\u00a0\u00a0<docking_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false minimize_ligand = 10/>\u00a0\u00a0\u00a0\u00a0<final_sidechain chain = X cutoff = 6.0 add_nbr_radius = true all_atom_mode = false/>\u00a0\u00a0\u00a0\u00a0<final_backbone chain = X cutoff = 7.0 add_nbr_radius = false all_atom_mode = true Calpha_restraints = 0.3/></LIGAND_AREAS><INTERFACE_BUILDERS>\u00a0\u00a0\u00a0\u00a0<side_chain_for_docking ligand_areas = docking_sidechain/>\u00a0\u00a0\u00a0\u00a0<side_chain_for_final ligand_areas = final_sidechain/>\u00a0\u00a0\u00a0\u00a0<backbone ligand_areas = final_backbone extension_window = 3/></INTERFACE_BUILDERS><MOVEMAP_BUILDERS>\u00a0\u00a0\u00a0\u00a0<docking sc_interface = side_chain_for_docking minimize_water = true/>\u00a0\u00a0\u00a0\u00a0<final sc_interface = side_chain_for_final bb_interface = backbone minimize_water = true/></MOVEMAP_BUILDERS><MOVERS>\u00a0\u00a0\u00a0\u00a0<Transform name = \"transform\" chain = \"X\" box_size = \"8.0\" move_distance = \"5.0\" angle = \"360\" cycles = \"5000\" temperature = \"5\" initial_perturb = \"1.0\"/>\u00a0\u00a0\u00a0\u00a0<HighResDocker name = high_res_docker cycles = 1 repack_every_Nth = 1 scorefxn = ligand_soft_rep movemap_builder = docking/>\u00a0\u00a0\u00a0\u00a0<FinalMinimizer name = final scorefxn = hard_rep movemap_builder = final/>\u00a0\u00a0\u00a0\u00a0<InterfaceScoreCalculator name = add_scores chains = X scorefxn = hard_rep/>\u00a0\u00a0\u00a0\u00a0<FavorNativeResidue name = favor_native bonus = 1.0/>\u00a0\u00a0\u00a0\u00a0<ddG name = calculateDDG jump = 1 per_residue_ddg = 1 scorefxn = hard_rep/>\u00a0\u00a0\u00a0\u00a0<PackRotamersMover name = designinterface scorefxn = hard_rep task_operations = design_interface/></MOVERS><PROTOCOLS>\u00a0\u00a0\u00a0\u00a0<Add mover_name = transform/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = favor_native/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = designinterface/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = high_res_docker/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = final/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = calculateDDG/>\u00a0\u00a0\u00a0\u00a0<Add mover_name = add_scores/></PROTOCOLS></ROSETTASCRIPTS>S1 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigC).Each datapoint corresponds to one of 5 000 models generated for each round of the docking. Some outliers with high total and/or interface scores are not shown for better visibility of the majority of the data. Insets show overlays of the ligand positions in the 50 best models of the corresponding round of docking . After the third round, docking converged on a single binding pose solution ((TIF)Click here for additional data file.S3 FigThe protocol starts with a small ligand position perturbation followed by optimization of the protein:ligand interface. Amino acids are allowed to change their identity only if it results in a significant decrease in energy, defined through a \u201cfavor native\u201d bonus (1 REU in the used protocol). The sequence design step is followed by reevaluation of the ligand position using high resolution docking protocol. The final model is scored and saved for further analysis.(TIF)Click here for additional data file.S4 FigRepresented are data for 35\u201340 cells from the same field of view across the whole ligand concentration range. Box whiskers indicate standard deviation, horizontal lines within boxes indicate median values, circles indicate outliers.(TIF)Click here for additional data file.S5 Fig1-109 or (B) DiB-RM-split110-177-TagBFP constructs. The signal from green and blue channels, correspondingly. Scale bars are 25 \u03bcm.Widefield fluorescence images of HEK293 cells transiently transfected with (A) NeonGreen-DiB-RM-splitN(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigA) using the same DiB1 model that was generated for the initial docking but with deleted E/F loop (-6 amino acids) or (B) using the DiB1 crystal structure-based DiB-RM model starting from the initial (\u201cold\u201d) starting position of the ligand. The M739 chromophore from the co-crystal structure is shown as yellow sticks. All docked chromophores are shown as green lines.Rerun of the chromophore M739 docking ((TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S2 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file.S3 MoviePlays at 30 fps (acquisition speed 30 Hz).(MP4)Click here for additional data file."}
+{"text": "Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants\u2019 occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants\u2019 tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants\u2019 emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant\u2019s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant\u2019s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world. Early on 2020 major viral diagnostic efforts were deployed, including the development of news strategies aiming at (1) reducing the diagnostics time, (2) decrease the costs per assay, (3) providing population-scale solutions, but also (4) presenting a high sensitivity and specificity, notably to discriminate asymptomatic cases. While costs and required time per assay were early on addressed by the development of immune-based tests5. These efforts rely on (1) the incorporation of DNA molecular barcodes during sample preparation; (2) pooling barcoded samples for an \u201cin-one-run\u201d diagnostics assay by the use of massive parallel DNA sequencing; (3) stratification of diagnostics\u2019 outcome with the help of bioinformatics processing. While such strategies demonstrated to up-scale the diagnostics power beyond several thousand candidates per assay, they require to count with a DNA sequencing platform where large instruments (Illumina Sequencers) are driven by specialized technical engineers. As consequence, this strategy remains applicable to highly developed countries (due to the prohibitive costs of the required DNA sequencers), which in addition requires a dedicated pipeline strategy for transporting collected samples towards sequencing centers.With the aim of taking advantage of the viral nucleic acid targeting diagnostics but at the same time increasing the number of candidates\u2019 through-put, strategies based on the use of massive parallel DNA sequencing for population-scale diagnostics were proposed6.Beyond the diagnostics requirements, the identification of the highly transmissible SARS-CoV-2 variant B.1.1.7\u2014originated in the south of England\u2014has strongly accelerated the worldwide interest in tracking SARS-CoV-2 variants\u2019 occurrence. More recently, two other extremely infectious variants, namely the P.1 issued on the Brazilian city of Manaus and the B.1.351 initially detected In South Africa, were described, and unsurprisingly further others are expected to be discovered, notably due to the long period of time on which the pandemic situation is lasting7). Indeed, this technology is widely used for SARS-CoV-2 whole-genome sequencing, notably by the use of the amplicon sequencing protocol developed by the ARTIC Network (https://artic.network/ncov-2019)8. Furthermore, a Covid-19 diagnostics strategy combining Loop-mediated isothermal Amplification (LAMP) with Nanopore sequencing has been also described9.Herein, we describe a proof of concept study for population-scale diagnostics and variants\u2019 tracking using massive parallel DNA sequencing performed with the MinION Oxford Nanopore Technology (ONT). ONT MinION sequencers are well-known for their portability, their reduced cost, and their simplicity of use; thus, representing a suitable strategy for its implementation even in remote places over the world  providing the outcome of the diagnostic in a real-time mode during sequencing; allowing to earn time for diagnostics report, essential in the current context of the pandemic situation. Finally, we have also demonstrated that our strategy is able to perform SARS-CoV-2 variants\u2019 detection across multiple candidates, notably by targeting the SPIKE gene instead of covering the whole viral genome Fig.\u00a0. This ta10.In summary, the proposed strategy satisfies the current need for counting with a population diagnostic assay, and discriminates between the various SARS-CoV-2 variants, which are recognized to potentially present variable infection properties as well as clinical outcomesWith the aim of performing SARS-CoV-2 diagnostics over multiple candidates with the MinION sequencer, we combined a regular reverse-transcription assay, with a particular PCR amplification allowing the generation of long DNA concatemers. This strategy allows to target short amplicons, like those used on quantitative real-time PCR strategies, but at the same time take advantage of the long fragments sequencing capabilities of the Oxford Nanopore technology.Reverse transcription (RT) is performed with a Covid-19 specific primer, presenting in addition, a unique molecular barcode (20nt length) and a common sequence adapter   and a reverse-oriented sequence for the common Gibson sequence adapter (30nt length) is used together with a Gibson primer . Hence, we have developed a computational pipeline dedicated to (1) collect available fastq files; (2) retrieve Gibson adapter sequences within sequenced molecules; 3) split them into monomers; (4) evaluate the presence of cDNA & PCR molecular barcodes in their flanking regions; (5) align the inner monomer sequences towards the SARS-CoV-2 reference genome; and (6) display the aligned read counts per cDNA/PCR barcode combinations. This pipeline is setup to be reinitiated in an automated time interval (e.g. every 10\u00a0min) such that newly available sequences can be cumulated to the previous analyses, thus providing a real-time view of aligned read counts associated with cDNA/PCR barcode combinations  were used for mimicking variable viral RNA copies per assay. Specifically, subsequent dilutions of the synthetic Wuhan-Hu-1 (GeneBankID: MN908947.3) SARS-CoV-2 RNA control sample has been used for reverse transcription, followed by a quantitative PCR assay targeting a region on the E gene of SARS-Cov-2, defined by primer sequences described previously (E-Sarbeco)For Nanopore DNA sequencing-based diagnostic, as described on Fig.\u00a0Subsequent dilutions of the synthetic SARS-CoV-2 RNA control sample were used for reverse transcription, performed with defined barcoded E-Sarbeco primers , in addition to a negative control sample devoid of viral RNA material (BC37)  we can afford to combine diagnostic and variants\u2019 tracking into a single assay, which per se represents a major progress relative to the current strategies.In contrast to the aforementioned diagnostics effort targeting a short amplicon (E-Sarbeco), variants\u2019 tracking over the whole SPIKE gene uses a random-hexamer sequence for the reverse transcription step, and four primers targeting the SPIKE gene at different regions (~\u20091\u00a0kb distance among each primer) Fig.\u00a0A. Like i12 , human RNA\u2014issued from cell lines in culture\u2014has been combined with the synthetic viral RNA samples, such that the random-hexamer primers used for the reverse transcription assay could also provide means to count with a positive control for sample collection. Furthermore, for addressing variants tracking performance, in addition to the Wuhan-Hu-1 SARS-CoV-2 RNA control sample, we have used synthetic viral RNA samples corresponding to the highly transmissible SARS-CoV-2 variant B.1.1.7, first described in the UK in December 2020, as well as the strain HF2393 described as a particular clade circulating in France since January 202012 Fig.\u00a0C.To perform diagnostics and variants\u2019 tracking assay over multiple pseudo-candidates, we have used eight barcoded random-hexamer primers for reverse transcription BC37-BC44) in presence of different viral RNA titers (consecutive dilutions) and issued from the aforementioned synthetic viral RNA strains  revealed a single common mutation, D614G, previously described within the French strain HF23936. As consequence, counting with methodologies for Covid-19 diagnostics but also variants\u2019 tracking on a population scale remains essential.Despite the current vaccination efforts, the pandemic situation anticipates the propagation of the recently described variants, as well as the emergence of new others, notably due to the major discrepancies between countries to access to optimal vaccination programsCurrently, diagnostics and variants\u2019 tracking are performed in two steps, which per se delays the time for obtaining a complete diagnostic. Furthermore, variants\u2019 tracking relies on full genome sequencing of SARS-CoV-2, which represent an expensive and labor-intensive effort, incompatible with population-scale assays. Finally, most of the variants\u2019 tracking efforts, relying on the use of large and expensive sequencing instruments, requires to centralize samples processing to dedicated institutions, which are rather absent on poor countries.To address all these pitfalls, we propose herein a methodology able to (1) perform SARS-CoV-2 diagnostic and variants\u2019 tracking at the same time; (2) shorten the time for accessing to the outcome of the diagnostic/variants\u2019 detection by the use of a real-time tracking system; (3) take advantage of the compact and affordable Oxford Nanopore MinION DNA sequencing instrument, notably for deploying the aforementioned diagnostics and variants\u2019 tracking platform anywhere over the world.Within this study we have validated the proposed Nanopore DNA sequencing-based diagnostic, by targeting a region on the E gene of SARS-Cov-2 (E-Sarbeco) on a total of 64 pseudo-candidate samples Fig.\u00a0. Importa13. Illumina instruments being a short fragments sequencing approach, HiSpike requires 42 primer pairs generating amplicons of 400 nucleotides of length for covering the SPIKE region. In contrary, our proposed strategy, based on the use of long DNA fragments, compatible with the Nanopore DNA sequencing technology, relies on 4 primers targeting the SPIKE region, one random primer for the cDNA step and a common Gibson sequence. In fact, a strategy for SARS-CoV-2 diagnostic and variants\u2019 surveillance, dedicated to be deployed anywhere on the world does not only requires to count with low cost instruments, but also involve a reduced number of reagents for allowing its use on a long-standing manner.The proposed variants\u2019 tracking focus on the analysis of the SPIKE gene instead of the whole SARS-CoV-2 genome. In fact, the SPIKE gene has shown to concentrate the majority of the currently discovered variants, which can be rationalized by the fact that it codes for the protein required for the interaction with the human cells. Noteworthy, most of the current vaccines against the Covid-19 are also based on the SPIKE region. Hence, concentrating variants\u2019 tracking towards this gene appears an optimal compromise to increase the number of candidates to screen at once. This philosophy is shared by a recent work describing the HiSpike method, performing variants\u2019 detection within the SPIKE gene with the help of the small Illumina MiSeq instrument14. In all cases, new mutations on the gene SPIKE were observed, further supporting our strategy for targeting this genomic region for variants surveillance.During the time of the final revision of this article, new variants derived from the lineage of B.1.1.7 has gained major attention notably due to the fact that they are vastly spreading in the UK, USA, and other locations For all these reasons, we are convinced that our proposed strategy, accompanied by the dedicated computational solution RETIVAD, can represent a game-changer approach for tracing variants\u2019 progression in the following months.Wuhan-Hu-1 (GeneBankID: MN908947.3); Twist Biosc. ID: Ctrl2.France/HF2393/2020 (EPI_ISL_418227); Twist Biosc. ID: Ctrl7.England/205,041,766/2020 (EPI_ISL_710528); Twist Biosc. ID: Ctrl14 (B.1.1.7).The following Synthetic SARS-CoV-2 RNA control samples, delivered at a concentration of 1 million copies per microliter has been used in this study:11, has been used:Primers used for reverse transcription are composed by (1) a fragment of the Gibson sequenced (redGibs: GAAGAACCTGTAGATAACTCGCTGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1\u201396 kit (EXP-PBC096) and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the reverse primer sequence provided by Corman et al.E_Sarbeco_R: ATATTGCAGCAGTACGCACACAFor SPIKE-centered variants tracing, the target sequence has been replaced by a random hexamer, notably to decrease the number of required primers during the assay.The following barcoded primers targeting the E-Sarbeco region were used for reverse transcription:redGibsBC33_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCAGACTTGGTACGGTTGGGTAACTATATTGCAGCAGTACGCACACAredGibsBC34_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGGACGAAGAACTCAAGTCAAAGGCATATTGCAGCAGTACGCACACAredGibsBC35_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCTACTTACGAAGCTGAGGGACTGCATATTGCAGCAGTACGCACACAredGibsBC36_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTATGTCCCAGTTAGAGGAGGAAACAATATTGCAGCAGTACGCACACAredGibsBC37_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCAATATTGCAGCAGTACGCACACAredGibsBC38_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAAATATTGCAGCAGTACGCACACAredGibsBC39_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCATATTGCAGCAGTACGCACACAredGibsBC40_E_Sarbeco_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCATATTGCAGCAGTACGCACACAThe following barcoded random-hexamer primers were used for reverse transcription:redGibsBC37_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGCTTGCGATTGATGCTTAGTATCANNNNNNredGibsBC38_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTACCACAGGAGGACGATACAGAGAANNNNNNredGibsBC39_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCCACAGTGTCAACTAGAGCCTCTCNNNNNNredGibsBC40_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTTAGTTTGGATGACCAAGGATAGCCNNNNNNredGibsBC41_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTGGAGTTCGTCCAGAGAAGTACACGNNNNNNredGibsBC42_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTACGTGTAAGGCATACCTGCCAGNNNNNNredGibsBC43_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTCTTTCGTTGTTGACTCGACGGTAGNNNNNNredGibsBC44_Rhexamer_R: GAAGAACCTGTAGATAACTCGCTGTAGTAGAAAGGGTTCCTTCCCACTCNNNNNN11, has been used:For PCR amplification a second set of barcoded primers were designed presenting the following structure: (1) A reverse-complementary Gibson fragment (revGibs: ACAGCGAGTTATCTACAGGTTCTTCAATGT), (2) a barcode sequence issued from the ONT PCR Barcoding Expansion 1\u201396 kit (EXP-PBC096), and (3) the target sequence. For targeting the E-Sarbeco region within the SARS-CoV-2 genome, the forward primer sequence provided by Corman et al.E_Sarbeco_F: ACAGGTACGTTAATAGTTAATAGCGTThe following barcoded primers targeting the E-Sarbeco region were used in this study:revGibs-_BC01_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTAAGAAAGTTGTCGGTGTCTTTGTGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC02_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTCGATTCCGTTTGTAGTCGTCTGTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC03_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGAGTCTTGTGTCCCAGTTACCAGGACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC04_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCGGATTCTATCGTGTTTCCCTAACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC05_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTCTTGTCCAGGGTTTGTGTAACCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC06_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCTCGCAAAGGCAGAAAGTAGTCACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC07_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTGTGTTACCGTGGGAATGAATCCTTACAGGTACGTTAATAGTTAATAGCGTrevGibs-_BC08_E_Sarbeco_F: ACAGCGAGTTATCTACAGGTTCTTCAATGTTTCAGGGAACAAACCAAGTTACGTACAGGTACGTTAATAGTTAATAGCGTFor SPIKE-centered variants tracing, the reverse-complementary Gibson sequence has been excluded, notably because the PCR amplification performed over random-hexamer generated cDNA templates generate large fragments (>\u20091\u00a0kb). In contrary, the amplicon product issued from E-Sarbeco targeting oligonucleotides gives rise to short amplicons\u2014compatible with quantitative PCR assays, thus requiring concatemerization prior nanopore sequencing.With the aim of covering the SPIKE gene (~\u20093.8\u00a0kb), the following four primers targeting the SPIKE region with\u2009~\u20091\u00a0kb interval within them were designed:Spike1_F: AGGGGTACTGCTGTTATGTCTSpike2_F: TGCACTTGACCCTCTCTCAGSpike3_F: GCAGGCTGTTTAATAGGGGCSpike4_F: TGCAGACATATGTGACTCAACAThe following Spike targeting primers including the corresponding barcode sequences were used for this study:BC01_Spike_1_F: AAGAAAGTTGTCGGTGTCTTTGTGAGGGGTACTGCTGTTATGTCTBC02_Spike_1_F: TCGATTCCGTTTGTAGTCGTCTGTAGGGGTACTGCTGTTATGTCTBC03_Spike_1_F: GAGTCTTGTGTCCCAGTTACCAGGAGGGGTACTGCTGTTATGTCTBC04_Spike_1_F: TTCGGATTCTATCGTGTTTCCCTAAGGGGTACTGCTGTTATGTCTBC01_Spike_2_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCACTTGACCCTCTCTCAGBC02_Spike_2_F: TCGATTCCGTTTGTAGTCGTCTGTTGCACTTGACCCTCTCTCAGBC03_Spike_2_F: GAGTCTTGTGTCCCAGTTACCAGGTGCACTTGACCCTCTCTCAGBC04_Spike_2_F: TTCGGATTCTATCGTGTTTCCCTATGCACTTGACCCTCTCTCAGBC01_Spike_3_F: AAGAAAGTTGTCGGTGTCTTTGTGGCAGGCTGTTTAATAGGGGCBC02_Spike_3_F: TCGATTCCGTTTGTAGTCGTCTGTGCAGGCTGTTTAATAGGGGCBC03_Spike_3_F: GAGTCTTGTGTCCCAGTTACCAGGGCAGGCTGTTTAATAGGGGCBC04_Spike_3_F: TTCGGATTCTATCGTGTTTCCCTAGCAGGCTGTTTAATAGGGGCBC01_Spike_4_F: AAGAAAGTTGTCGGTGTCTTTGTGTGCAGACATATGTGACTCAACABC02_Spike_4_F: TCGATTCCGTTTGTAGTCGTCTGTTGCAGACATATGTGACTCAACABC03_Spike_4_F: GAGTCTTGTGTCCCAGTTACCAGGTGCAGACATATGTGACTCAACABC04_Spike_4_F: TTCGGATTCTATCGTGTTTCCCTATGCAGACATATGTGACTCAACAReverse transcription assays were performed using SuperScript\u2122 IV Reverse Transcriptase ) with a standard reaction volume of 20ul including 0.5ul of super RNAseIn , 1ul DTT (0.1\u00a0M), 4 ul SSIV buffer (5X), 0.5 ul dNTP (10\u00a0mM each), 1 ul of synthetic SARS-CoV-2 RNA, 1 ul of 2uM gene-specific-barcoded oligonucleotide  or 1 ul of 50uM random hexamer-barcoded oligonucleotide . For assays including random hexamer-barcoded primers, 1ul of human RNA  were added to the assay together to the synthetic SARS-CoV-2 RNA and prior adding the random hexamers.Synthetic SARS-CoV-2 RNA was added at a copy number of 1 million , 100 thousand (d.f: 0.1), 10 thousand (d.f: 0.01) and 1 thousand (d.f: 0.001) per reaction respectively. Note that these consecutive dilutions give rise after downstream dilutions to the number of copies of 10 thousand, 1 thousand, 100 and 10 respectively, analyzed by quantitative PCR Fig.\u00a0B.Multiple reverse transcription assays\u2014each of them performed in presence of a defined barcoded primer\u2014were incubated at 50\u00a0\u00b0C during 30\u00a0min, then at 80\u00a0\u00b0C 10\u00a0min. Barcoded cDNA samples were pooled (8 samples at once) and cleaned with SPRIselect reagent  with a ratio of 0.5x. Cleaned cDNA is recovered on 80ul . 40ul of this pooled & cleaned cDNA will be used for PCR amplification , while the remaining material is used for quantitative PCR evaluation.Barcoded-cDNA material has been validated by quantitative PCR assay with the following primer sequences:Primers targeting region on E gene of SARS-Cov-2 :Human_RNASE_P-F: AGATTTGGACCTGCGAGCGHuman_RNASE_P-R: GAGCGGCTGTCTCCACAAGTQuantitative PCR assays were performed with QuantiTect SYBR Green PCR Kit  over a 1/10 diluted cDNA material.PCR amplification targeting the E-Sarbeco amplicon is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 2.5ul of 1uM barcoded-E-Sarbeco targeting primer (BC1-8), 2.5ul of 1uM short Gibson primer (AGAACCTGTAGATAACTCGCTGT) and 5ul of the pooled & cleaned cDNA.98\u00a0\u00b0C 30\u00a0s98\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 20\u00a0s72\u00a0\u00b0C 30\u00a0sRepeat 2\u20134 steps 25\u00d798\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 20\u00a0s72\u00a0\u00b0C 2\u00a0minRepeat 6\u20138 steps 20\u00d772\u00a0\u00b0C 10\u00a0minKeep at 12\u00a0\u00b0CFor short-amplicon E-Sarbeco PCR amplification the following thermal cycling program is used:For PCR amplification targeting SPIKE and E-Sarbeco on random-hexamers generated cDNA material the assay is performed with a pool of barcoded SPIKE primers and shortGibson. For 100ul mix 10ul of barcoded primer Spike1 (10uM), 10ul of Spike2 (10uM), 10ul of Spike (10uM), 10ul of Spike4 (10uM) and 40ul of ShortGibson (10uM) completed with distilled water is prepared. Then the PCR amplification is performed on 25ul final volume including 12.5ul of Phusion Hot Start II High-Fidelity PCR Master Mix (ThermoFisher ref: F565), 1.25ul of pooled barcoded-SPIKE & shortGibson primers  and 5ul of pooled & cleaned cDNA.98\u00a0\u00b0C 30\u00a0s98\u00a0\u00b0C 10\u00a0s61\u00a0\u00b0C 45\u00a0s72\u00a0\u00b0C 2\u00a0minRepeat 2\u20134 steps 39\u00d772\u00a0\u00b0C 10\u00a0minKeep at 12\u00a0\u00b0CThe following thermal cycling program is used:In both cases (E-Sarbeco Concat-PCR or SPIKE PCR amplification), PCR products were verified by TapeStation automated electrophoresis .Multiple PCR assays are performed in presence of different barcoded primers, such that the combination with the barcoded cDNA material allows to generate a large complexity. Specifically, for E-Sarbeco targeting assay, 8 barcoded contact-PCR assays are pooled (8\u2009\u00d7\u200925\u2009=\u2009200ul) and cleaned with SPRIselect reagent  with a ratio of 0.5\u00d7. Similarly, for SPIKE and E-Sarbeco PCR assays issued from random hexamer cDNA material, 4 barcoded PCR assays for each target regions  were pooled (4\u2009\u00d7\u20092\u2009\u00d7\u200925\u2009=\u2009200ul) and cleaned with SPRIselect reagent  with a ratio of 0.5x.Pooled & cleaned material is recovered on 50ul and used for Nanopore sequencing library preparation (Nanopore Ligation Sequencing Kit: SQK-LSK109). DNA libraries were sequenced on Nanopore FLO-MIN106D flowcells (R9) with the MinION Mk1C instrument .https://pypi.org/project/regex) with maximum 4 mismatches accepted by default); (2) split sequenced reads on monomers\u2014delimited by the presence of the identified Gibson sequences; (3) search for barcode sequences introduced during the reverse transcription ; (4) search for barcodes introduced during the PCR amplification ; (5) collect the sequences between the cDNA and PCR-associated barcodes for their alignment towards the SARS-CoV-2 reference genome . Considering that steps (3\u20135) are performed within the retrieved monomers in step (2), RETIVAD matches cDNA/PCR barcode combinations to aligned outcomes, thus providing a diagnostic outcome for the associated pseudo-candidate. Due to the low number of sequences retrieved at intervals of 10\u00a0min, and the relative short size of the SARS-CoV-2 genome (~\u200929\u00a0kb), RETIVAD is able to provide such diagnostic outcome within such 10\u00a0min interval. Hence, at the next round of data collection, RETIVAD reiterates on the aforementioned steps over the newly collected fastq files and appends the outcome to the previous results. The continuous gain on aligned read-counts per region of interest  is visualized within scatterplot view (png file) updated during the processing  software. RETIVAD collects each ten minutes the fastq files generated during sequencing from the Mk1C instrument and (1) search for Gibson sequences within the sequenced reads (regex query (racon package (version 1.4.20) is used for raw de novo DNA assembly, followed by the alignment with Minimap2 (version 2.17). Medaka (version 1.2.5) is used to create a consensus sequence and variant calls from the aligned data . RETIVAD generates BAM files per cDNA/PCR barcode combination harboring base conversion information and their associated coverage . Furthermore, summary files per cDNA/PCR barcode combination are also generated, providing the genomic position in which the base conversion has been detected, the identity of the converted bases as well as their associated coverage. Like in the case of the diagnostics processing, variants detection is performed in real time, thus providing the possibility to the user to visualize the results and decide whether sequencing requires still to be performed or whether the collected information is sufficient to conclude the diagnostics/variants tracing for the corresponding candidates.In addition to the diagnostic outcome, RETIVAD has been designed for performing real-time variants detection. For it, the RETIVAD generates a final summary report for the diagnostics outcome when no new fastq files are available after 40\u00a0min from the last data collection and processing.Supplementary Information."}
+{"text": "HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.Heterozygous mutations in  Summary: A novel established mouse model carrying a heterozygous splicing human mutation in the Hnf1b gene exhibits phenotypes similar to those of patients with renal cysts and diabetes disease. HNF1B gene are the cause of a complex human syndrome known as renal cysts and diabetes , characterized by early onset of diabetes and developmental abnormalities of the kidney, genital tract and pancreas, as well as a variety of renal, liver, pancreas and biliary dysfunctions . Intragenic mutations map predominantly in the DNA binding domain (exon-2 and exon-4), while whole-gene deletions (1.3\u2005Mb deletion at chromosome 17q12) account for up to 50% of the HNF1B variants. HNF1B mutant carriers exhibit a highly variable phenotype, both between and within families. No clear genotype-phenotype correlations were observed for the type or location of mutations, and haploinsufficiency has been the main underlying disease proposed mechanism.More than 150 eletions . These mHNF1B mutations is severe non-diabetic renal disease. HNF1B mutations are also the most common monogenic causes of developmental kidney disease, being part of a spectrum of malformations known as congenital anomalies of the kidney and urinary tract  branching and the induction of nephrogenesis  . Moreoveutations , this moSp2/+Hnf1b] exhibited bilateral cysts and tubular dilatations as well as glomerular cysts from embryonic day (E)15, along with rare cases of genital tract abnormalities and extrarenal manifestations similar to those described in human HNF1B mutant carriers. Unlike previous mouse mutants heterozygous for a null allele, in which the HNF1B protein levels remained either unchanged or even increased  ncreased , the HNFHnf1b ablation were relatively insensitive. Postnatal analyses revealed several renal abnormalities, ranging from few clusters of glomerular cysts and microcysts to hydronephrosis and, rarely, multicystic dysplasia. Further urine proteomic analyses uncovered a particular signature of differentially excreted peptides in Hnf1bSp2/+ mice, exhibiting several similarities to the urinary peptide signature reported in pediatric RCAD patients  was sensitive to reduced HNF1B levels at different embryonic stages, whereas the previously identified targets upon patients .Hnf1b dosage in this mouse model differentially affects the expression of target genes, leading to the onset of disease phenotypes.Our results highlight that reduced Fig.\u00a0S1A), thus reproducing the c.544+1G>T (<IVS2nt+1G>T) human mutation  using RNA from Hnf1bSp2/+ heterozygotes and wild-type (WT) kidneys and primers located in exon-1 and exon-3. Sequence of the PCR products in Hnf1bSp2/+ mutants indicated the production of the two expected Hnf1b spliced isoforms A and B and four additional novel transcripts, present at low levels, corresponding to the isoforms A and B in which were deleted either exon-2 or the last 32\u2005bp of exon-2 through the activation of a near cryptic splice donor site . The same pattern of alternative splicing in Hnf1bSp2/+ heterozygous mice was observed at different stages .To define the consequences of this mutation on Fig.\u00a0S1C), variants lacking the last 32\u2005bp of exon-2 have not been described  using primers located in the ATG translational site and the last 32\u2005bp of exon-2, thus allowing the detection of transcripts produced only by the normal Hnf1b allele (Table\u00a0S2). We found that, in Sp2/+Hnf1b, the Hnf1b transcripts were significantly decreased by 30-42% relative to WT levels, in particular during embryonic stages. In adults, the reduction in transcript levels was highly variable and modest without reaching significance . Thus, these truncated proteins are either very unstable or not produced as previously described in several dominant mutations associated with premature termination codons (PTCs) (see Discussion). Unlike previously characterized heterozygous mutants, we found a significant decrease in HNF1B protein levels in the Sp2/+Hnf1b mutants, not only at embryonic stages but also in adults , although in some cases mutant embryo kidneys exhibited dilated Bowman's capsules . Subsequently, from E15.5, we reproducibly observed bilateral cysts, including glomerular and tubular cysts in the cortico-medullar regions . Up to E14.5, heterozygous embryos developed apparently normally . A summary of histological renal phenotypes observed from E18.5 to postnatal day (P)0 is shown in Table\u00a0S1A.We then examined the influence of mouse strain susceptibility on the disease phenotype by backcrossing the om E15.5 G-N and a kidneys ; Fig.\u00a0S4Table\u00a0S1B). Heterozygous mutants in both backgrounds were able to reproduce, but they were less fertile than WT littermates. Further postnatal analysis revealed rare cases of genital tract abnormalities  in either C57BL/6N or 129/sv backgrounds. Heterozygous mutants also exhibited several pancreatic dysfunctions, including glucose intolerance and pancreatitis, a phenotype that will be described elsewhere  or attenuate (129/sv) the phenotype, further contributing to variability in phenotypic manifestations.These data show that Hnf1b is known to be required for early UB branching and initiation of nephrogenesis . Notably, the expression of Pax2, the transcription of which in the collecting system depends on Hnf1b , or at later stages . Nascent nephrons appeared normally induced as indicated by the presence of normally shaped comma- and S-shaped bodies  and WT1-stained glomerular podocytes . Mutant kidneys without overt hydronephrosis were examined to visualize the collecting duct network and medullar nephron tubules. The expression of HNF4A, a marker of early PTs and a target of HNF1B , suggesting defective differentiation of PTs. Notably, HNF4A staining, which correlated with the reduced levels of HNF1B , in contrast to HNF4A expression, which showed only a partial restoration at E17.5  or P0 . Consistent with these observations, the mature PT marker LTA was not expressed up to E16.5 . However, Sp2/+Hnf1b PT clusters that were LTA+ both at E17.5 and P0 exhibited an unequal distribution and remained reduced in size . Interestingly, the Na-K-Cl co-transporter , expressed in the thick ascending limb (TAL) of the loop of Henle, stained most of Sp2/+Hnf1b medullar cysts and tubular dilatations at E17.5 and P0 . Distal tubules labeled by SLC12A3 (NCC) showed only rare and mild dilatations (data not shown).The expression of HNF1A, restricted to mature PTs, was similarly reduced at E16.5 B\u2032. Its eto E16.5 F\u2032 and beSp2/+Hnf1bcollecting ducts, stained either by AQP2  or pancytokeratin (CK) , were mainly devoid of dilatations. Intriguingly, they were not stained by Dolichos biflorus lectin (DBA), either during development or in postnatal life , uncovering impaired polarization of glycoconjugates in mutant collecting duct cells. This observation evoked an altered differentiation status of collecting duct cells, because previous studies have shown that UB cells begin to express DBA-binding glycoconjugates once they differentiate into stalks   .Sp2/+Hnf1b non-cystic glomeruli displayed the normal layer of podocytes .Without evidence of urinary tract obstruction, we further examined HNF1B, HNF4A and WT1 immunostainings on serial embryonic sections, focusing on glomeruli with different degrees of Bowman's capsule expansion. We reproducibly observed that cystic glomeruli were surrounded by a decreased number of PTs that were often dilated or cystic H. Immunoodocytes F,I. In mHNF1B human mutant fetuses showing a very severe cystic renal phenotype  exhibited a global decrease and partial disorganization in E15.5 heterozygous kidneys, both in dilated and non-dilated tubules K,M. In mFig. S6A). However, at later stages, the number of proliferating cells in Sp2/+Hnf1b normal tubular structures exhibited a 1.84-fold increase relative to WT tubules. Consistent with the enhanced proliferation associated with cystic expansion in autosomal dominant polycystic kidney disease . No changes were observed in the number of apoptotic cells assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (not shown).Further analysis of proliferation, using the mitosis marker phosphorylated histone H3, showed comparable numbers of proliferating cells in E14.5 renal tubules of heterozygous mutants and WT  on WT and heterozygous mutant kidneys at E14.5  and at disease stages  as detailed in the Materials and Methods.P-value <0.05. Remarkably, several genes were differentially expressed with log2FC>\u22121 at all stages (Table\u00a0S3), while only a few differentially expressed upregulated genes were detected. We found an increased number of downregulated genes from E14.5 to E17.5 followed by a partial decrease at P1, in agreement with a partial restoration of the expression of several PT markers. Accordingly, the numbers of downregulated genes shared between stages increased to reach 107 genes between E17.5 and P1 .Because heterozygous mutants still expressed HNF1B, we considered an absolute fold-change cutoff value of > or <0.5 log2FC, with an adjusted Table\u00a0S3, File\u00a06). At E17.5 and P1, downregulated genes included drug-metabolizing enzymes  and more than 50 SLC-transmembrane-transporter genes .Downregulated genes with greater fold changes were predominantly expressed in early and maturing PTs and to a lesser extent in primitive loops of Henle and distal tubules (Table\u00a0S4).Gene ontology (GO)-term analyses  highlighHnf4a, Tmem27 (Cltrn), Cubn, Spp1, Spp2, Pah, Kcnj1, Pdzk1 and several SLC-transmembrane transporter genes, representing the subset of targets that appeared particularly sensitive to reduced HNF1B protein levels (Table\u00a0S3). Interestingly, many of the genes downregulated at E17.5/P1 and regulated by HNF1B were also found strongly decreased in Hnf4a mutant P1 kidneys . By contrast, the expression of previously identified targets strongly reduced upon Hnf1b ablation, including Wnt9b, Pax2, Pkd2, Bicc (Bicc1), Tg737 (Ift88), Crb3, Kif12, Cys1, Glis2 and Glis3, were either modestly decreased or not affected. These results were further validated by qRT-PCR . Interestingly, the well-known targets Umod, Tmem27 and Pkhd1 (Table\u00a0S3).The regulatory sequences of most downregulated genes contained HNF1 consensus binding sites. Consistently, specific HNF1B recruitment to the regulatory sequences of many of them has been reported by chromatin immunoprecipitation (ChIP)-PCR or ChIP sequencing . This ocular phenotype remains undefined. It may be linked to the Rd8 mutation of the Crb1 gene present in the strain C57BL/6N , with the lacZ gene and the SV40 polyadenylation sequence and neomycin resistance cassette, replacing the first exon of Hnf1b (Hnf1btm1Sce) and C57BL/6N (C57BL/6N-Hnf1btm1Sce) backgrounds are available at EMMA  together with the phenotype description. The +/\u2212Hnf1a mice were provided by Frank Gonzalez    and mainAnimal care and the experimental protocols were approved by and conducted in accordance with French and European ethical legal guidelines and the local ethical committee for animal care , respecting the 3R rules.Paraffin-embedded human fetal tissues were obtained from family members with informed consent approved by the Ethics committee of Debr\u00e9 Hospital , as previously reported in Fbp and Spp2 cRNA probes were generated by PCR . Embryos and postnatal kidneys up to 2\u00a0months of age were fixed with 60% ethanol/11% formaldehyde and 10% acetic acid. Adult kidneys (>2\u00a0months) were fixed in alcoholic Bouin (Duboscq-Brasil) solution and paraffin sections were used for H&E histological analysis and immunohistochemistry. Antibody staining on paraffin sections was performed as described  and washed in ice-cold PBS. WT and heterozygous mutant embryos were from the same litter. Adult kidneys were cut sagittal: one-half was used for histology, one-quarter for RNA extraction and the other quarter for protein extraction. Total RNA was extracted using an miRNeasy Mini Kit (Qiagen). Samples were treated with Dnase1 on the columns according to the manufacturer's instructions, and 250-500\u2005ng was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) and the Step-One Plus system (Applied Biosystems), as described . The priFig.\u00a0S1. PCR products were resolved in 2% agarose/TBE ethidium bromide gels and photographed using a GELDOC documentation system. Densitometry quantification was performed with ImageJ software. Primer sequences used were Gapdh for normalization and, for Hnf1b, vATG and v695 (Table\u00a0S2).Total RNA from microdissected kidneys was extracted and subjected to semiquantitative RT-PCR as described  with theHnf1b allele are listed below:The cDNA sequences of spliced isoforms and the encoded truncated proteins from the mutated CGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGAgATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLSEDPWRAAKMIKGYMQQHNIPQREVVDVTGLNQSHLSQHLNKGTPMKTQKRAALYT*VQPDTAAaAGTTCAACCAGACAGTCCAGAGCTCTGGAAACATGACAGACAAAAGCAGTCAGGATCAGCTGCTGTTTCTCTTTCCAGAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQPDSPELWKHDRQKQSGSAAVSLSRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*TAAaAGTTCAGTCAACAGAACCAGGGGCCTGGGCAGTCGGAGGACACCTGCTCCGAGCCCACCAACAAGAAGATGCGCCGCAACCGGTTATGGTGTCCAAGCTCACGTCGCTCCAGCAAGAACTCCTGAGTGCCCTGCTGAGCTCCGGAGTCACCAAGGAAGTGCTGATCCAGGCCTTGGAGGAGTTACTGCCGTCCCCGAATTTCGGGGTGAAGCTGGAGACACTGCCCCTGTCCCCCGGGAGCGGGGCGGATCTCGACACCAAGCCGGTTTTCCATACTCTCACCAATGGCCACGCCAAGGGCCGCTTGTCTGGGGACGAGGGCTCAGAGGACGGCGACGACTATGACACTCCTCCCATCCTCAAAGAGCTCCAGGCGCTCAACACCGAGGAGGCCGCGGAGCAGCGGGCCGAGGTGGACCGGATGCTCAGMVSKLTSLQQELLSALLSSGVTKEVLIQALEELLPSPNFGVKLETLPLSPGSGADLDTKPVFHTLTNGHAKGRLSGDEGSEDGDDYDTPPILKELQALNTEEAAEQRAEVDRMLRVQSTEPGAWAVGGHLLRAHQQEDAPQPV*Note that the STOP codons of putative encoded proteins are followed by a purine, which is predicted to positively influence translational termination efficiency .GTACGTCAGAAAGCAACGGGAGATCCTCCGACCGAGGACCCGTGGAGGGCTGCCAAAATGATCAAGGGATACATGCAACAGCACAATATCCCCCAGAGGGAGGTGGTCGATGTCACAGGCCTGAACCAATCCCACCTCTCTCAACACCTCAACAAGGGCACCCCCATGAAGACCCAGAAGAGAGCTGCCCTGTACACTTGUnderlined text shows the 32\u2005bp of exon-2 spliced out of mouse exon-2.Human embryonic kidney (HEK) 293 cells were maintained and transiently transfected as described , with eig. The cellular pellet was resuspended into ice-cold HNB buffer , frozen and thawed four times in liquid N2, and centrifuged for 10\u2005min at 11,180 g. The supernatant containing the whole-cell extracts was frozen in liquid N2 and kept at \u221280\u00b0C.Transfected cells were washed with ice-cold PBS plus Roche protease inhibitors, scraped, transferred into Eppendorf tubes and centrifuged for 5\u2005min at 1006 n=3) different litters were pooled, frozen in liquid N2 and lysed in ice-cold lysis buffer using 23\u2005g and 26\u2005g syringes. Adult kidney pieces were frozen in liquid N2, reduced to a fine powder under liquid N2 and further lysed in lysis buffer using a syringe. Lysis buffer contained 15% glycerol, 10\u2005mM Tris-HCl pH 7.4, 150\u2005mM NaCl, 5\u2005mM EDTA, 1% NP40, 3\u2005mM sodium pyrophosphate and 50\u2005mM sodium fluoride, with complete protease inhibitor cocktail (Roche) added before using. Samples were centrifuged for 15\u2005min at 18,894 g at 4\u00b0C, and the supernatant was frozen in liquid N2 and kept at 80\u00b0C. Protein concentration was determined using a Pierce\u2122 BCA Protein Assay kit. Whole-cell extracts containing 20-30\u2005\u03bcg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE . After the proteins were transferred onto a 0.2\u2005\u00b5m nitrocellulose membrane (Bio-Rad) and blocked with TBST , immunoblotting was performed by overnight incubation in TBST 1% skim milk buffer with a rabbit polyclonal antibody against HNF1B (1:500) previously validated (Table\u00a0S7). Positive HNF1B bands were detected by chemiluminescence . Western lightning Plus-ECL Perkin Elmer was used to detect \u03b1-tubulin. Images were captured with G-BOX Syngene Europe, Chemiluminescence Image Capture software and quantified by Image J. WT and HNF1B proteins normalized by \u03b1-tubulin expression were quantified from each litter. A WT sample was assigned a 100% value and the other samples of the same litter WT and heterozygotes were referred to as a percentage of this value. This allowed the comparison of different litters of a given embryonic stage. Statistical significance was determined using unpaired Student's t-test, using Prism 6.00 . P<0.05 was considered significant.The two kidneys of each embryo of at least three . Twenty-four-hour urine samples collected under mineral oil to avoid evaporation were obtained at baseline in individual metabolic cages, after 2-3\u2005days habituation. Each 24\u2005h, animals were weighed and food intake, water intake, urine volume and fecal weight recorded. Blood was sampled by retro-ocular puncture, in general 2\u2005weeks after being in the metabolic cages, and plasma samples were kept at \u221280\u00b0C. The urinary concentration ability was tested after 22\u2005h of water deprivation. Urinary creatinine, urea and electrolytes, plasma urea, creatinine, Mg2+, AST and ALT were measured on an Olympus AU400 Chemistry Analyzer . Osmolality was measured using a vapor pressure osmometer .Urine and plasma were obtained from age- and sex-matched heterozygous and WT mice . They were housed in a light- and temperature-controlled room with t-test or unpaired Student's t-test with Welch correction were used for statistical analysis. P<0.05 was considered significant.Data are presented as mean\u00b1s.e.m. Unpaired Student\u2019s Sp2/+Hnf1b and 18 WT mice from the age of 3, 8, 12 and 17\u2005months. Urine from WT and mutant mice  was collected by spontaneous voiding or after animals were placed individually in metabolic cages as described. The total volume of 24-h urine was aliquoted and frozen at \u221280\u00b0C. A 150-\u00b5l sample of mouse urine was diluted with the same volume of urea buffer . Then, 150\u2005\u00b5l of the urine samples was ultrafiltrated, desalted, lyophilized and resuspended for proteome analysis as described  online coupled to a micrOTOF II MS , as described . For norHnf1bSp2/+ mutants and WT embryos in the C57BL/6N background were microdissected from the same litter. This requirement limited the number of samples used in general to two WTs and two heterozygous mutants, independently of the sex. Note that we found a similar phenotype in males and females. The stages analyzed included E14.5  and at disease stages E15.5, E17.5 and P1. Note that at E15.5 we performed deep sequencing from pooled samples of three WT and three Sp2/+Hnf1b (six kidneys each sample). RNA-seq of E14.5, E15.5 and P1 samples was performed at the Alexander Fleming Institute, Genomics Facility, Greece; RNA-seq of E17.5 samples was performed at Fasteris, Switzerland (Table\u00a0S8).The two kidneys from heterozygous \u00ae mRNA DIRECT\u2122 Micro Kit (ThermoFisher Scientific). mRNA was digested with RNase III, purified, hybridized and ligated to Ion Adaptors (ThermoFisher Scientific), reverse transcribed, barcoded and amplified, using an Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific). RNA-seq was performed on an Ion Proton\u2122 System (ThermoFisher Scientific), according to the manufacturer's instructions. The prepared libraries were quantified and pooled together in duplicates at the required concentration. The pools were then processed on a OneTouch 2 instrument and enriched on a OneTouch ES station. Templating was performed using an Ion PI\u2122 Template OT2 200 Kit (ThermoFisher Scientific) and sequencing with an Ion PI\u2122Sequencing 200 Kit on Ion Proton PI\u2122 chips (ThermoFisher Scientific) according to commercial protocols. The resulting RNA-seq BAM files were analyzed with the Bioconductor package metaseqR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).RNA from microdissected kidneys was extracted by Tryzol, using a Qiagen miRNA mini kit for the extraction of total RNA and miRNAs. The quality of the RNA samples was assessed on an Agilent Bioanalyzer system using an RNA 6000 Nano Kit (Agilent Technologies) and RNA with a Ring higher than 8 was used. Then, 1-2\u2005\u03bcg of total RNA was used for mRNA isolation using a DynabeadsmetaseqR , applyinin vitro samples to generate a library of short inserts (the DNA Colonies Template Library). The library was sequenced on an Illumina HiSeq 2000. For each lane, 130-150 million DNA colonies producing pass filter sequences were assured. The read lengths are 1\u00d750\u2005bp or 1\u00d7100\u2005bp for single-read runs using the forward sequencing primer. The inserts can also be sequenced from both ends using a \u2018forward\u2019 and a \u2018reverse\u2019 sequencing primer, generating paired reads of 2\u00d7100\u2005bp. The data were processed using bioinformatics tools to extract biologically useful information. To homogenize mRNA-seq comparisons, RNA-seq files were all analyzed as described above (High-throughput DNA sequencing using Illumina technology consisted of processing ed above ."}
+{"text": "Photoimmunotherapy is one of the most promising strategies in tumor immunotherapies, but targeted delivery of photosensitizers and adjuvants to tumors remains a major challenge. Here, as a proof of concept, we describe bone marrow mesenchymal stem cell-derived nanovesicles (NVs) displaying anti-PD-L1 antibodies (aPD-L1) that were genetically engineered for targeted drug delivery.The high affinity and specificity between aPD-L1 and tumor cells allow aPD-L1 NVs to selectively deliver photosensitizers to cancer tissues and exert potent directed photothermal ablation. The tumor immune microenvironment was programmed via ablation, and the model antigen ovalbumin (OVA) was designed to fuse with aPD-L1. The corresponding membrane vesicles were then extracted as an antigen\u2013antibody integrator (AAI). AAI can work as a nanovaccine with the immune adjuvant R837 encapsulated. This in turn can directly stimulate dendritic cells (DCs) to boast the body's immune response to residual lesions.aPD-L1 NV-based photoimmunotherapy significantly improves the efficacy of photothermal ablation and synergistically enhances subsequent immune activation. This study describes a promising strategy for developing ligand-targeted and personalized cancer photoimmunotherapy.The online version contains supplementary material available at 10.1186/s12951-022-01266-3. Immunotherapy is a novel strategy for tumor therapy that works via an activated immune system \u20133. HowevPhotoimmunotherapy has shown value versus conventional immunotherapies. Recent studies have shown that photothermal-based immunotherapy can induce tumor cell death via high temperatures while simultaneously activating the tumor immune response \u201316. The Actively targeting nanoplatforms to the tumor microenvironment often requires conjugation of targeting ligands. Chemical covalent linkage of receptors or ligands to deliver photosensitizers or adjuvants may cause a loss of tumor targeting and lead to limited photoimmunotherapy. Antibodies can facilitate immunoregulation and binding to specific markers on tumor cells \u201321. NatuCell membrane-derived nanovesicles (NVs) are biogenic nanocapsule structures obtained by crushing or squeezing selected cell membranes \u201327. NVs To optimize the curative effect of malignant tumors, we describe here a novel method based on cell membrane-derived biomimetic nanovesicles displaying aPD-L1 for photoimmunotherapy , and regulation of the immune checkpoint. Thus, it is an immunotherapy for residual and distal lesions. Accordingly, the model antigen ovalbumin (OVA) served as a tumor-associated antigen and was designed to be co-expressed with aPD-L1 on the membrane of MSCs and the corresponding membrane vesicles. OVA was extracted as an antigen\u2013antibody integrator (AAI) and is a promising multifunctional nanoplatform. With immune adjuvant (R837) encapsulated in AAI (AAI-R837), antigens and immune adjuvants are delivered to antigen-presenting cells (APCs) in a spatio-temporal way for follow-up immunotherapies. AAI can be a carrier for targeted delivery and can also carry antigen signal to APCs to promote the mature differentiation of DCs, thus boosting the immune response to tumors. Afterwards, AAI-R837 can work as a nanovaccine to facilitate immunotherapy. In this treatment strategy, the multi-functional nanoplatform was used in a combined way for photoimmunotherapy. The material achieved satisfactory inhibitory effects on malignant melanomas.A plasmid containing aPD-L1 fragments was first constructed to prepare aPD-L1 displaying MSCs. Then a lentiviral vector was used to infect MSCs extracted from the bone marrow cavity of C57BL/6 embryonic mice with the aPD-L1 displayed on the outside of the cell membrane Fig.\u00a0b. SubseqTo test the binding ability of aPD-L1 NVs to the B16F10 melanoma cells, NVs and aPD-L1 NVs labeling fluorescent dye FITC were incubated with B16F10 cells for 3\u00a0h and then imaged with confocal laser scanning microscopy (CLSM). Significant fluorescence was observed in the periphery of B16F10 cells after being incubated with FITC-aPD-L1 NVs: They were five-fold brighter than FITC-NVs Fig.\u00a0g. These 2, 5\u00a0min) was used to irradiate aPD-L1 NVs-ICG and free ICG at different concentrations; real-time detection used a NIR thermal imager  was implemented on the B16F10 melanoma cells with or without aPD-L1 NVs-ICG. The aPD-L1 NVs-ICG had good photothermal killing effects on B16F10 melanoma cells  test was used to verify the safety of different concentrations of aPD-L1 NVs-ICG on DC2.4 cells  and B16F10 melanoma cells (a representative cancer cell). The aPD-L1 NVs-ICG have no obvious toxic effects on DC2.4 cells and B16F10 melanoma cells Fig.\u00a0d. In addlls Fig.\u00a0e. The sulls Fig.\u00a0f.Inflammation-induced PD-L1 propagates expression in the tumor microenvironment, thus inhibiting the antitumor cytotoxic T-cell response \u201341. TherThe drug distribution in vivo was monitored by an IVIS Lumina II imaging system. After 12\u00a0h and 24\u00a0h of administration, the aPD-L1 NVs-ICG had a significant fluorescence signal at the tumor site, and metabolites changed more slowly in vivo than free ICG and NVs-ICG  was used to irradiate the initial tumor on the left side for photothermal treatment, and the NIR thermal imager was used to monitor the temperature change of tumor site in real time. The results show that tumors treated with aPD-L1 NVs-ICG after laser irradiation, plus the warming capacity of the melanoma itself, led to a surface temperature of 60 \u2103 that could ablate the tumor . The data indicated that the content of CD11c\u2009+\u2009CD86\u2009+\u2009increased compared to PBS-treated groups Fig.\u00a0e. HoweveFurthermore, after being co-incubated for 48\u00a0h, the cell culture medium was collected, and the content of immune-related cytokines  was detected by ELSA Fig.\u00a0f\u2013i. The Encouraged by the results in vitro, we further investigated the effect of AAI-R837 on the immune response in vivo. First, we verified the process of AAI uptake by DCs and migration to lymph nodes in vivo. AAI, free NVs, and OVA labeled with fluorescent dye Cy5.5 were injected subcutaneously into the right leg of mice, and the migration process of drugs in vivo was observed by an IVIS Lumina II imaging system. The injected free Cy5.5 and Cy5.5-OVA rapidly diffused and metabolized in the body although there was a certain concentration of inguinal lymph nodes in the early stage. The product was metabolized quickly over time Fig.\u00a0a. Cy5.5-Next, photoimmunotherapy was done on the tumor. After that, various immunological materials were injected subcutaneously at the root of the tail on 13, 15, and 17 d as a vaccine for immunotherapy on remnant tumors Fig.\u00a0d. After The immune effect after treatment was also studied. Three days after the final immunization, distal tumors, serum, and lymph nodes were collected, and immune cells and cytokines related to the immune response were detected. A cell suspension was prepared by homogenized the material, and the content of mature DCs in lymph nodes, as well as CTLs in tumor was analyzed by FCM Fig.\u00a0g, h. CelIn summary, we constructed AAI that simultaneously express antibodies and tumor-related antigens using the cell membrane of bone marrow mesenchymal stem cells. Nano-vesicles expressing aPD-L1 can specifically bind to PD-L1 receptors on cancer cells and can be utilized to deliver photosensitizers to tumor sites for efficient photothermal therapy. Meanwhile, the aPD-L1 expressed on the modified nanocapsule can also specifically bind to the PD-L1 receptor on the DCs. In short, this design takes advantage of specific binding at the immune checkpoints. This provides a promising strategy for targeted drug delivery. Intriguingly, the immune environment in the tumor region was programmed via photothermal ablation. This led to improved immunotherapy to arrest the residual and distal tumor. Additionally, antibodies and antigens were co-expressed on AAI-R837 and delivered the immune adjuvant to the antigen presenting cells. Antigen signal was sent to the DCs correspondingly and the powerful immune response of the immune system was elicited. The experimental results indicated that the combined treatment of photoimmunotherapy on the melanoma model not only had a significant inhibiting effect on the initial tumor, but also inhibited distant tumor. The low immunogenicity of the materials extended the in vivo circulation time of the nanocarrier, and its biological safety suggests clinical applications.Imiquimod (R837) and Indooyanine gree (ICG) were purchased from Invivogen and BBI Life Science Corporation, respectively. Ovalbumin (OVA), calcein acetoxymethyl ester  and propidium iodide (PI) were purchased from Sigma-Aldrich Inc. FITC was purchased from MedChemExpress LLC and Cy5.5 was purchased from APExBIO Technology LLC. Anti-mouse CD274 , FITC-anti-mouse CD86, PE-anti-mouse CD11c, TITC-anti-mouse CD8 and PerCP-anti-mouse CD3\u03b5 for flow cytometry were purchased from Biolegend Inc. Basement Membrane Matrix was purchased from Becton, Dickinson and Company. TNF-\u03b1, IL-6, IL-12 and IFN-\u03b3 ELSA kit were purchased from Beyotime Biotech Inc.2. To make aPD-L1 be expressed on membrane of MSCs, the aPD-L1 gene with a signal peptide, flag tags, aPD-L1 ScFv and transmembrane sequence were synthesized. While to make aPD-L1 and OVA be co-expressed on membrane of MSCs, plasmids was constructed containing signal peptide, flag tags, aPD-L1 ScFv, transmembrane sequence as well as an intramembrane fragment containing an OVA sequence.DNA sequence of membrane-located signal peptide (from integrin beta 1):ATGAATTTGCAACTGGTTTCCTGGATTGGATTGATCAGTTTGATTTGTTCTGTATTTGGCCAAACAGATAAADNA sequence of Flag tag:GACTACAAGGACGACGACGACAAGDNA sequence of aPD-L1 ScFv:GCCCAGGCCGCCCTGACCCAGCCCAGCAGCGTGAGCGCCAACCTGGGCGGCACCGTGAAGATCACCTGCAGCGGCGGCAGCGGCAGCTACGGCTGGTACCAGCAGAAGGCCCCCGGCAGCGCCCCCGTGAGCCTGATCTACGACAACACCAACAGGCCCAGCGACATCCCCAGCAGGTTCAGCGGCGCCCTGAGCGGCAGCACCGCCACCCTGACCATCACCGGCGTGCAGGCCGAGGACGAGGCCGTGTACTACTGCGGCAGCAGGGACAGCAGCAACGCCGGCAGCGTGTTCGGCGCCGGCACCACCCTGACCGTGCTGGGCCAGAGCAGCAGGAGCAGCGGCGGCGGCGGCAGCAGCGGCGGCGGCGGCAGCGCCCTGACCCTGGACGAGAGCGGCGGCGGCCTGCAGACCCCCGGCGGCGCCCTGAGCCTGGTGTGCAAGGCCAGCGGCTTCACCTTCAGCGACAGGGGCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGCCATCAGCAGGAGGGGCAGCACCACCACCTACGCCCCCGCCGTGAAGGGCAGGGCCACCATCACCAGGGACAACGGCCAGAGCACCGTGAGGCTGCAGCTGAACAACCTGACCGCCGAGGACACCGCCACCTACTTCTGCGCCAAGAACGACGACAGCGTGGGCATCGTGACCACCAGCACCATCGACGCCTGGGGCCACGGCACCGAGGTGATCGTGAGCAGCACCAGCGGCCAGGCCGGCCAGCACCACCACCACCACCACGGCGCCTACCCCTACGACGTGCCCGACTACGCCAGC.DNA sequence of transmembrane fragment:TTATGGGTCATCCTGCTGAGTGCTTTTGCCGGATTGTTGCTGTTAATGCTGCTCATTTTAGCACTGTGGDNA sequence of OVA:ATGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAGATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAG.AGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCTTAA.Bone marrow mesenchymal stem cells were extracted from the bone marrow of suckling C57BL/6 mice (Seven days after birthed) referring to the established method . The micThen, Lentivirus vector encoding corresponding sequence was applied to infect MSCs. Infected MSCs displaying aPD-L1 with or without OVA were cultured in C57BL/6 mouse bone marrow mesenchymal stem cell complete culture medium maintained in 20% FBS and 1% Penicillin and streptomycin.6 in a petri dish, the medium in the culture dish with MSCs was removed, then the MSCs were scraped away by cell scraping, and were collected into the centrifuge tube with phosphate buffer solution (PBS). After 5% deoxysodium cholate, a surfactant, was added to the cell suspension (v:v\u2009=\u20091:100), MSCs were broken by a low power ultrasound  for 10 to 20\u00a0s using the ultrasonic crusher , and the protease inhibitor PMSF (10\u00a0mg/mL) was added (v:v\u2009=\u20091:200) to worked solution immediately. To remove the cytoplasm and nucleus, the suspending liquid was centrifuged at 3500\u00a0rpm (4\u00a0\u2103) for 5\u00a0min. Supernatant was collected for further centrifugation at 15,000\u00a0rpm(4\u00a0\u2103) for 20\u00a0min to acquire nanoscale membrane vesicles, then resulting vesicles were quantified by BCA kit (Beyotime Biotech Inc.) and dissolved in PBS and stored at 4\u00a0\u2103 for standby application.In the preparation of nanovesicles, when the number of cells reaches 5\u2573106 MSCs, the ultrasonic crushing instrument (20 w) was used for ultrasonic concussion for three times at 4\u00a0\u2103, each session lasts 5 to 10\u00a0s and interrupted at least 10\u00a0s. After resulting sample was centrifuged at 12,000\u00a0rpm (4\u00a0\u2103) for 20\u00a0min, the content of ICG in the supernatant was detected by a microplate analyzer, and the loading efficiency of ICG was calculated. NVs loading drugs were dispersed in PBS and stored at 4\u2103 for further study. When loading the immune adjuvant, 100 \u03bcL R837 (0.5\u00a0mg/mL) was added to NVs suspension, and the loading was carried out according to the same method mentioned above. The loading efficiency of R837 was determined by HPLC (Waters 1525)-UV visible detector at 325\u00a0nm. Acetonitrile was used as the mobile phase. ICG encapsulation and the UV absorption spectra of NVs-ICG were measured by microplate reader.After 200 \u03bcL ICG aqueous solution (1\u00a0mg/mL) was mixed with NVs suspension come from 5\u00d7102) for 5\u00a0min. During the heating of each solution, an NIR thermal imaging camera was used to record the temperature changes.The expression of aPD-L1 on the MSCs membrane was verified by immunofluorescence. The morphology and structure of NVs were characterized by fetenay transmission electron microscopy (F20). The dynamic particle size and electric potential of NVs was measured with Zeta sizer Nano-ZS . The existence of aPD-L1 on the NVs was detected with coimmunoprecipitation (CO-IP) assay and Western blot (WB) analysis. In order to study photothermal performance of acquired materials, aPD-L1 NVs-ICG and ICG with different concentrations were dispersed in ultrapure water, then the samples were irradiated with 808\u00a0nm laser (1\u00a0W/cm4 cells/well) and cultured for 24\u00a0h. FITC-labeled aPD-L1 NVs or NVs were incubated with the cancer cells for 3\u00a0h, and cells in the other group were incubated with antiPD-L1 antibodies (10\u00a0\u03bcg/mL) for 2\u00a0h before the addition of aPD-L1 NVs to the culture medium. The NVs that were not bound to the cells were washed with PBS for three times, then the nuclei were stained with DAPI for 15\u00a0min. After the excess dye was washed and added new medium to the wells, confocal microscopy was performed under a confocal laser scanning microscope (LSM780) with 60\u2009\u00d7\u2009oil-immersion lenses. The binding of AAI to DC2.4 cells was observed by the same method.To exam the ability of aPD-L1 NVs to binding to tumor cells, B16F10 cells were seeded in confocal culture dish (3\u2009\u00d7\u2009104 cells/well) for 24\u00a0h, respectively. After the cells were washed with PBS for three times, fresh medium containing different concentrations of aPD-L1 NVs-ICG and AAI-R837 was added for training another 24\u00a0h. The cell relative viability was detected by MTT assay and the cytotoxicity of the nanomedicine was determined.DC2.4 cells and B16F10 cells were selected to determine the cytotoxicity of the nanomedicine by standard MTT assay. DC2.4 cells and B16F10 cells were cultured in 96-well plates (1\u2009\u00d7\u2009104 cells/well) for 24\u00a0h. After washing the cells with PBS for three times, fresh medium containing 100\u00a0\u03bcg/mL of aPD-L1 NVs-ICG was added. Treated cells were irradiated with 808\u00a0nm laser (1.0\u00a0W/cm2) for 5\u00a0min. Viability of B16F10 cell was then assessed using standard MTT procedure. In order to intuitively evaluate the relative viability of B16F10 cells after different treatments, 2\u00a0\u03bcmol/L calcein acetoxymethyl ester  and 4 \u03bc mol/L propidium iodide (PI) were used to stain the living and dead cells for 15\u00a0min, respectively. After washed with PBS, the cells were observed with inverted fluorescence microscope .To study the effect of photothermal therapy on B16F10 Cells with aPD-L1 NVs-ICG, B16F10 cells were cultured in 96-well plates(1\u2009\u00d7\u200910Bone marrow derived dendritic cells (BMDCs)were extracted from the bone marrow of 6\u2009~\u20098\u00a0weeks old C57BL/6 mice according to the established method . BMDCs w50\u00a0\u03bcg of ICG, 100\u00a0\u03bcg of NVs-ICG and aPD-L1 NVs-ICG (containing 50\u00a0\u03bcg of ICG) dispersed in 100 \u03bcL PBS were injected into subcutaneous melanoma burdened C57BL/6 mice via tail vein. Fluorescence imaging system  was used to obtain fluorescence images at 0, 6, 12, 24 and 48\u00a0h after injection, respectively. At 48\u00a0h, the main organs and tumors of the mice were collected, and fluorescence imaging and intensity records were collected.NVs, OVA and AAI were labeled with Cy5.5, subsequently, and 50 \u03bcL normal saline with equivalent 10\u00a0\u03bcg of OVA or approximately 50\u00a0\u03bcg of vesicular proteins injected into unilateral foot pad of C57BL/6 mice. Fluorescence imaging in vivo was obtained carrying out the system of fluorescence images at different time points . The left and right inguinal lymph nodes of all mice were collected at 96\u00a0h after injected, fluorescence images were obtained and the fluorescence signal intensity was calculated using the imaging system.6 OVA-expressing B16F10 cells were suspended in PBS and subcutaneously injected into the left posterior back of C57BL/6 mice to challenged primary tumor. On 6 d, 3\u2009\u00d7\u2009106 OVA-expressing B16F10 cells were subcutaneously injected into the right posterior back of C57BL/6 mice to challenged distant tumor. On 9 d after first inoculation, primary tumor was about 200 mm3 in size, various formulations were injected into the tail vein containing equivalent 4.5\u00a0mg/kg of ICG or approximately 100\u00a0\u03bcg of vesicular proteins (five mice per group). After 12\u00a0h, the primary tumor was irradiated by 808\u00a0nm laser (0.75\u00a0W/cm2) for 8\u00a0min, and the temperature of tumor surface was monitored by near-infrared thermal imaging instrument. On 13, 15 and 17 d after challenge of the first tumor, mice treated with photothermal therapy were subcutaneously injected with different immune drugs containing equivalent 10\u00a0\u03bcg of OVA or 10\u00a0\u03bcg of R837 or approximately 50\u00a0\u03bcg of vesicular proteins for immunotherapy of distant tumor (five mice per group). The body weight of the mice was recorded using electronic balance, and the tumor size and volume were measured using a vernier caliper every other day. The calculation formula (V) was V\u2009=\u2009d2*D/2, d and D was mm in the shortest and longest diameters of the tumors respectively. Animals were euthanized when they showed signs of impaired health or tumors larger than 2cm3.Female C57BL/6 mice (6\u2009~\u20098\u00a0weeks) were purchased from Shanghai slake laboratory animal co., LTD and used according to the agreement approved by committee of Xiamen University Laboratory Animal Center. The mice were randomly divided into groups. 3\u2009\u00d7\u200910To test the immune response in the treated mice, the mice were sacrificed and serum, tumor, and lymph nodes were collected on 3 d after photothermal treatment or final immunization. The levels of TNF-\u03b1, IL-6, IL-12 and IFN-\u03b3 in serum were detected by ELISA kit according to the instructions. The tissues were homogenized to prepare single-cell suspension, and tumor cells were labeled with anti-mouse CD3a and CD8 antibodies, and lymph node cells were labeled with anti-mouse CD11c and CD86 antibodies. Then, the content of CD8\u2009+\u2009T lymphocytes and mature DC cells were detected by flow cytometry.The main organs  and tumor tissues of mice in each treatment group were collected and fixed in 4% paraformaldehyde. Hematoxylin and eosin staining were performed before electron microscopy.All the statistical figures are mean\u2009\u00b1\u2009standard deviation, P values were calculated by Student's t test . All statistical analyses were performed using the IBM SPSS statistics 20.Additional file 1: Figure S1. (a) The standard curve of R837 was detected by HPLC; (b) The standard curve of ICG was measured by ultraviolet spectrophotometer; (c) Analysis of drug loading efficiency using nanometer delivery platform. Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808\u00a0nm wavelength . Figure S2. The temperature change curve of the aPD-L1 NVs-ICG and ICG with corresponding concentration under the near infrared (NIR) laser irradiation at 808\u00a0nm wavelength . Figure S4. (a) Temperature change curve of the tumor under different treatments under 808\u00a0nm laser irradiation . (b)Tumor volume growth curve for irradiated distant secondary tumors (in right flank) in mice after PTT with different drugs. (c) Survival curves of the mice after PTT with different drugs. Images of Hematoxylin and eosin (H&E) staining of irradiated primary tumors and distant secondary tumors sections after various treatments, respectively. Scale bar: 100\u00a0\u00b5m. (b)Weight curve after PTT with the different substances. Images of H&E staining of the main viscera after PTT, respectively. Scale bar: 100\u00a0\u00b5m. Figure S5. Productions of IL-6 (a), IL-12 (b), TNF-\u03b1 (c) in serum from the mouse determined by ELISA after treated with PTT for 3 d. P values were calculated by Student's t test . Figure S6. Cell viability of the different concentrations of AAI-R837 on B16 cells and DCs after co-incubation for 24\u00a0h. Figure S7. The curve of fluorescence signal intensity in inguinal lymph nodes with time after subcutaneous injection of various drugs. Figure S8. (a) Images of Hematoxylin and eosin (H&E) staining of distant secondary tumors sections after photoimmunotherapy, respectively. Scale bar: 100\u00a0\u00b5m. (b) Weight curve after photoimmunotherapy with the different substances. (c) Images of H&E staining of the main viscera after photoimmunotherapy, respectively. Scale bar: 100\u00a0\u00b5m."}
+{"text": "Clubionazilla-group is a relatively small species group, distributed exclusively in East Asia, with only three species clearly documented so far.The Clubionahooda Dong & Zhang, 2016, which was previously placed in the C.trivialis-group, is assigned to the C.zilla-group in the present paper. A new spider of the C.zilla-group from Jiugong Mountain in China is described under the name of C.jiugong sp. nov. Detailed descriptions and photographs of the new species are provided. Clubionazilla D\u00f6nitz & Strand, 1906 was reported by C.zilla-group, was established to accommodate C.zilla. The C.zilla-group was rede\ufb01ned by Anaclubiona by Anaclubiona was rejected by zilla-group. The genus Anaclubiona Ono, 2010 is currently considered as a junior synonym of Clubiona by The female of zilla-group can be easily recognised by its tiny body (with body length 1.8\u20133.6 mm), in association with the characteristic genital organs. The male palp has a developed and occasionally branched embolic apophysis. The female epigyne has a pair of guide pockets (or hoods) or a transverse hood near the copulatory openings. The zilla-group is a relatively small taxon, with only three species having been clearly documented: C.zilla widespread in Japan, C.minima  endemic to Honshu in Japan, C.tanikawai Ono, 1989 from Ryukyu Is. in Japan and Hunan and Taiwan in China. C.hooda Dong & Zhang, 2016 was assigned to the trivialis-group in the original publication  and both sexes possess certain characters associated with the zilla-group. Therefore, it is very likely they are the opposite sexes of the same species. Based on that, as well as the DNA barcoding data, we matched the female and male together. This species is new to science and is described under the name of C.jiugong sp. nov. The aim of the current paper is to describe the new species, providing detailed morphological descriptions and illustrations.While examining spiders collected from Jiugong Mountain, Hubei Province, China Fig. A, we fouSpecimens in this study were collected by hand collecting from leaf-litter in Mt. Jiugong, Hubei. Spiders were fixed and preserved in 95% ethanol. Specimens were examined with an Olympus SZX7 stereomicroscope, details being studied with an Olympus CX41 compound microscope. Female epigynes and male palps were examined and illustrated after being dissected. Epigynes were removed and cleared in warm lactic acid before illustration. The vulva was also imaged after being embedded in Arabic gum. Photos were made with a Cannon EOS70D digital camera mounted on an Olympus CX41 compound microscope. The digital images were taken and assembled using Helifocus 3.10.3 software package . The disA DNA barcode was also obtained for the species matching. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced for two specimens, using the primers LCOI1490 (5\u2019-GGTCAACAAATCATAAAGATATTG-3\u2019) and HCOI2198 (5\u2019-TAAACTTCAGGGTGACCAAAAAAT-3\u2019). For additional information on extraction, amplification and sequencing procedures, see All measurements were obtained using an Olympus SZX7 stereomicroscope and given in millimetres. Eye diameters are taken at the widest point. The total body length does not include chelicerae or spinnerets length. Leg lengths are given as total length . Most of the terminologies used in text and figure legends follow All specimens are deposited Museum of Guizhou Education University, Guiyang, Guizhou, China .Yu & Zhongsp. n.9B74C4BF-3DDC-5DB0-98D9-7D1EDCFDB51436f4d405-32d2-4119-97b6-9c3758c20f22Type status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0274; individualCount: 1; sex: male; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020606GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 3; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenType status:Holotype. Occurrence: recordedBy: Qianle Lu; individualID: YHCLU0275; individualCount: 1; sex: female; lifeStage: adult; behavior: foraging; preparations: whole animal (EtOH); associatedSequences: MZ020605GenBank: ; Taxon: order: Araneae; family: Clubionidae; genus: Clubiona; specificEpithet: jiugong; scientificNameAuthorship: Yu & Zhong; Location: continent: Asian; country: China; countryCode: CHN; stateProvince: Hubei; county: Tongshan; locality: Jiugongshan Nature Reserve; decimalLatitude: 29.39; decimalLongitude: 114.65; Identification: identifiedBy: Hao Yu; dateIdentified: 2020-07; Event: samplingProtocol: by hand; samplingEffort: 10 km by foot; year: 2020; month: 7; day: 4; Record Level: institutionCode: MGEU; basisOfRecord: Preserved SpecimenMale 5'CTTGATCTGCTATAGCAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTAGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCTATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCTCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTGTTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGATCCAGCTGGAGGGGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' 5'TTTGATCTGCTATAGTAGGAACAGCTATAAGTGTTATAATTCGTATAGAATTGGGACAATCTGGAACATTTTTAGGAGATGATCATTTATATAATGTAGTAGTTACAGCTCATGCTTTTGTTATAATTTTTTTTATAGTAATACCAATTTTAATTGGAGGTTTTGGAAATTGAATAATTCCTATGATATTAGGAGCAGCTGATATAGCTTTTCCTCGTATAAATAATTTAAGTTTTTGATTATTACCCCCTTCGTTATTTATATTATTTATATCTTCTATAGCTGAAATAGGTGTGGGAGCAGGGTGAACTATTTATCCTCCTCTTGCATCTAGTATAGGTCATACAGGAAGAGCTATAGATTTTGCTATTTTTTCGTTACATCTAGCTGGAGCTTCTTCTATTATAGGGGCTGTAAATTTTATTACTACTATTATTAATATACGATATATTGGGATGAGAATAGAAAAAGTTCCATTATTTGTTTGGTCTATTATAATTACTGCAGTACTCTTATTATTATCATTACCTGTATTAGCAGGTGCTATTACTATATTATTGACTGATCGAAATTTTAATACATCTTTTTTTGACCCAGCTGGAGGAGGAGATCCTATTTTATTTCAGCATTTATTTTGATTTTTTGG3' , but is consistently separable by its genitalia. Male of the new species resembles that of C.hooda (C.hooda) and by the EPA originating from the prolateral portion of the tegulum, pointed to the retrolateral side  and differ from C.zilla by: (1) copulatory openings closely spaced and partly fused, situated at the medial portion of epigynal plate posterior margin ; (2) the proximal half of copulatory ducts close together  near the copulatory openings in female (C.zilla-group species with tiny bodies (less than 3.6 mm), C.trivialis-group is median sized clubionids . Although we have not examined the types of C.hooda, the species was well described with high-quality illustrations: developed embolar part apophysis in male and the paired epigynal hoods in female . The present study follows Anaclubiona as a synonym of Clubiona, rather than resurrecting the generic status of the zilla-group. Consequently, we temporarily place the new species in Clubiona sensu lato and assign them to C.zilla-group.The genus"}
+{"text": "Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a  Saccharomyces cerevisiae (ATR in humans)  . Mec1 th humans) . In addi humans) .rad53 or mec1 mutant cells , and finally firing factors, DNA polymerases, and accessory factors were added to initiate DNA replication. We followed replication progression by separating the products on alkaline agarose gels to visualise incorporation of radiolabelled dCTP. Rad53 inhibits late origin firing in vivo by phosphorylating two substrates: Dbf4 and Sld3 . To detely shown , whilst ly shown . As showly shown . Howeverly shown .To test whether Rad53 also inhibited Sld3, we took a similar approach by pre-incubating Sld3/7 with Rad53 prior to addition to the replication reaction. Similar to DDK, pre-incubation of Sld3/7 with Rad53 and ATP resulted in reduced mobility of Sld3 in SDS-PAGE  and inhiNext, we wanted to determine whether Rad53 could affect replication elongation. We pre-incubated the elongation factor mix  with Rad53 and ATP, then added this to reactions after MCM loading, DDK phosphorylation, and firing factor addition. We stopped the reactions at early time points so that any effects of elongation could be more easily seen by the size of the leading strand replication products. KD  are non-essential proteins known to directly stimulate replication fork rate . Their iKD , indicatecreased . This leecreased . These dKD was detected in Mrc1 immunoprecipitates  as a measure of CMG helicase activity, Devbhandari and Remus have recently shown that more U* product is generated in reactions containing M/C/T than in reactions lacking M/C/T . This co17AQ protein, which cannot be phosphorylated by Mec1  that indeed exhibited faster replication than wild-type Mrc1 after Rad53 phosphorylation . Unfortunately, these mutants \u2014 especially Mrc119A \u2014 did not stimulate replication to the rate of unphosphorylated wild-type Mrc1 . This is likely due, in part, to the fact that these mutants exhibited defects in promoting faster replication in the absence of Rad53 indicating that they are not completely functional . Moreover, even Mrc119A was still inhibited slightly by incubation with Rad53 (compare lanes 5 and 6), suggesting that additional sites not mutated in this construct can still be phosphorylated by Rad53 and inhibit Mrc1 function. Cells expressing 19AMRC1 as the sole copy of MRC1 were not sensitive to the replication stress agent hydroxyurea (HU)  . Another8D) was unable to stimulate replication even in the absence of Rad53  were identified in a phosphoproteomics screen of MMS-treated cells , whilst the mrc1\u2206, rad9\u2206 double mutant was completely defective in Rad53 phosphorylation (lanes 12\u201315). mrc1\u0394 rad9\u0394 mutants are inviable  or MMS (0.006%), although they did not promote additional survival to higher concentrations (8 mM HU and 0.01% MMS)  relative to MRC1 wild type  . Similar01% MMS)  arguing 1\u0394 cells . MRC18D ild type . These dOur results show that, in addition to its role in checkpoint activation upstream of Rad53, Mrc1 also has a role downstream of the checkpoint, as a substrate of Rad53. Phosphorylation of Mrc1 by Rad53 prevents Mrc1-dependent stimulation of CMG unwinding, leading to a reduced replication fork rate. We suggest that linking these two roles could allow Rad53 to slow replication speed specifically at a stressed or damaged fork, and, therefore, act efficiently at stochastic fork stalling events without initiating a global checkpoint response. But under more severe replication stress, where more Rad53 is active, Rad53 could phosphorylate Mrc1 at all forks to slow replication globally.Using a novel assay to measure DNA unwinding activity, we found that unwinding by CMG is not very synchronous, as evidenced by shallow unwinding curves. Moreover, sites as close as 1 kb from the origin were not completely unwound after 50 min. In the presence of Mrc1, unwinding was faster and the curves were steeper, suggesting more synchronous unwinding leading to even the site 2 kb from the origin approaching 100% unwinding by 30 min. In single-molecule experiments, CMG frequently paused and backtracked while unwinding DNA . The abi8D mutant showed normal checkpoint activation supporting the idea that it remains bound to replication forks. However, we note that Rad53 can target more than these eight sites and full phosphorylation may affect more functions and protein-protein interactions than those disrupted in the Mrc18D mutant.A recent structure of CMG bound to M/C/T suggests the C-terminus of Mrc1, containing the majority of its Rad53 phosphorylation sites, may contact Cdc45 and Mcm2 . Other wmrc1\u2206 cells treated with HU, suggesting that Mrc1 has some role in restraining CMG at stalled forks . The peptides were synthesised using FMOC for temporary \u03b1-amino group protection. Protecting groups used were Pbf for arginine, OtBu for glutamic acid and aspartic acid, Trt for asparagine, glutamine, histidine, and cysteine, tBu for serine, threonine and tyrosine, and Boc for lysine and tryptophan. Each amino acid was coupled by activating its carboxylic acid group with DIC in the presence of HOBT. Individual aliquots of amino acids were spotted on to a cellulose membrane which has been derivatised to have 8 to 10 ethylene glycol spacers between the cellulose and an amino group. Synthesis was accomplished by cycles of coupling of amino acids, washing then removal of the temporary \u03b1-amino protecting group by piperidine followed by more washing. Once the required number of cycles of coupling and deprotection and washing had been completed, the membranes were treated with a solution of 20 ml containing 95% TFA, 3% TIS, and 2% water for 4 hr. Following this treatment, membranes were washed four times with DCM, four times with ethanol, and twice with water to remove side chain protecting groups and TFA salts and once again with ethanol for easier drying. Just prior to kinase assay, membranes were washed extensively in reaction buffer, then incubated with 80 nM Rad53, 10 \u00b5M ATP, and 0.02 \u00b5Ci/\u00b5l \u03b3exo- , 20 nM GINS, 15 nM CDK, 20 nM Csm3/Tof1, 20 nM Mrc1, 20\u201330 nM Sld3/7, 20 nM Mcm10, 20\u201350 nM Sld2, and 350 nM RPA. At each time point, 2.5 \u00b5l of reaction was added to a tube containing 5 \u00b5l replication buffer and 1 \u00b5l MseI (NEB) for 3 min, and then the reaction was quenched with the addition of EDTA. Samples were then deproteinated with SDS and proteinase K followed by column clean-up (QIAquick PCR purification kit) according to manufacturer\u2019s instructions. The final elution was done with 300 \u00b5l 10 mM Tris pH 8. Then qPCR was performed in triplicate using 4 \u00b5l sample in 8\u20139 \u00b5l reaction with FastStart Universal SYBR Green Master Mix (Roche) and primers flanking each MseI cassette  was then fit with a spline and normalised to the unwound DNA in the last timepoint of the closest site (200 bp from origin). The unwinding rate was then calculated by integrating the spline and averaging over the last three sites .AGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGGACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGGGGAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTGGCCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTCGATTTTTTTATGTTTAGTTTCGCGGACGACGGTTTCGAGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGAGGCGGTGTTCGCCGAGATCGGCTCGCGCAAAGCCGAGTTGAGCGAACTAAACATAAAAATACAGCATCAGATGGTAGGCCTCCTGGCGCCGCACCGGCCTCAGCATCCGGTACCTCAGCTGGCCACATCACTGTCTTTCTTATGACGGTACTACCGGTGTTCACTGCACCAAGGTAACACTCATTAAATTAAGGTTAAATTAATCTACACAATTCTCTTTTGCTATTGGTACCGGATTCTCCAGCTCTGACTTCAGCGTCTCTGAAGGAATCTTTGCAGGTGCTTACGCTTACTACCTAAACTACAATGGTGTTGTCGCTACTAGTGCCGCTTCTTCAACCACTGGATCTGGTCCTAGGGCTTCGGTCCGCCCCTACTTCAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAATTCGGTTAAGTTAAGTTAAGGTTAAAGAAGCTAACGCCAGGGTTTTCCCAGTCACGATTCGGTGCTTCCGTTACCGGTTCAACTGCTTCCACTTCATGGGCTACTTTTTGGACCGGAACGGCTGGTACTATCGGCCTGGTATCATCCTTTACCGAAGCAACATCTGTTTACACTACAACACTAGACCAAGCACAGTCGTAGTTTCTTGTTCAGAGATGACTCCAATGGTAACGTCTATACCATTACCACAATCATTAAGTTAAATTAAGTTAATCAAACCGTTCCATGCTCATCCACTACCGCCACTATTACTTCTTGTGATGAAACTGGATGTCACGTTAGTACATCAACCGGTGCTGTTGTAACTGAAACCGTTTCTTCCAAGGCATACACAACTGCCAAAGTAACTCGTTGTGACGCCAATCAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCATGGTTAAAGTTAACTTAAATTAAGGTACGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAGGCTCAATGTAACTTAGCCACTGTCAATTGGGAATGTTCCAGGGATTCATGGACAACAACTGCAACTGGAGTATCATACACCACTGTCACCGTAACCCACTGTGACGACAATGGCTGTAACACCAAGACTAAGCTCCTGAAGCTACCACCACAACTATCGCCCACCAGGACCACCGTCACCTTTAGTGATGACAATGAAGGTAAGACCTTGGGTGAGTCTGGTCCAGCGGAGGGCCACTACTGTTTCTCCAAAGACATACACCACCGCTACTGTTACTCAGGGGGATAAAAATGCCTGCCTCACCAAGACTGTCACTTCTGAATGTCCTGAAGAAACTTCAGCAACTACTACTGTCACTTCTGAGGGTTCTAAAGCAACCTCATTGAGTCGACGCGGGGGCGACGATTAACCTTAACGTTAAGTTAAGCTAGCACGACTACGCATCCCTCTGACTACTTCTCGGGGTGGGACTATACTGGTACCGATACGGGCTGTGATGACAACGATGTGTAGAACTGGGACAATCAGATCTGAGGCCCCTGAAGCCACAACGGGTACTGTTTCTAACAACAGATACAACATGGAGGGCCAACATTGTCACAATAGAAGCTCCGCCAGAAACAGTAGAAACTTCAGAAACCAGTGCTGCCCCTAAGGACATACACTACTGCCACTGGTTACTCAATGGTTTAGAGGGTGGTTGCCACGTCAAGATAATCACCTCTAAAATACCTGAAGCTACTTCAACCGTCACGGGTGCTTCTCCAAAACGGCCTTACATAGCCGGATACAGTGACTTTGACAGGTTTGCGGGGCACAGCAATGACTTGCATAGCTGCGTGCGGGGGAAGGAACTCTTGCGTCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCATTATGGTTAACTTAAGTTAATTTAAGCTATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAGTGAGTATCTCTGCTTGACGACCCCTTGGCGCAGAGGTGCTGGCCGCGTGCTAAGTTGAAGCGGCTGCACTGCTGCAAGGTCCGTCACGGAGGCGTCGGACCGGCAGGAGCACTAGCCCATCGACCCGTACGGGAACACTCTATATCGCTCTCGGACGGACATTCTGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTGGAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG.600 0.5 and arrested with 20 \u00b5g/ml of alpha-factor for 2\u20133 hr at 25\u00b0C. Cells were washed two times with YPD and then resuspended in YPD + 200 mM HU. Cells were then harvested at the indicated times, and protein was extracted with 10% trichloroacetic acid. Extracts were then processed by 3\u20138% Tris-acetate SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Flag  and anti-Rad53  antibodies.Log-phase yeast cultures in YPD were diluted to OD600 0.3 and arrested with 20 \u00b5g/ml of \u0251-factor for 3 hr at 25\u00b0C. Cells were washed two times with YPG and then resuspended in YPG + 200 mM HU + 200 \u03bcg/ml BrdU. After 1 hr, cells were washed two times with YPG and then resuspended in YPG + 2 mM thymidine. Cells were harvested at the indicated times, and DNA was extracted with phenol-chloroform. DNA was run on a 0.8% alkaline agarose gel, then alkaline transferred to a positively charged nylon membrane with the VacuGene XL Vacuum Blotting System (Amersham), and immunoblotted with anti-BrdU antibody .Log-phase yeast cultures in YP-raffinose were centrifuged and resuspended in YP-galactose to ODK227A,K339A was constructed by PCR with mutated oligos on pET21b-RAD53  and the 3xFLAG tag between the BamHI and NotI sites of pRS40N and then was cut with Blp1 to modify the already integrated codon-optimised MRC1 at the his3 locus. pVP14 (MRC117AQ) contains all S/T residues followed by Q sites mutated to A as in 14A) contains the following mutations: S911A, S918A, S920A, T952A, S957A, T996A, T997A, S1006A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM25 (MRC119A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, T952A, S957A, S958A, T996A, S997A, S1006A, S1010A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A. pAWM18 (Mrc141A) contains the following mutations: T882A, S911A, S918A, S920A, S924A, T932A, S937A, T952A, S957A, S958A, S961A, T963A, S965A, T967A, S969A, T970A, T971A, S972A, T974A, T977A, T996A, S997A, S1006A, S1010A, S1013A, T1015A, T1027A, S1033A, T1036A, S1039A, S1040A, S1043A, T1045A, T1050A, T1060A, T1063A, T1079A, T1081A, S1083A, S1089A, S1093A. pAWM17 (Mrc18D) contains the following mutations: S911D, S918D, S920D, T952D, S957D, T996D, S997D, S1006D. pAWM47 and pAWM48 contained base pairs 1424\u20133288 of MRC1 between XhoI and BamHI and 127 bp of the 3\u2019 UTR of MRC1 between BamHI and NotI. The 3xFLAG tag was added with flanking BamHI sites, and the plasmids were integrated into yeast after cutting with XbaI. The template used in the CMG helicase assay was made by modifying a plasmid with a pBlueScript vector and contained a synthetic origin with either two ORC binding sites 70 bp apart (pAWM36) or no ORC binding sites (pAWM37) (Yeast strains are listed in 1b-RAD53 . Mrc1 fr53 (MRC1  and modi(pAWM37)  and a sy public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:S. cerevisiae  in blocking both initiation of DNA replication and replication fork progression via inhibition of Mrc1 activation of the replicative DNA helicase. The paper elegantly utilizes the power of biochemical reconstitution of complete DNA replication with purified proteins and its regulation by checkpoint kinases.This paper addresses an important role for the DNA replication checkpoint kinase Rad53 in the yeast Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Bruce Stillman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Oscar M Aparicio (Reviewer #3).Thank you for submitting your article \"Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.We all felt that this is a very strong paper that confirms the effect of Rad53 kinase on Sld3 and Dbf4 activities in initiation of DNA replication, but goes further and identifies Mrc1 and Mcm10 as substrates for the kinase in regulating replication fork progression and CMG activity. The results are clear and support the conclusions.Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try. Essential revisions:The reviewers made a number of suggestions, but only a subset of these are listed below and we ask that you consider adding the requested data.(1) Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.KD  does not slow replication. However, we have now included new data  showing that neither wild type Rad53 nor Rad53KD co-immunoprecipitate appreciably with Mrc1. Under the same buffer conditions \u2014 identical to the buffer conditions in the replication reactions \u2014 Csm3/Tof1 co-immunoprecipitate nearly stoichiometrically. We think that, together, these results indicate that Rad53 inhibition of Mrc1 is likely via phosphorylation and not binding. We have added this point in the section \u201cRad53 inhibition of replication elongation via Mrc1 and Mcm10\u201d on page 6.The reviewer correctly points out that Rad53 might inhibit Mrc1 by binding and not just by phosphorylating Mrc1, as has been suggested for another Rad53 target, DDK . We think this is unlikely because pre-incubation of Mrc1 with Rad53(2) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? It would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1 and cause the same phenotype.It is, of course, very difficult to exclude the possibility that some other kinase(s) may also inhibit Rad53. Nonetheless, we think this is an interesting point, especially given the fact that DDK has been implicated in Mrc1 regulation in fission yeast. We, therefore, tested whether the two main replicative kinases, DDK and CDK, can phosphorylate and inhibit Mrc1. In a new Figure 2 \u2014figure supplement 1B we show that neither DDK nor CDK were able to inhibit Mrc1 in replication elongation. We have added this point to section \u201cRad53 inhibition of replication elongation via Mrc1 and Mcm10\u201d on page 6.(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression in vivo to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Drosophila melanogaster deoxyribonucleoside kinase (dmdNK) and the human equilibrative nucleoside transporter (hENT1) which together allow efficient BrdU incorporation and rapid chasing of BrdU from nucleotide pools . In these experiments, cells are released from \u03b1 factor into HU in medium containing BrdU. This results in a smear of short, labelled replication intermediates from early firing origins on alkaline agarose gels detected by \u2018western\u2019 blotting with anti-BrdU antibody. We can then estimate fork rates from the extension of this labelled smear to larger sizes after HU is washed out and BrdU is replaced with thymidine. We have done this in +RAD53 cells carrying wild-type MRC1 or 8D.MRC1 The replication intermediates of 8DMRC1 cells were shorter and were extended at a slower rate consistent with the idea that 8DMRC1 promotes slower replication forks in vivo . We have added this to the \u201cMrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity\u201d section on page 11. Unfortunately, this approach requires synchronisation with HU, and therefore cannot be used with rad53 mutants because of the extensive fork collapse and continued origin firing in HU . Rad53 becomes rapidly dephosphorylated after release from HU, so the latter part of the curve in the new Figure 6E represents forks moving in the absence of active Rad53. Although we recognise this does not exactly address the reviewer\u2019s question, it is probably the best we can do at the moment, and it shows that the 8D mutant does indeed have slower forks in vivo.This is a good point, but not a trivial request. We include new data that we believe at least partially addresses this issue. We previously showed that we could do \u2018pulse-chase\u2019 experiments in vivo using yeast strains that express the Reviewer #1 (Recommendations for the authors):1. Page 10 and Figure 5E. The observation that Mrc119A can still be inhibited by Rad53 kinase, albeit less than the wild type Mrc1 protein, might suggest (1) that there are additional phosphorylation sites (as suggested by the authors) or (2) that Rad53 binds directly to Mrc1 and this interaction may partially block its stimulation of CMG. Can the authors test if the Rad53 binds to purified Mrc1? The reason for asking this question is that it is possible that both Mrc1 and Rad53, which are both known to be located at active DNA replication forks, create a solid-state system, feedback control at the replication fork for Mec1 activating Rad53 kinase and Rad53 phosphorylating Mrc1.Addressed in Essential revisions.Reviewer #2 (Recommendations for the authors):The key point that needs to be addressed, in my opinion, is whether the phenomena identified in the biochemical studies are reflective of the in vivo situation. I offer below a few comments that should help addressing this issue.(1) How selective is the effect of Rad53 on the Mrc1-dependent replisome slowdown in the biochemical assays? I ask this since recombinant Rad53 is highly active and it would be useful to exclude the possibility that any active kinase could phosphorylate Mrc1. Alternatively, does the effect of Rad53 on in vitro DNA replication dependent on its two FHA domains?Addressed in Essential revisions.(2) While I do have sympathy with the authors for their struggle in identifying the key Rad53 target sites on Mrc1, I feel they should nevertheless provide evidence that one or more of the sites mutated in the Mrc1-8D variant are indeed phosphorylated in cells, in a Rad53-dependent manner, in response to DNA replication stress.8D  have been detected by proteomics from cells treated with MMS  showing they are indeed phosphorylated in response to replication stress. Another phosphoproteomics screen identified nearby residues  that were also dependent on the presence of Rad53 . We have added this to the \u201cMrc18D slows fork rate in vitro and in vivo and partially rescues rad53 mutant sensitivity\u201d section on page 11. It would be very difficult/impossible to get the kind of mass spec coverage we would need to validate all of the sites, even in vitro.Apologies \u2013 we realise that we neglected to include some important references related to previous phosphoproteomic studies. Three of the eight sites in Mrc1(3) In addition to the viability assays shown in Figure 6, it would be useful if the authors could provide an assay that monitors replication fork progression to ensure that the Mrc1-8D protein restrains fork progression even in the absence of Rad53.Addressed in Essential revisions.Reviewer #3 (Recommendations for the authors):I would really appreciate a figure showing the locations of the 8/14/19 mutations. I feel these should be added to the diagram in S3B.We have now added figures with the location of the mutations to new Figure 5 \u2014figure supplement 1B.I suggest the authors construct and test an 8A version. If this allele exhibits more limited effects than 14A, it will strengthen the inference that the 8D changes are not just breaking the protein.14A mutant is nearly wild-type in its ability to promote replication speed in vitro, it would be very surprising if the Mrc18A mutant, which is effectively a subset of the 14A mutant wasn\u2019t also like wild type. Because we thought it would be highly unlikely the 8A mutant would work less well than the 14A mutant and because of time constraints we decided not to test this mutant.Because the Mrc1I think a bit more in vivo analysis of these mutants would be useful and appreciated, especially by experts such as bulk analysis of DNA content during S phase progression. One would predict that the 8D allele would cause slow replication even without drugs. However, it might have little effect given the apparently intact checkpoint signaling and presumably intact origin firing control.8D strains. Because S phase progression by FACS analysis reflects both the state of origin firing and replication fork speed, we instead used a system using BrdU incorporation and replication intermediate size measurements to determine replication fork rates in these cells. This experiment is described in Essential revisions.The reviewer makes a great suggestion to look at replication fork rate in the Mrc1One set of easy experiments that seems to be missing is more analysis of the 14A and 19A alleles in vivo. These alleles might be predicted to exhibit sensitivity to HU and MMS. Should effects be noted, it will probably be important to examine Rad53 activation in these mutants similar to analyses in 6B and C to rule out possible effects on transducing the signal to activate Rad53 as opposed to defects in receiving effector signals from activated Rad53. It is possible that the 14A and 19A mutations will have no sensitivities due to incomplete penetrance or redundant mechanisms, but worth a try.19A mutant in response to hydroxyurea and didn\u2019t detect any phenotype . As the reviewer points out, this could be due to incomplete penetrance  or redundant mechanisms. We have added this to the section \u201cIdentification of Rad53 phosphorylation sites in Mrc1\u201d on page 10.We tested the sensitivity of the Mrc1"}
+{"text": "Correction to: BMC Genomics 22, 771 (2021)https://doi.org/10.1186/s12864-021-08057-4Hsp23_E2 and Prx2540-1_E, were not entered correctly into this table.Following the publication of the original article , the corThe correct sequence of YB320 is 5\u2019-TAGTTGGGGATGTCTTCGAATGTACATATGTTCCAAATCG -3\u2019 and of YB362 is 5\u2019- TAGTTGGGGATGTCTTCCATTTAGCTCATCTCCACGCTAG -3\u2019.The correct Additional file Additional file 23: Table S10. Excel file with description of synthetic DNA fragments (oligos and gene blocks)."}
+{"text": "Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs. LAMP2A targets cytosolic proteins to extracellular vesicles to modulate interorgan communication. Exosomes are nanosized vesicles of 40 to 160 nm in diameter that are secreted by most cell types to the extracellular space , referred to as multivesicular bodies (MVBs)  machinery  the LAMP2A gene in a human cell line with normal chromosome number, ARPE-19. LAMP2A originates from the alternative splicing of the LAMP2 gene, which contains nine exons, including three different splice variants of the exon 9  . All splExtracellular vesicles (EVs) from the media supernatants of WT and LAMP2A KO ARPE-19 cells were isolated for mass spectrometry (MS)\u2013based proteome analysis. Note that there is still a lack of consensus on specific markers for exosomes, and the most common method used for exosome isolation, ultracentrifugation, is unable to separate exosomes from other populations of small EVs such as microvesicles. Following EV guidelines . TEM of 6 cells confirmed the presence of membrane proteins of endosomal origin, CD63 and LAMP2A, and the cytosolic proteins associated to EVs Flotillin-1 (FLOT1) and HSC70, as well as the absence of the endoplasmic reticulum marker calnexin and the plasma membrane marker Na+- and K+-dependent adenosine triphosphatase  of 2.5 \u03bcg of whole-cell lysates and 100% of isolated sEVs secreted by 40.0 \u00d7 10P < 0.05) in LAMP2A KO sEVs and that 203 of the down-regulated proteins (67%) contained a putative KFERQ motif . Accordingly, the identified proteins were enriched in EV markers to the same level as in previously published EV studies (fig. S1D) . InitialRQ motif . These fTo address the mechanisms involved in the targeting of proteins containing KFERQ motifs to EVs, we used chimeric proteins consisting of a fluorescent protein, such as mCherry, and the KFERQ motifs of \u03b1-synuclein (VKKDQ) and ribonuclease A (KFERQ) separated by a peptide spacer , based oWe hypothesized that HSC70 is involved in the targeting of proteins into nascent EVs by binding and delivering proteins containing KFERQ-like motifs to endosomes. To assess whether HSC70 has a role in the loading of proteins into sEVs, we used Pifithrin-\u03bc (Pifi), a compound that acts by inhibiting the interaction of HSC70 with its substrates  and a deWhile some EVs such as microvesicles originate from shedding of the plasma membrane, exosomes are formed by the inward invagination of the endosomal limiting membrane followed by fusion of the endosome with the plasma membrane. To investigate whether ExoSignal-tagged proteins are present in endosomes, we used the constitutively active mutant of Rab5 (Q79L) that blocks the conversion of early endosomes (EEs) into LEs, resulting in the formation of very large hybrid endosomes that accumulate large ILVs . LAMP2A Subsequently, we isolated endosomal compartments using a discontinuous sucrose gradient, according to protocol . The mat2 showed that HIF1A is present in sEVs only when cells express LAMP2A (fig. S4A). Isolation of sEVs from 769-P cells, KO for HIF1A and the ubiquitin ligase Von Hippel\u2013Lindau (AA) showed that only WT HIF1A is present in sEVs (fig. S4B). Moreover, subcellular fractioning using an OptiPrep linear gradient showed that endogenous HIF1A localizes to endosomal fractions and that LAMP2A KO decreases HIF1A presence in those fractions (fig. S4C). We next incubated endosomal fractions with glutathione S-transferase (GST) (no KFERQ motif) and HIF1A-GST, in the presence or absence of recombinant HSC70 and adenosine triphosphate (ATP), followed by trypsin treatment to degrade recombinant HIF1A protein unprotected by the endosomal membrane  independently of LAMP2A , which expresses mCherry in endothelial cells, showed that GFP-ExoSignal was present in the lumen of the blood vessels and inside endothelial cells (Tg(mpeg1:mCherry), which expresses mCherry in macrophages, we observed that macrophages moving within the caudal plexus incorporated GFP-ExoSignal , expressing GFP in endothelial cells, with sEVs from WT cells, we were able to increase the number of vascular branches, including longer blood vessels with greater volume, in the retinal vascular network . The targeted proteins contain amino acid sequences, biochemically related to the KFERQ motif, a pentapeptide sequence previously involved in two selective forms of autophagy: chaperone-mediated autophagy (CMA) , 7.5 \u03bcM Pifi (Merck), and 50 nM bafilomycin A1 (Apollo Scientific).The human cell lines ARPE-19  and 769-P  were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) with glutamine (Biowest) supplemented with 10% fetal bovine serum  and penicillin-streptomycin . Cells were cultured at 37\u00b0C under 5% CO+,K+-ATPase, dilution of 1:1000 ; goat anti-GAPDH, dilution of 1:2000 ; mouse anti-ExoC2, dilution of 1:500 ; rabbit anti\u2013histone 3, dilution of 1:1000 ; anti-Vps4b, dilution of 1:500 , goat anti-GFP, dilution of 1:1000 , goat anti-Rab31, dilution of 1:500 ; mouse anti\u2013syntenin-1 ; and horseradish peroxidase (HRP)\u2013conjugated secondary goat anti-mouse , goat anti-rabbit , rabbit anti-goat , and goat anti-rat , dilution of 1:5000. The following were also used: Alexa Fluor 568\u2013conjugated donkey anti-goat , Alexa Fluor 488\u2013conjugated donkey anti-goat , Alexa Fluor 488\u2013conjugated donkey anti-rabbit , Alexa Fluor 546\u2013conjugated donkey anti-mouse , donkey anti-rabbit Cy3 , and donkey anti-mouse Cy5, dilution of 1:250. The following were also used: dextran, Alexa Fluor 488, 10,000 molecular weight (MW), anionic, fixable ; dextran, Alexa Fluor 647, 10,000 MW, anionic, fixable ; wheat germ agglutinin, Alexa Fluor 488 conjugate ; cathepsin B assay kit ; DAPI ; Protein G\u2013Sepharose ; Dynabeads Protein G ; nitrocellulose membranes ; ECL ; protease inhibitor cocktail ; OptiPrep iodixanol density media ; ONE-Glo Luciferase Assay System ; and Pierce BCA Protein Assay Kit .The following antibodies were used: mouse anti-LAMP2 clone H4B4, dilution of 1:1000 (WB) and 1:100 ; rabbit anti-LAMP2A, dilution of 1:500 (WB) and 1:100 ; rabbit anti-LAMP2B, dilution of 1:500 ; mouse anti-actin, dilution of 1:2000 ; goat anti-HIF1A, dilution of 1:1000 ; goat anti-CD63, dilution of 1:1000 ; mouse anti-CD63, dilution of 1:100 ; rabbit anti\u2013Flotillin-1, dilution of 1:500 ; goat anti-CTSB/cathepsin B clone S-12, dilution of 1:500 ; mouse anti-Alix, dilution of 1:500 ; mouse anti-TSG101, dilution of 1:500 ; goat anti-GST, dilution of 1:500 ; goat anti-EEA1, dilution of 1:500 ; goat anti-mCherry, dilution of 1:500 (WB) and 1:100 ; rabbit anti-DsRed, dilution of 1:100 ; mouse anti-m6pR, dilution of 1:100 ; rat anti-HSC70 clone 1B5, dilution of 1:1000 ; goat anti-Rab27, dilution of 1:500 ; goat anti-tubulin, dilution of 1:2000 ; mouse lamin B1, dilution of 1:500 ; goat anti-calnexin, dilution of 1:1000 ; goat anti\u2013NaThe sEV protein solution containing SDS and dithiothreitol (DTT) was loaded onto filtering columns and washed exhaustively with 8 M urea in Hepes buffer (m/z) was followed by higher-energy collisional dissociation (HCD) fragmentation and Orbitrap detection of the 15 most intense ions observed in the MS scan. Target value in the Orbitrap for MS scan was 1,000,000 ions at a resolution of 70,000 at m/z 200. Fragmentation in the HCD cell was performed at normalized collision energy of 31 eV. Ion selection threshold was set to 25,000 counts, and maximum injection time was 100 ms for MS scans and 300 and 500 ms for MS/MS scans. Selected sequenced ions were dynamically excluded for 45 s.Peptide samples were analyzed by nano\u2013LC-MS/MS (Dionex RSLCnano 3000) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific). Briefly, the samples (5 \u03bcl) were loaded onto a custom-made fused capillary precolumn  with a flow of 5\u03bcl/min for 7 min. Trapped peptides were separated on a custom-made fused capillary column  packed with ReproSil-PurC18 3-\u03bcm resin  with a flow of 300 nl/min using a linear gradient from 92% A (0.1% formic acid) to 28% B (0.1% formic acid in 100% acetonitrile) over 93 min followed by a linear gradient from 28 to 35% B over 20 min at a flow rate of 300 nl/min. Mass spectra were acquired in positive ion mode, applying automatic data-dependent switch between one Orbitrap survey. MS scan in the mass range of 400 to 1200 mass/charge ratio  transformation; (ii) removing common MS contaminants followed by log2 (x + 1) transformation and quantile normalization; and (iii) removing common MS contaminants followed by log2 (x + 1) transformation, quantile normalization, and abundance filtering to optimize overall Gaussian distribution of the quantitative values. For simplicity, only the quantile-normalized quantitative data are presented here. Statistical differences were calculated by using the R package limma (https://rshine.einsteinmed.org/).Statistical and bioinformatics analyses of MS data were performed in the statistical programming language R. Quantitative data from MaxQuant and VEMS were analyzed in R statistical programming language. IBAQ and protein spectral counts from the two programs were preprocessed by three approaches: (i) removing common MS contaminants followed by logFor this work, we used the plasmids pT81 HRE (x3)-Luciferase  (https://zlab.bio/guide-design-resources).ARPE-19 cells were transduced with the lentiviral vector pCW-Cas9  protein] and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro [American Type Culture Collection (ATCC)]. Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200The sequences were synthesized and cloned into pUC57 (GeneCust). PA-mCherry and PA-mCherry ExoSignal were subcloned into pLenti6 (Thermo Scientific) using a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific) according to the manufacturer\u2019s instructions.Human LAMP2A was synthesized into pUC57 plasmid with the sequence 5\u2032-CAAGTTTGTACAAAAAAGCAGGCTCTCGAGCAatgGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTGTGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCAGATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGCACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGACCTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTCCTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTTTTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATGTTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTTCCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACAACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGCTGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAATACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAGTATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGTATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAGTTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTAAGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCTCAAGACTGCAGTGCAGATGACGACAACTTCCTTGTGCCCATAGCGGTGGGAGCTGCCTTGGCAGGAGTACTTATTCTAGTGTTGCTGGCTTATTTTATTGGTCTCAAGCACCATCATGCTGGATATGAGCAATTTTAGGGTACCACCCAGCTTTCTTGTACAAAGTGGACGCGT-3\u2032. Human LAMP2A was subcloned into pLenti6 (Thermo Scientific), a Gateway adapted vector using BP/LR Clonase II (Thermo Scientific), according to the manufacturer\u2019s instructions.P2A-GFP-ExoSignal was synthesized and subcloned into the p3E plasmid with the sequence 5\u2032-TACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAACTTTCTTGTACAAAGTGGAGATCTatgGGAAGCGGACGTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTCGAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGAAAGCTTTGTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAACGACAATGAGGTACCGTCGACCAACTTTATTATACAAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTA-3\u2032. Subsequently, the sequence was subcloned into a pDestTol2pA5 plasmid (gift from C.-H. Yuhm\u2019s laboratory) according to the manufacturer\u2019s instructions (Thermo Scientific), along with p5E-Ubi  and pME-mCherry (gift from C.-H. Yuhm\u2019s laboratory).g for 10 min, and used. For TSG101, VPS4b, CD63, and Alix, pLKO.1 plasmids with shRNA sequences were obtained from the RNAi Consortium . Control used was a nontargeting sequence . The target sequences used were as follows: TSG101 , VPS4b , CD63 , and Alix . For Syntenin-1 target, pLKO.1 plasmids with shRNA sequences were obtained from Sigma Mission library .All lentiviral particles were produced by cotransfection of pLenti6 and pMD 2.G (VSV-G protein) and psPAX2 (Rev and Pol proteins) into a producer cell line, 293STAR RDPro (ATCC). Recombinant viral particles were harvested 48 days later, cleared for cell debris by centrifugation at 3200For the knockdowns, Rab27a and Rab27b, as well as Rab31 microRNA (miRNA)\u2013expressing vectors, were constructed by inserting specific nucleotide sequences into pcDNA6.2-GW/EmGFP-miR plasmid harboring a Pol II promoter obtained from Thermo Scientific. These sequences are fused with GFP coding sequence. The synthesized oligonucleotides were annealed and ligated into pcDNA6.2-GW/EmGFP-miR according to the manufacturers\u2019 instructions. For Rab27a and Rab27b, tandem miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: Rab27a miRNA1, 5\u2032-AAACTTTGCTCATTTGTCAGG-3\u2032; Rab27a miRNA2, 5\u2032-TTAACTGATCCGTAGAGGCAT-3\u2032; Rab27b miRNA1, 5\u2032-ATTGACTTCCCTCTGATCTGG-3\u2032; and Rab27b miRNA2, 5\u2032-TTTCCCTGAAGATCCATTCGG-3\u2032. For Rab31, miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: miRNA1, 5\u2032-TTTCTTTGCAGGAAACGTCCC-3\u2032 and miRNA2, 5\u2032-TAAACTGAAGGCCATGTTGCG-3\u2032. Viral particles were produced, and cells were infected as described before  in 0.1 M phosphate buffer (pH 7.4). Cells were processed for ultracryomicrotomy and contrasted as described . Samples were washed with phosphate-buffered saline (PBS) and fixed with 1% GA for 5 min. After washing with distilled water, grids were contrasted with uranyl oxalate (pH 7) for 5 min and transferred to methylcellulose-uranyl acetate for 10 min on ice. Observations were carried out using a Tecnai G2 Spirit BioTwin electron microscope (FEI) at 80 kV.g for 10 min, followed by centrifugation at 16,500g for 20 min. From this pellet, the microvesicle fraction was collected. To remove larger particles, the supernatant was filtered with a 0.22-\u03bcm filter unit, after which it was ultracentrifuged at 120,000g for 70 min, using an SW 32 Ti rotor. The resulting pellet was washed with PBS, and after ultracentrifugation, exosomes were resuspendedin PBS.Exosomes derived from 40 million cultured cells were isolated from 40 ml of conditioned medium. Cells were cultured in exosome-depleted medium for 48 hours, prepared in accordance with the work of Lasser and colleagues  containing the exosomes was collected using a syringe with a 18-gauge needle. Exosomes were then transferred to a new ultracentrifuge tube, diluted in 35 ml of PBS, and centrifuged overnight at 100,000g and 4\u00b0C. The resulting pellet was resuspended in 100 \u03bcl of PBS, and protein quantification was performed.For MS, an extra step of purification was introduced using a 30% sucrose cushion . Cells were lysed by sonication (three rounds of 3 s at 4\u00b0C) and centrifuged at 1000g for 5 min (2\u00d7). Continuous 5 to 30% OptiPrep gradients were prepared in working solution using a gradient mixer. Postnuclear supernatant (PNS) was added to the top of 9-ml gradients and centrifuged at 100,000g for 16 hours using a 70.1 Ti rotor. Sequential fractions were collected . Fractions were recovered by ultracentrifugation at 100,000g for 30 min in an SW 32 Ti rotor. For WB, fractions were collected in PBS and further denaturated in Laemmli buffer.Continuous density gradients were performed as described previously with adaptations. Briefly, 8 \u00d7 10et al. was used (6), cultured in DMEM with 10% FBS, were washed with ice-cold PBS and collected with a cell scraper. Cells were collected to a 2-ml microfuge tube and centrifuged at 300g for 5 min at 4\u00b0C. Cell pellet was loosened using cold finger in a homogenization buffer . Samples were centrifuged at 1300g for 10 min at 4\u00b0C. Supernatant was discarded, and the pellet was gently resuspended with a wide-cut tip in three times the pellet volume of HB. The suspension was passed through a 25-gauge needle, attached to a 1-ml syringe, 10 times.For the separation of EEs and LEs, a protocol described by de Araujo g for 10 min at 4\u00b0C. The supernatant was collected and centrifuged again. The PNS that originated from the second centrifugation is used for organelle isolation. PNS (5%) is centrifuged at 4\u00b0C for 10 min at 16,000g. The recovered supernatant was collected for cytoplasmatic fraction, free of vesicles. For the remaining PNS, the sucrose concentration was adjusted to 40.6%. The PNS was then loaded at the bottom of an ultracentrifuge tube. The 40.6% solution was overlaid with 1.5 volumes of 35% sucrose solution and 1 volume of 25% sucrose solution, and the tube was filled to the top with HB. The gradient was centrifuged at 210,000g for 16 hours at 4\u00b0C, using a 70.1 Ti rotor. Each interface was collected. LEs are found in the 25%/HB interface. EEs are present in the 35%/25% interface. The endosomal fractions were diluted in 35 ml of HB solution and centrifuged at 100,000g for 1 hour in an SW 32 Ti rotor. The organelle pellets were resuspended in an appropriate buffer depending on the downstream procedures.The homogenate was then diluted in HB (1 part homogenate to 0.7 parts HB) and centrifuged at 16002PO4, pH 7.25 was adjusted with KOH) buffer according to protocol ] for 45 min at 37\u00b0C. After the incubation period, samples were cooled on ice for 1 min followed by a 30-min incubation with trypsin at 37\u00b0C. Samples were put back to ice and denaturated in Laemmli buffer, heated at 95\u00b0C for 5 min, and resolved on a 10% SDS\u2013polyacrylamide gel electrophoresis (PAGE) gel. The gel was transferred to a nitrocellulose membrane for 75 min at 100 V. The membranes were blocked with TBS-T containing 5% skim milk, followed by incubation with antibodies against the proteins of interest.Recombinant proteins GST (SICGEN) and GST-HIF1A  were incubated with 5 \u03bcg of freshly isolated vesicles and recombinant protein HSC70  in Mops buffer  and ATP regeneration buffer  and sonicated three times, 1 s each at 4\u00b0C. Afterward, samples were centrifuged at 15,000g for 10 min, and pellets were discarded. All samples were incubated with 2 \u03bcg of the antibody against the protein of interest overnight at 4\u00b0C. Subsequently, 30 \u03bcl of Protein G\u2013Sepharose was added to the sample, and incubations proceeded at 4\u00b0C for 2 hours. Beads were washed three times with lysis buffer containing 0.15% NP-40, denatured with Laemmli buffer, and boiled at 95\u00b0C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Cells were collected from dishes with ice-cold PBS using a cell scraper and centrifuged at 15,0002PO4; pH 7.25 was adjusted with KOH). Magnetic beads were incubated with anti-LAMP2A or anti-LAMP2B antibodies for 1 hour. Subsequently, the endosomes were incubated with the magnetic beads for an additional hour at 4\u00b0C. Immunoprecipitates were then gently washed three times with KPBS. The samples were denatured with Laemmli buffer and boiled at 95\u00b0C for 5 min. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.Afterward, endosomal immunoprecipitation was adapted from protocol , 150 mM NaCl, 10 mM iodoacetamide, 2 mM PMSF, and 1\u00d7 cocktail inhibitor from Sigma-Aldrich]. Samples were then centrifuged at 4\u00b0C for 10 min at 1000g. Supernatants were removed to a new 1.5-ml microfuge tube, and pellet (nuclear pellet) was lysed with Laemmli buffer. Supernatant was centrifuged at 4\u00b0C for 10 min at 16,000g. The resultant pellet (vesicular pellet) and the second supernatant (cytoplasm) were also lysed with Laemmli buffer. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% nonfat milk in TBS-T and probed for the proteins of interest.To evaluate the presence of exosomal HIF1A in the nuclei of receiving cells, exosomes were isolated from ARPE-19 cells that were under hypoxia with 300 \u03bcM CoClHIF1A activity was measured by transducing cells with the reporter gene Luciferase under the control of the HRE, using ONE-Glo Luciferase Assay System (Promega), according to the manufacturer\u2019s specifications.casper, Tg(kdrl:mCherry), or Tg(mpeg1:mCherry) embryos, at 100-cell stage, were injected in the YSL with pUbi-mCherry-p2A-GFP-ExoSignal in the presence or absence of MOs at 150 nM concentration, with a microinjector under a stereomicroscope. We used a Syntenin-a MO oligonucleotide directed against the translation start site [5\u2032-ACAACGACATCCTTTCTGCTTTCA-3\u2032 (Tg(fli1a:EGFP) zebrafish with a microinjector under a stereomicroscope. At 5 dpf, embryos were fixed with 4% PFA overnight at 4\u00b0C. Embryos were washed in PBS and mounted in glycerol mounting media. Images were acquired in a Zeiss LSM 710 confocal microscope using a 40\u00d7 1.2 C-Apochromat water-immersion objective. When required, optical slices were acquired, and 3D reconstruction of the images and quantifications were performed using Imaris software.Zebrafish lines were maintained in a circulating system with 14-hour day and 10-hour night cycle periods at 28\u00b0C. The mating and spawning of zebrafish were incited by the change of dark to light. Embryos were collected before they start independent feeding, at 5 days postfertilization (dpf). Therefore, no ethical approval was necessary according to the Council Directive 2010/63/EU on the protection of animals used for scientific purposes  with Tukey\u2019s multiple comparisons tests, using GraphPad Prism 8.0 software (GraphPad Software). For comparison between two groups, the paired"}
+{"text": "Vagal sensory neurons (VSNs) form an important body-to-brain connection, navigating visceral organs along the rostral\u2013caudal axis of the body and crossing the surface\u2013lumen axis of organs into appropriate tissue layers6. The brain can discriminate numerous body signals through VSNs, but the underlying coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer and stimulus modality\u2014three key features of an interoceptive signal\u2014in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs\u00a0in mice using multiplexed projection barcodes reveals a \u2018visceral organ\u2019 dimension composed of differentially expressed gene modules that code organs along the body\u2019s rostral\u2013caudal axis. We discover another \u2018tissue layer\u2019 dimension with gene modules that code the locations of VSN endings along the surface\u2013lumen axis of organs. Using calcium-imaging-guided spatial transcriptomics, we show that VSNs are organized into functional units to sense similar stimuli across organs and tissue layers; this constitutes a third \u2018stimulus modality\u2019 dimension. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial manner and facilitate the complex projection of VSNs in the brainstem. Our study highlights a multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival Single-cell profiling of vagal sensory neurons from seven organs in mice and calcium-imaging-guided spatial transcriptomics reveal that interoceptive signals are coded through three distinct dimensions, allowing efficient processing of multiple signals in parallel using a combinatorial strategy. As a key body\u2013brain axis in interoception, VSNs in the nodose and jugular ganglia transmit numerous signals from visceral organs in the respiratory, cardiovascular, gastrointestinal, endocrine and immune systems into the brainstem6. Signals communicated through VSNs are precisely discriminated in the brain for highly specific responses5, yet how this is achieved remains unclear. Extensive data show that VSNs are highly heterogeneous in multiple characteristics, including developmental origins, electrical properties, response patterns, molecular identities, terminal morphologies, sensory mechanisms, anatomical connections and physiological roles19. However, despite the accumulation of a large amount of data over the past seven decades describing the complexity of VSN characteristics, little is known about how such heterogeneity and diversity facilitates interoceptive coding at a systems level.Sensing the body\u2019s internal state is a critical life-ensuring process that maintains physiological homeostasis, provides motivational drivers and shapes our thoughts and emotions20. Similarly, the same bioactive signal (such as serotonin) released from different tissue layers within the intestine communicates different organ information to the brain21. We reasoned that a faithful coding of these features in VSNs is necessary for accurate signal discrimination in the brain. Here we developed several techniques to determine whether and how these essential features of interoceptive signals are coded in VSNs.The physiological role of an interoceptive signal can be specified by three important features: visceral organ; tissue layer; and stimulus modality. Stimuli that differ in these features represent different body changes. For example, stretching the arterial wall implicates an increase in blood pressure, whereas stretching the stomach wall signals food ingestion23. Whether a viscerotopic map exists in the nodose ganglion, where most visceral-organ-innervating VSNs are located6, is a subject of debate27. Using retrograde adeno-associated virus (AAVrg), we show that VSNs labelled from various visceral organs are largely non-overlapping  along the spinal cordWe therefore hypothesize that visceral organs are coded by specific genes in VSNs. We developed a single-cell sequencing approach named \u2018Projection-seq\u2019 for unbiased, high-throughput genetic and anatomical dissection of complex neural circuits Fig. . EngineePhox2b+ placode-derived nodose VSNs18  were grouped into 12 subpopulations (A\u2013L) and 52 clusters (A1\u2013L2) on the basis of differentially expressed genes (DEGs) using Seurat and visualized using uniform manifold approximation and projection (UMAP)  Fig. . VSNs laAP) Fig. . The disAP) Fig. . VSN cluAP) Fig. , showingAP) Fig. . VSNs thAP) Fig. . Togethe32. Of note, it seems that VSNs are primed for the functions of their target organs. For example, the stomach contains several anatomically and functionally distinct subregions: the proximal region similar to the oesophagus and the distal region similar to the intestine33. Accordingly, UPB-stomach+/UPB-oesophagus+ VSNs are genetically similar to oesophagus VSNs, whereas UPB-stomach+/UPB-duodenum+ VSNs are closer to duodenum VSNs , cardiovascular (heart), gastrointestinal  and exocrine\u2013endocrine (pancreas) systems Fig. . Gut VSN6. Our discovery of a genetic \u2018visceral organ\u2019 trajectory is in accordance with this long-standing observation, suggesting that VSNs might follow morphogen gradients to their target spots. Indeed, signalling gradients formed by secreted morphogens help to establish an anterior\u2013posterior patterning34 and specify peripheral targets for vagal motor neurons35. Together, our data show that instead of a topographic organization, VSNs use a genetic trajectory to code visceral organs along the body\u2019s rostral\u2013caudal axis.Notably, we further identified a genetic trajectory that represents visceral organs along the body\u2019s rostral\u2013caudal axis Fig. . The relGpr65, Sst, Trpv1, Drd2 and Agtr1a  and examined the innervation of tdTomato+ sensory fibres in whole-mount cleared organs. Notably, VSNs along this second trajectory project to different tissue layers   Fig.  and calced organ . The tis17, it is unclear how they contribute to interoceptive coding. Systematic analyses of VSN terminals within the lung, heart, oesophagus, stomach, duodenum and colon in Vglut2tdT mice revealed that there is a marked similarity between many stereotypical VSN ending types in the same tissue layer across visceral organs ; and (6) bud endings wrapped around specialized sensory epithelial cell clusters. Our data thus suggest that VSN ending structures are predominantly organ-independent but tightly associated with tissue layers.We then asked whether VSNs form organ-specific ending structures in various tissue layers. Although VSN endings have been extensively characterized in individual organsans Fig. , includi+ VSNs by nodose infection of AAV-FLEX-tdTomato in corresponding Cre mice; and (3) determined the sensory structures formed by DEG+ VSNs in the target organ or region using whole-organ clearing and volumetric imaging , we (1) identified enriched VSN clusters and their DEGs on the basis of Projection-seq data; (2) labelled DEGing Fig. . With thing Fig. , showing38. Together, our results reveal a two-dimensional genetic representation of the body\u2019s internal space in VSNs that codes the precise anatomical location of interoceptive signals  biological processes for neuron development, cell\u2013cell signalling and synaptic signalling or ion transportation  mice. VSN identity was then determined post hoc in cryo-sectioned nodose ganglia using RNAscope against 21 marker genes that together faithfully represent VSN subpopulations  responsive VSNs were unambiguously registered from six mice  Fig. . First, ice Fig. . VSNs reice Fig. . The vCaPiezo2+ VSNs (55.1%) were activated significantly faster than Piezo2\u2212 K/L-VSNs . Uts2b+Vip+Glp1r+Cckar+ G-VSNs represented a large fraction (39.2%) of intestinal stretch responders   Fig. , suggestTo better quantify the relationship between VSN characteristics, we systematically mapped all characterized connections among the five VSN characteristics Fig. . In this47. Consistently, VSN central projections labelled using AAVrg-tdTomato from respiratory, cardiovascular and digestive systems were largely segregated, whereas vagal afferents from functionally related organs terminated in more overlapping areas  guidelines. All mice were age- and gender-matched adults (older than 8 weeks) and no differences between sexes were observed.Glp1r-ires-Cre (029283), Gpr65-ires-Cre (029282), Npy2r-ires-Cre (029285), P2ry1-ires-Cre (029284), Agtr1a-Cre (030553), Calb2-ires-Cre (010774), Nts-Cre (017525), Vglut2-ires-Cre (016963), Sst-ires-Cre (013044), Twist2-Cre (008712), Vip-ires-Cre (010908), Piezo2-GFP-ires-Cre (027719), Trpv1-Cre (017769), Pvalb-Cre (017320), Vglut1-ires2-Cre (023527), Chat-ires-Cre (031661), lox-ChR2 (024109), lox-tdTomato (007914), and Snap25-2A-GCaMP6s-D (025111) were from the Jackson Laboratory. Drd2-Cre (032108-UCD) mice were from the Mutant Mouse Resource and Research Center (MMRRC). lox-L10-GFP mice were described before12.Wild-type C57BL/6J (000664), 12\u20131013 viral genomes per ml) were generated at the UNC Vector Core. Plasmids have been deposited to Addgene.UPB sequences were cloned from coding sequences of hChR2 , hM3Dq  and hM4Di  and inserted right before the SV40 poly (A) of AAVrg-CAG-tdTomato-WPRE-SV40  using the In-Fusion HD Cloning Kit . Projection-seq AAVs (at titre\u2009>\u200910UPB sequences (5\u2032\u20133\u2032):UPB-oesophagus :ACAGCACCATCCTCAACTCCACCAAGTTACCCTCATCGGACAACCTGCAGGTGCCTGAGGAGGAGCTGGGGATGGTGGACTTGGAGAGGAAAGCCGACAAGCTGCAGGCCCAGAAGAGCGTGGACGATGGAGGCAGTTTTCCAAAAAGCTTCTCCAAGCTTCCCATCCAGCTAGAGTCAGCCGTGGACACAGCTAAGACTTCTGACGTCAACTCCTCAGTGGGTAAGAGCACGGCCACTCTACCTCTGTCCTTCAAGGAAGCCACTCTGGCCAAGAGGTTTGCTCTGAAGACCAGAAGTCAGATCACTAAGCGGA.UPB-stomach :ATGGACTATGGCGGCGCTTTGTCTGCCGTCGGACGCGAACTTTTGTTCGTTACTAATCCTGTGGTGGTGAACGGGTCCGTCCTGGTCCCTGAGGATCAATGTTACTGTGCCGGATGGATTGAATCTCGCGGCACGAACGGCGCTCAGACCGCGTCAAATGTCCTGCAGTGGCTTGCAGCAGGATTCAGCATTTTGCTGCTGATGTTCTATGCCTACCAAACCTGGAAATCTACATGCGGCTGGGAGGAGATCTATGTGTGCGCCATTGAAATGGTTAAGGTGATTCTCGAGTTCTTTTTTGAGTTTAAGAATCCCTCTATGCTCTACCTT.UPB-duodenum :AATGGCAGCTCGGGCAATCAGTCCGTGCGCCTGGTCACGTCATCATCCCACAATCGCTATGAGACGGTGGAAATGGTCTTCATTGCCACAGTGACAGGCTCCCTGAGCCTGGTGACTGTCGTGGGCAACATCCTGGTGATGCTGTCCATCAAGGTCAACAGGCAGCTGCAGACAGTCAACAACTACTTCCTCTTCAGCCTGGCGTGTGCTGATCTCATCATAGGCGCCTTCTCCATGAACCTCTACACCGTGTACATCATCAAGGGCTACTGGCCCCTGGGCGCCGTGGTCTGCGACCTGTGGCTGGCCCTGGACTGCGTGGTGAGCAACGCCTCCGTCATGAACCTTCTCATCATCAGCTTTGACCGCTACTTCTGCGTCA.UPB-colon :ATCGATGGGCCTTAGGGAACTTGGCCTGTGACCTCTGGCTTGCCATTGACTGCGTAGCCAGCAATGCCTCTGTTATGAATCTTCTGGTCATCAGCTTTGACAGATACTTTTCCATCACGAGGCCGCTCACGTACCGAGCCAAACGAACAACAAAGAGAGCCGGTGTGATGATCGGTCTGGCTTGGGTCATCTCCTTTGTCCTTTGGGCTCCTGCCATCTTGTTCTGGCAATACTTTGTTGGAAAGAGAACTGTGCCTCCGGGAGAGTGCTTCATTCAGTTCCTCAGTGAGCCCACCATTACTTTTGGCACAGCCATCGCTGGTT.UPB-pancreas :AAGATGGCAGGCCTCATGATTGCTGCTGCCTGGGTACTGTCCTTCGTGCTCTGGGCGCCTGCCATCTTGTTCTGGCAGTTTGTGGTGGGTAAGCGGACGGTGCCCGACAACCAGTGCTTCATCCAGTTCCTGTCCAACCCAGCAGTGACCTTTGGCACAGCCATTGCTGGCTTCTACCTGCCTGTGGTCATCATGACGGTGCTGTACATCCACATCTCCCTGGCCAGTCGCAGCCGAGTCCACAAGCACCGGCCCGAGGGCCCGAAGGAGAAGAAAGCCAAGACGCTGGCCTTCCTCAAGAGCCCACTAATGAAGCAGA.UPB-lung :ACAGGACACCGGGTGCAGTGGCTGCGCTATGCAGAGTGGCTGCTCACTTGTCCTGTCATCCTTATCCGCCTGAGCAACCTCACCGGCCTGAGCAACGACTACAGCAGGAGAACCATGGGACTCCTTGTCTCAGACATCGGGACTATCGTGTGGGGGGCTACCAGCGCCATGGCAACCGGCTATGTTAAAGTCATCTTCTTTTGTCTTGGATTGTGCTATGGCGCGAACACATTTTTTCACGCCGCCAAAGCATATATCGAGGGTTATCATACTGTGCCA.UPB-heart :AGGACACTTCCAATGAGTCCAGCTCAGGCAGTGCCACCCAGAACACCAAGGAACGCCCAGCCACAGAGCTGTCCACCACAGAGGCCACCACGCCCGCCATGCCCGCCCCTCCCCTGCAGCCGCGGGCCCTCAACCCAGCCTCCAGATGGTCCAAGATCCAGATTGTGACGAAGCAGACAGGCAATGAGTGTGTGACAGCCATTGAGATTGTGCCTGCCACGCCGGCTGGCATGCGCCCTGCGGCCAACGTGGCCCGCAAGTTCGCCAGCATCGCTCGCAACCAGGTGCGCAAGAAGCGGCAGATGGCGGCCCGGGAGCGCAAAGTGACACGAACGATCTTTGCCATTCTGCTGGCCTTCATCCT.51 represents a powerful genetic tool in VSN studies54. AAVrg-tdTomato and AAVrg-GFP were purchased from the UNC vector core. AAVrg-FLEX-tdTomato (28306-AAVrg), AAVrg-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (84445-AAVrg) and pAAVrg-hSyn-Con/Fon hChR2(H134R)-EYFP (55645-AAVrg) were purchased from Addgene. All viruses contained 1012\u20131013 viral genomes per ml, and 0.05% Fast Green FCF  was occasionally used to facilitate visualization.Retrograde adeno-associated virus (AAVrg)\u22121) and buprenorphine (1.5\u2009mg\u2009kg\u22121). Lung: virus  was injected through a tracheal catheter into the lung using a Hamilton syringe. Heart: the mouse was ventilated  using a mouse ventilator  via an intubated angiocatheter. The heart was gently exposed via thoracotomy. Virus (5\u2009\u03bcl) was injected intramurally (20\u2009nl per second) using a Nanoject III injector at multiple sites covering most of the area of the heart. Stomach, duodenum, colon and pancreas: the target organ was gently exposed via an abdominal incision. Virus was injected intramurally at multiple sites covering most of the area . Oesophagus: the cervical oesophagus underneath the trachea was surgically exposed via a neck incision. The abdominal oesophagus and the oesophageal sphincter were gently exposed via an abdominal incision. Virus was injected into oesophageal muscularis and serosa layers .In all surgeries, mice were anaesthetized with 1\u20132% isoflurane on a heating pad, followed by subcutaneous injections of meloxicam  were mixed and co-injected into the stomach in wild-type mice  and reverse-transcribed into cDNA using the SuperScript IV First-Strand Synthesis System . Primer sets used : CATCGATACCGTCGACACAGGACACCGGGTGCAGTG (forward); CTGCTCGAAGCGGCCGCTGGCACAGTATGATAACCCTCG (reverse). UPB4 (5\u2032\u20133\u2032): CATCGATACCGTCGACATCGATGGGCCTTAGGGAAC (forward); TGCTCGAAGCGGCCGCAACCAGCGATGGCTGTGCC (reverse).17. Approximately 5,000\u201310,000 VSNs were loaded in each channel of the 10X microfluidic device to target 3,000\u20136,000 cells as an output of one sample. Single-cell cDNA libraries were prepared at the Yale Center for Genomic Analysis (YCGA) and sequenced using an Illumina NovaSeq S4 sequencer at 150\u2013300 million reads to achieve a fine sequencing depth of 30,000\u201350,000 reads per cell. For Projection-seq, 30 age- and gender-matched wild-type mice (divided into four samples) were first injected with different Projection-seq AAVs into thoracic and abdominal organs . Vagal ganglia were collected seven days later. Of the examined VSNs, 29.5% (102/346) were tdTomato+. VSNs were sequenced as mentioned above.For control scRNA-seq, vagal ganglia (left and right) were collected from 40 age- and gender-matched C57BL/6J wild-type mice (10 mice per sample) and VSNs were acutely isolated and enriched using previously described methods55, and 42 cell clusters identified using the top 30 principal components (PCs) were visualized using UMAP56  were re-clustered into 67 populations with the top 100 PCs  were re-clustered into 52 clusters with the top 100 PCs and visualized with UMAP separately , and consistently more Sprr1a+ VSNs were observed after AAVrg injection  or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics). The following quality control metrics were applied to filter low-quality cells in scRNA-seq: number of genes per cell\u2009>\u2009500; number of genes per cell\u2009<\u20098,000; percentage of mitochondria genes\u2009<\u200910%. A total of 56,575 cells were sequenced . Control scRNA-seq and Projection-seq data were then integrated and processed using the R package Seurat v.3tely Fig.. DEGs fotely Fig.. VSN cluPrdm12+ (2.5%) neural-crest-derived jugular VSNs and 4,791 out of 14,590 (32.8%) Phox2b+ placode-derived nodose VSNs59 were recognized as UPB-positive (expression level\u2009>\u20090.8), suggesting that VSNs retrogradely labelled from the seven major visceral organs in our study mainly originate from the nodose but not the jugular ganglia. We therefore focused on nodose VSNs. After removing E-VSNs, 3,539 out of 4,609 UPB-marked nodose VSNs (76.8%) expressed a single barcode , and 740 out of 4,609 (16.1%) VSNs expressed were marked by two UPBs. A correlation matrix . DEGs for organ-specific VSNs, or between thoracic  and abdominal  VSNs were identified using the Wilcoxon rank-sum test . For example, Pou4f1, which is essential for DRG neuron specification32, is preferentially expressed in lung VSNs  using the Upstream Analysis (Upstream Regulators) module. For cell\u2013cell interaction analysis between organ-innervating VSNs and various organ cell types , lung63 , colon64 , duodenum65  and pancreas60 . Cell\u2013cell interactions between organ-innervating UPB+ VSNs and indicated organ cell types  were then analysed using the CellPhoneDB66  Python package. GO pathway analyses of DEGs in heart, lung, gut and pancreas VSNs, and along the tissue trajectory .Further analysis was performed for the Projection-seq dataset. A total of 42 out of 1,701 organ . The organ position score for VSN clusters  as the weight value for each organ, expressed as \u03a3(Porgan-cluster\u2009\u00d7\u2009Positionorgan)/\u03a3Porgan-cluster. The organ trajectory score of an organ-specific VSN , and the tissue layer index for DEG+ VSNs \u2009\u00d7\u2009Indextissue)  Fig. .12. In brief, mice were anaesthetized with 1\u20132% isoflurane and maintained on a heating pad. Both left and right vagal ganglia were surgically exposed. A virus mix containing a 1:1 dilution of AAV9-FLEX-tdTomato and AAV5-CAG-GFP with 0.05% (w/v) Fast Green FCF was injected  using a Nanoject III injector (Drummond). Mice were euthanized four weeks after surgery for tissue collection (see \u2018Histology and immunochemistry\u2019).Vagal ganglia injection was performed as previously describedRNAscope HiPlex assays were performed following the manufacturer\u2019s protocol (Advanced Cell Diagnostics). Vagal ganglia were acutely dissected and freshly frozen in cryo-embedding medium (OCT). Cryosections  were cut using a cryostat (Thermo Fisher Scientific), mounted onto Superfrost Plus slides (Thermo Fisher Scientific), and stored at \u221280\u2009\u00b0C until use. Slides were immediately immersed into fresh 4% paraformaldehyde (PFA) in RNase-free PBS for 60\u2009min at room temperature, followed by dehydration with 50%, 70% and 100% ethanol. Samples were then digested with protease IV for 30\u2009min at room temperature. After hybridization with designed probes , followed by 25\u2009ml cold 4% PFA. Fast Green FCF injected organs  and transcardially perfused with 15\u2009ml cold PBS (pH 7.4) containing 10\u2009U\u2009mlFor brain samples, 40-\u03bcm cryosections were mounted onto Superfrost Plus slides. Cryosections were washed (3\u00d7 PBS), permeabilized , blocked ) and incubated with primary antibodies  diluted in blocking buffer for 2\u2009h at room temperature. Then, the slides were washed (3\u00d7 PBST), and incubated with fluorophore-conjugated secondary antibodies , 1:1,000, Jackson ImmunoResearch) diluted in blocking buffer for 2\u2009h at room temperature. After incubation, the samples were washed (3\u00d7 PBST), and mounted with Fluoromount-G with DAPI before imaging with the Leica SP8 confocal microscope.72 and stained with the following protocol unless specifically mentioned. In brief, the dissected organ was immersed into 1/2-water-diluted reagent-1  with shaking at 37\u2009\u00b0C for 3\u20136\u2009h, followed by reagent-1 (R1) with shaking at 37\u2009\u00b0C for 7 days. R1 was replaced fresh every two days. Next, the tissue was washed (3\u00d7 PBS/0.01% NaN3), blocked  and incubated with primary antibodies  in blocking buffer with shaking for 7 days at room temperature. Samples were then washed  and incubated with fluorophore-conjugated secondary antibodies diluted in blocking buffer with shaking for five days at room temperature. After antibody incubation, the samples were washed and immersed in 1/2-PBS-diluted reagent-2  overnight at room temperature, and then reagent-2 (R2) at 37\u2009\u00b0C for 2 days. The samples were finally immersed in oil for at least 1\u2009h and flattened to approximately 500\u2009\u03bcm in a custom-built imaging chamber and imaged using the Leica SP8 confocal microscope as described above, with a 10\u00d7 objective  or a 40\u00d7 objective . Some heart samples were similarly processed but not flattened and imaged with a LaVision Vltramicroscope II light-sheet microscope at the CNNR Imaging Core at Yale University , 1:1,000) overnight, respectively, and treated with R2 for one day. Cleared ganglia were imaged with the Leica SP8 confocal microscope.tdT-GCaMP6s mice were continuously anaesthetized (1\u20132% isoflurane/oxygen) during the experiment. A tracheal tube was inserted for air injection. The upper oesophagus and pyloric sphincter were cannulated and flushed with saline multiple times to remove residual food particles. The duodenum (around 0.5\u2009cm below the pyloric sphincter) was cannulated with a bundle of six PE-10 tubing for separate delivery of saline, water and glucose (1\u2009M), Ensure , 10\u00d7 PBS and 150\u2009mM HCl (pH\u2009=\u20090.84). Left vagal ganglia were exposed and immobilized on a stable platform12. During calcium imaging, a series of stimuli were delivered to the same mice in the following sequence: (1) lung inflation for 20\u2009s with 600\u2009ml\u2009min\u22121 flow , twice with a 2-min interval; (2) small intestine stretch via fast injection of 600\u2009\u03bcl saline through the duodenal cannula; stomach stretch with 100\u2009\u03bcl, 300\u2009\u03bcl and 600\u2009\u03bcl saline through the oesophageal cannula for 30\u2009s (duration precisely controlled by closing or opening of the pyloric cannula); (4) small intestine infusion with 100\u2009\u03bcl saline, water, 1\u2009M glucose, Ensure, 10\u00d7 PBS and 150\u2009mM HCl in sequence with a 3-min interval between infusions. GCamp6s fluorescence was measured from two focal planes 15\u2009\u03bcm apart using a two-photon microscope . The imaging frequency for each plane was 1.72\u2009s per frame. At the end, electrical stimulation was applied to the vagus nerve and a z-stack of the ganglia was taken for both GCaMP and tdTomato  signals for cell registration.Gpr65Trpa1 (R1T1), Runx3 (R1T2), Uts2b (R1T3), Gabra1 (R1T4), Slit2 (R1T5), Kcng1 (R1T6), Piezo2 (R1T7), Ddc (R1T8), Vip (R1T9), Trpv1 (R1T10), tdTomato (R1T11), Gpr65 (R1T12), Chodl (R2T1), Glp1r (R2T2), Grm5 (R2T3), Slc17a7 (R2T4), P2ry1 (R2T5), Tmc3 (R2T6), Car8 (R2T7), Nts (R2T8), Cckar (R2T9) and Calca (R2T11). The following criteria were used to categorize VSN subpopulations  to generate successive in vivo imaging planes that resemble RNAscope sections. tdTomato\u2212 neurons were then registered on the basis of their relative distance and depth to their neighbouring tdTomato+ cells  was defined as the average GCaMP6s fluorescence over a 10-frame period  before stimulus induction and neuronal activity was calculated as \u0394F/F. Cells were coded as responsive to a given stimulus if the maximum GCaMP6s fluorescence was more than 100% above baseline during stimulus period . Peak response was identified as the maximum \u0394F/F within the stimulus period. To compare adaptation rates in lung stretch-sensitive VSNs, GCaMP6s traces were aligned at activation frame  reached 10% of the peak response  reached 10% of peak response.Regions of interest were manually extracted from GCaMP images. The stimulus induction frame was set as 0 unless specifically mentioned. Baseline signal . For quantitative analyses of MEs and IMAs in the indicated gastrointestinal regions; alveoli, longitudinal and patch-terminals in the lung; and varicose endings and IMAs in the heart .Four types of gut VSN ending types, three types of heart VSN ending types and five types of lung VSN ending types were classified on the basis of their morphologies and locations using whole-mount preparations in Vglut2ice Fig. ; n\u2009=\u20094\u20137Piezo2), H4 (Drd2) and I3/I4 (Agtr1a). In Piezo2tdT mice, most cardiac afferents were varicose surface endings. In Drd2tdT mice, cardiac afferents densely innervated myocardium with both branched and parallel IMAs. Agtr1a+ VSNs predominantly formed flower-spray endings in the heart and the aortic arch. Our results thus reveal the identity of various VSN cardiac ending types  bud ending wrapping around NEBs  near the bronchial bifurcation with branch endings  and examined IMAs around pyloric sphincters in P2ry1tdT and Calb2tdT mice. Most (75.0%) of the P2ry1+ IMAs around the oesophageal sphincter were pIMAs and all Calb2+ IMAs were cIMAs , H2 (Vip) and C4 , whereas all afferent types in Vglut2tdT mice were enriched in region 8 around the pyloric sphincter. Both Sst+ and Gpr65+ VSNs formed MEs on the stomach, as reported16, with Sst+ endings mainly in the antrum and Gpr65+ endings more evenly distributed across the stomach, indicating that F1-VSNs form MEs. Both Glp1r+ and Piezo2+ VSNs predominantly formed IGLEs, suggesting that C4-VSNs form stomach IGLEs. Vip+ VSNs exhibited pIMA morphology in region 8, suggesting that H2-VSNs also form pIMAs  for each ending type  in each stomach region , calculated as the innervation intensity of corresponding ending types in various stomach regions normalized by the innervation intensity of the same ending type in the entire stomach in Vglut2tdT mice  of each ending type  in each stomach region  was calculated as the percentage of corresponding dual-labelled VSNs forming this ending type normalized to the percentage of stomach-UPB single-labelled VSNs forming this ending type, expressed as \u03a3/\u03a3. We then calculated the total variance between anatomically and Projection-seq-derived innervation intensities in all stomach regions across all ending types, expressed as \u03a3 (SFnME- \u2212 AFnME-)2 + \u03a3 (SFnpIMA- \u2212 AFnpIMA-)2 + \u03a3 (SFncIMA- \u2212 AFncIMA-)2 + \u03a3 (SFnIGLE- \u2212 AFnIGLE-)2. The trial with the lowest variance was defined as the best fit. AFafferent type-stomach region and SFafferent type-stomach region for this condition were plotted together , Vip (H3/G2/G5), Glp1r (G2/G5), and Agtr1a (I5) , suggesting that both F- and G-VSN clusters form MEs. By contrast, Agtr1a+ neurons representing I5-VSNs primarily formed IGLEs in the duodenum , C5\u2013C8 VSNs also form oesophageal IMAs. Probably owing to low infection efficiency, no apparent oesophageal ME or IGLE clusters were revealed. Notably, among all Cre lines examined, afferents in NTStdT mice preferentially formed oesophageal IGLEs , expressed as (number of connections \u2212 minimum number of possible connections)/(maximum number of possible connections \u2212 minimum number of possible connections), for each pair of characteristics. We next calculated the statistical variance of connections per variable for both characteristics (v1 and v2). As both variances contribute to the correlation equally, the correlation index was finally calculated as (1\u2009\u2212\u2009C)/((v1\u2009+\u20091)\u2009\u00d7\u2009(v2\u2009+\u20091)).Correlations among various VSN characteristics, including 7 visceral organs , 4 tissue layer types , 6 VSN ending types , 11 VSN subpopulations and 4 response patterns , were plotted in Fig. Mouse line and organ combinations were selected on the basis of Projection-seq anterograde tracing results: lung alveoli ending , lung NEB ending , oesophageal IGLE , oesophageal IMA , heart IMA , stomach IGLE , stomach mucosal ending , stomach IMA , duodenal IGLE , duodenal mucosal ending  and colon IGLE . Organ injection of AAVrgs is described in \u2018AAV infection of visceral organs\u2019. Brain processing and imaging is described in \u2018Histology and immunochemistry\u2019.Bregma-subnucleus) was calculated as average fluorescence in the sub-nucleus minus background fluorescence measured in a region in the sub-nucleus with no fluorescence-labelled vagal fibres; (2) the area of each sub-nucleus (ABregma-subnucleus) was measured; (3) the total fluorescence (TFBregma) was calculated as \u03a3 (FIBregma-subnucleus\u2009\u00d7\u2009ABregma-subnucleus); and (4) the percentage innervation (PI) of a sub-nucleus at a certain Bregma was calculated as FIBregma-subnucleus\u2009\u00d7\u2009ABregma-subnucleus/TF\u2009\u00d7\u2009100. The correlation variance between VSNs labelled from various visceral organs 2 and a phylogenetic tree was generated based on the correlation variance matrix using the seqlinkage function in MATLAB.Area innervated by vagal afferents retrogradely labelled from various visceral organs at different Bregma levels  were measured using Fiji (ImageJ) , heart (n\u2009=\u20093); varicose: heart (n\u2009=\u20095), aorta (n\u2009=\u20096); pIMA: colon (n\u2009=\u20093), heart (n\u2009=\u20096); cIMA: stomach (n\u2009=\u20094), oesophagus (n\u2009=\u20093); epithelial: stomach (n\u2009=\u20094), lung (n\u2009=\u20095); bud: lung (n\u2009=\u20095), oesophagus (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20092; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. n\u2009=\u20093), stomach (n\u2009=\u20094), duodenum (n\u2009=\u20093), colon (n\u2009=\u20093), aorta (n\u2009=\u20096); Extended Data Fig. n\u2009=\u20093; Extended Data Fig. n\u2009=\u20094; Extended Data Fig. Piezo2 (n\u2009=\u20099), Drd2 (n\u2009=\u20096), Agtr1a (n\u2009=\u20093), Npr2r (n\u2009=\u20092); Extended Data Fig. n\u2009=\u20094; Extended Data Fig. Npy2r (n\u2009=\u20093), Trpv1 (n\u2009=\u20095), P2ry1 (n\u2009=\u20096), Agtr1a (n\u2009=\u20094), Piezo2 (n\u2009=\u20099), Vglut1 (n\u2009=\u20094), Pvalb (n\u2009=\u20093), Twist2 (n\u2009=\u20093); Extended Data Fig. P2ry1 (n\u2009=\u20094), Calb2 (n\u2009=\u20093), Glp1r (n\u2009=\u20095), Vip (n\u2009=\u20095), Gpr65 (n\u2009=\u20097), Sst (n\u2009=\u20095), Agtr1a (n\u2009=\u200912); Extended Data Fig. Glp1r (n\u2009=\u20093), Agtr1a (n\u2009=\u20094); Extended Data Fig. Trpv1 (n\u2009=\u20094), Agtr1a (n\u2009=\u20093); Extended Data Fig. Nts (n\u2009=\u20095), Trpv1 (n\u2009=\u20093), Agtr1a (n\u2009=\u20094), Gpr65 (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20095; Extended Data Fig. n\u2009=\u20093), heart (n\u2009=\u20093), oesophagus (n\u2009=\u20092), stomach (n\u2009=\u20093), duodenum (n\u2009=\u20093), colon (n\u2009=\u20093), pancreas (n\u2009=\u20093); Extended Data Fig. n\u2009=\u20093 for all groups.Representative images and experiments were repeated independently in multiple mice with similar results: Fig. Further information on research design is available in the\u00a0Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04515-5.Supplementary Figure 1Original source image for electrophoresis, related to Extended Data Fig. 2b. Red box indicates how the gel was cropped for the final figure.Reporting SummarySupplementary Table 1Detailed information for RNAscope probes."}
+{"text": "However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated. Here, we demonstrate that FTO expression is downregulated and inversely correlated with poor survival of lung adenocarcinoma patients. Mechanistically, Wnt signaling induces the binding of EZH2 to \u03b2-catenin. This protein complex binds to the LEF/TCF-binding elements at the promoter region of FTO, where EZH2 enhances H3K27me3 and inhibits FTO expression. Downregulated FTO expression substantially enhances the m6A levels in the mRNAs of a large number of genes in critical pathways, particularly metabolic pathway genes, such as MYC. Enhanced m6A levels on MYC mRNA recruit YTHDF1 binding, which promotes MYC mRNA translation and a subsequent increase in glycolysis and proliferation of tumor cells and tumorigenesis. Our findings uncovered a critical mechanism of epitranscriptome regulation by Wnt/\u03b2-catenin-mediated FTO downregulation and underscored the role of m6A modifications of MYC mRNA in regulating tumor cell glycolysis and growth.FTO removes the N6-methyladenosine (m The abundance and effects of m6A on RNA depend on the dynamic and integrated regulation by \u201cwriters\u201d and \u201cerasers\u201d, which add and remove the methylation, respectively, and \u201creaders\u201d, which recognize the modification8. The identified writers include methyltransferase-like (METTL) 3/14, Wilms tumor 1-associated protein (WTAP), RNA binding motif protein 15/15B (RBM15/15B), and KIAA1429, whereas erasers include fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). YT521-B homology (YTH) domain-containing proteins , heterogeneous nuclear ribonucleoprotein (HNRNP) protein families, and IGF2 mRNA binding proteins (IGF2BP) families are regarded as readers9. FTO, as the first m6A demethylase identified11, regulates the m6A modification of critical genes in different types of cancer, such as glioblastoma12, acute myeloid leukemia (AML)14, cervical squamous cell carcinoma (CSCC)15, and breast cancer16. However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated.N6-Methyladenosine : normal rabbit IgG (# 2729) (for immunoprecipitation and ChIP), \u03b2-catenin (#8480) (for immunoblotting and immunoprecipitation), FTO (#31687) (for immunoblotting and RIP), EZH2 (#5246) (for immunoprecipitation and ChIP), TCF4 (#2569) (for ChIP), H3K27me3 (#9733) (for ChIP), H3K9me2 (#4658) (for ChIP), HK2 (#2867) (for immunoblotting and IHC), and Ki-67 (#9449) (for IHC). A mouse monoclonal antibody against tubulin (T9026) (for immunoblotting) was purchased from Sigma-Aldrich . Rabbit antibodies recognizing c-Myc (ab32072) (for immunoblotting and IHC), Wnt-3a (ab219412) (for IHC) and FTO (ab124892) (for IHC) were purchased from Abcam . Rabbit antibody recognizing YTHDF1 (17479-1-AP) (for RIP and immunoblotting) was purchased from Proteintech . RIPA lysis and extraction buffer (89901) and Pierce IP lysis buffer (87787) were purchased from Thermo Fisher Scientific . Protein A/G plus-agarose (sc-2003) was purchased from Santa Cruz Biotechnology . Wnt-3a (5036-WN) was obtained from R&D Systems . Cycloheximide (CHX) (HY-12320) and nitro blue tetrazolium chloride (HY-15925) were purchased from MedChem Express . Agar (1182GR500) was purchased from BIO FROXX .http://xena.ucsc.edu/welcome-to-ucsc-xena/). Gene transcription estimates for each gene were analyzed with RNA-Seq using Expectation Maximization (RSEM) software.The clinical records and RNAseqV2 level 3 gene-level lung adenocarcinoma data were downloaded from TCGA (Forty pairs of frozen tissues for RNA extraction and 83 pairs of frozen tissues for immunohistochemistry (IHC) were obtained from patients with lung adenocarcinoma who underwent radical resections in the Department of Thoracic Surgery of the Cancer Hospital, Chinese Academy of Medical Sciences. The clinical features of the patients are summarized in Table S17. Section of lung adenocarcinoma TMA was stained with an antibody against FTO. The tissue sections were quantitatively scored according to the percentage of positive cells and staining intensity as described previously18. The following proportion scores were assigned to the sections: 1, 0\u20131%; 2, 2\u201310%; 3, 11\u201330%; 4, 31\u201370%; and 5, 71\u2013100%. The staining intensity was rated on a scale of 0\u20133: 0, negative; 1, weak; 2, moderate, and 3, strong. Then the proportion and intensity scores were combined to obtain a total score  as described previously18.Eighty-three pairs of frozen tissues from lung adenocarcinoma patients were formalin-fixed and paraffin-embedded. The tissue microarray (TMA) was constructed as previously described2 at 37\u2009\u00b0C in a humidified incubator. And all these cells were routinely tested for mycoplasma. When cells were 50% confluent, the medium was replaced with a fresh medium containing 0.5% serum for 1 day, and then Wnt-3a was added at a final concentration of 60\u2009ng/ml for cell stimulation.H322 and HEK 293T cells were grown in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin. H358 cells were grown in RPMI 1640 supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin. Cells were cultured in 5% CO19. The relative mRNA expression levels were calculated by the 2\u2212\u0394\u0394Ct method with normalization to ACTB or GAPDH; the PCR primers are listed in Table STotal RNA was isolated with TRIzol reagent  according to the manufacturer\u2019s instructions. RNA (1000\u2009ng) was reverse-transcribed into cDNA with a RevertAid First Strand cDNA Synthesis kit (Thermo); SYBR Green-based qRT-PCR was performed using a 7900HT fast real-time PCR system , as described previously20. The shRNA sequences are listed in Table SPlasmids containing transgenes and packaging plasmids were cotransfected into HEK 293T cells using Lipofectamine 3000 . Viruses were collected and concentrated after 48\u2009h. When tumor cells reached 50%-60% confluence, we infected the cells with concentrated virus and then selected them by antibiotic treatment21.Extraction of proteins with a modified buffer from cultured cells was followed by immunoprecipitation and immunoblotting with antibodies, as described previously22. The primers are listed as follows:ChIP assays were performed using the SimpleChIP\u00ae Enzymatic Chromatin IP kit  according to the manufacturer\u2019s instructions, as described previouslyFTO promoter Forward: GTTATCCTTCTTTGCTCACTATGC;binding site within the FTO promoter Reverse: CTGAGGAAGTGAACTGAGCTC.binding site within the 6A reporter assays, the DNA fragments of MYC-CDS containing the wild-type m6A motifs, as well as the mutated motifs (m6A was replaced by T) were inserted into the Xhol site of the pMIR-REPORT luciferase reporter vector. Dual-luciferase reporter assays were performed, as described previously in HEK 293T cells23.For the promoter-reporter assay, the wild-type DNA oligos of the FTO promoter and the mutated oligos with three LEF/TCF-binding element (TBE) deletions were inserted into the upstream region of the firefly luciferase of the PGL4.1 vector. For mSequences of wild type FTO promoter:CTTTGCTCACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CCTTTGTATTAGCTGGTTCCTTAATCTTTGGAGCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCACGGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTSequences of truncated FTO promoter:GGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CTAGCTGGTTCCTTAATCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCAC6A sites:MYC-CDS with wild-type mGGACTTGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTGCGGTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA6A sites:MYC-CDS with the mutated mGGTCTTGTTGCGGAAACGACGAGATCAGTTGAATCACAATCTTGATCAGCTACGGATCTCTTGTGCG.GTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA5 cells were seeded in a 6-well plate and maintained in a medium with 10% FBS for different periods of time. The cells were harvested and counted24.2\u2009\u00d7\u20091025. Briefly, for the bottom layer of agar, we deposited the mix of 1% agar and 2\u00d7 medium into each well of a six-well plate. For the upper layer of agar, we deposited the mix of 0.6% agar and a suspension of cells in each well (10000 cells/well). The cells were cultured for 21 days in a 37\u2009\u00b0C humidified cell culture incubator and then were stained with nitroblue tetrazolium chloride solution (200 \u03bcl/ well) and incubated overnight at 37\u2009\u00b0C.The assay was performed as described previously26.Migration and invasion assays were performed in the chamber coated with or without Matrigel matrix  according to the manufacturer\u2019s instructions as previously described6 Cells were seeded in 6\u2009cm plates. After incubation for 24\u2009h, the culture medium was replaced and the cells were arrested at the S phase by adding thymidine (2\u2009mM) for 24\u2009h. The thymidine was then removed, and the cells were washed by PBS and cultured in a fresh culture medium for 3\u2009h to release cells before nocodazole treatment (100\u2009ng/ml) for 12\u2009h to arrest cells in the G2/M phase. Then, the cells were washed with PBS and cultured in a fresh medium to release cells27.2\u2009\u00d7\u200910g for 5\u2009min, followed by washing with PBS. The cells were then fixed in 1\u2009ml ice-cold 70% ethanol at 4\u2009\u00b0C for 24\u2009h and centrifuged at 500 \u00d7 g for 5\u2009min. After being washed with PBS, the fixed cells were fully resuspended in 1\u2009ml of 2\u2009M HCl/0.5% Triton X-100 solution and incubated at room temperature for 30\u2009min. The cells were spun down and neutralized in 1\u2009ml of 0.1\u2009M Na2B4O7 solution . Then, the cells were spun down and washed with cold PBS containing 1% BSA and incubated with mouse anti-BrdU  at 4\u2009\u00b0C for 12\u2009h. Afterward, centrifuged cells were washed with cold PBS containing 1% BSA and incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG  in room temperature for 1\u2009h. Finally, the cells were spun down and washed with cold PBS containing 1% BSA, followed by resuspended in 300\u2009\u03bcl of PBS containing 5\u2009\u03bcg/ml DAPI for 15\u2009min at room temperature, and then cell cycle progression was analyzed by flow cytometry.One hour before harvesting cells at each time point, BrdU  (20\u2009\u03bcM) was used to label the cells. The cells were collected by centrifugation at 500 \u00d7 18.Cells were seeded in culture dishes, and the medium was changed when cells reached 50% confluence. After incubation for 12\u201324\u2009h, the culture medium was collected. The glucose levels were detected by a glucose colorimetric assay kit , and the lactate levels were detected by a lactate colorimetric assay kit  according to the manufacturer\u2019s instructions, and values were calculated as previously described6 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4\u20135 weeks). The width (W) and length (L) of the tumors were measured every three days, and the volume (V) of each tumor was calculated using the formula V\u2009=\u2009(W2\u2009\u00d7\u2009L/2). For lung colonization assays, 1\u2009\u00d7\u2009106 cells were injected into the tail vein of female NOD/SCID mice (6\u20137 weeks), and 6 weeks later the lung was removed and fixed with 10% formalin. Fixed lung tissues were embedded in paraffin and cut into consecutive sections. These sections were stained by hematoxylin and eosin (H&E)28. All animal experiments were approved by the Animal Care and Use Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences.Mice were randomized into several groups. For the subcutaneous implantation model, 1\u2009\u00d7\u200910A Magna RIP kit  was used to perform the RIP assays. A sufficient number of H322 cells were lysed by RIP lysis buffer, and the supernatant of the RIP lysate was incubated with specific antibodies on beads overnight at 4\u2009\u00b0C. After washing, RNA was extracted and analyzed by qRT-PCR.6A-seq was performed by Cloudseq Biotech Inc.  according to the published procedure with slight modifications1. Briefly, fragmented mRNA was incubated with an anti-m6A polyclonal antibody  in IPP buffer for 2\u2009h at 4\u2009\u00b0C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4\u2009\u00b0C for an additional 2\u2009h. The bound RNA was eluted from the beads with N6-methyladenosine  in IPP buffer, and then the RNA was extracted with TRIzol reagent (Thermo Fisher) following the manufacturer\u2019s instructions. Purified RNA was used for RNA-seq library generation with the NEBNext\u00ae Ultra\u2122 RNA Library Prep kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150\u2009bp paired-end sequencing on an Illumina HiSeq 4000 sequencer.m29. After that, clean reads of all libraries were aligned to the reference genome by Hisat2 software (v2.0.4)30. Methylated sites on RNAs (peaks) were identified by MACS software31. Differentially methylated sites were identified by diffReps32. These peaks identified by both software and overlapped with exons of mRNAs were selected. Raw counts of mRNA sequencing were got by HTSeq software (v0.9.1) and normalized by edgeR software.Paired-end reads were harvested from Illumina HiSeq 4000 sequencer and were quality-controlled by Q30. After 3\u2032 adapter-trimming and low-quality reads removal by cutadapt software (v1.9.3), clean reads of input libraries were aligned to reference genome (UCSC HG19) by STAR software6A kit  was used according to the manufacturer\u2019s instructions. m6A enrichment was analyzed by qPCR with specific primers, and data were normalized to input. Primer sequences were as follows:Total RNA was isolated with a miRNeasy kit  with DNase I digestion. mRNA was extracted from total RNA using a GenElute mRNA Miniprep kit . Then, a Magna MeRIP mMYC Forward: TTGCGGAAACGACGAGAACA;MYC Reverse: TCATAGGTGATTGCTCAGGACA.33.Cycloheximide (CHX) was added to the cell culture at a final concentration of 100\u2009\u03bcg/ml for 10\u2009min to stop translation. Cells were washed with cold PBS and lysed with lysis buffer. The cell lysate was loaded onto the top of a 10\u201350% sucrose gradient tube immediately, and the tube was centrifuged at 36,000\u2009rpm for 3.5\u2009h at 4\u2009\u00b0C. The sample was separated into 12 fractions by a fraction collector and measured at 254\u2009nm. RNA was extracted by TRIzol and subjected to qPCR analysis. The relative expression of MYC in each fraction was normalized to GAPDH, as well as to the inputt-tests to compare means between two groups; p\u2009<\u20090.05 was considered significant.All data are expressed as the mean\u2009\u00b1\u2009SD. We used two-tailed Student\u2019s 35. As a major type of non-small-cell lung carcinoma, lung adenocarcinoma (LUAD) remains one of the most aggressive and fatal tumor types36. To determine the expression of FTO in lung adenocarcinoma, we analyzed TCGA data and revealed that FTO expression levels were much lower in lung adenocarcinoma tissues than in their adjacent normal tissues  accounts for 85% of cases, is the most commonly occurring cancer and the leading cause of cancer deathues Fig. . To valiues Fig. . Consistues Fig. C, D. Kapues Fig. . These rTo determine the role of FTO in lung adenocarcinoma progression, we depleted FTO expression in H322 Fig.  and H358We next subcutaneously injected H358 cells with or without FTO depletion into athymic nude mice. As shown in Fig. FTO promoter sequence with PROMO software and identified three potential LEF/TCF-binding elements (TBE)  assays demonstrated that Wnt stimulation increased the binding of TCF4 to the promoter region of FTO  Fig. S that areFTO Fig. . LuciferRNA Fig.  and protRNA Fig.  levels oion Fig. . These r42. ChIP analyses with anti-H3K27me3 and anti-H3K9me2 antibodies showed that Wnt stimulation significantly enriched H3K27me3  27 (H3K27me3) and dimethylation of histone H3 K9 (H3K9me2) are marks of transcription repressionme3 Fig.  but not me3 Fig.  in the Tnin Fig.  and the ion Fig. , as deteion Fig. . These rWe next examined the clinical relevance of Wnt signaling component expression and FTO expression by TCGA analysis. We found that expression of a positive regulator of Wnt signaling Frizzled (FZD)9 and DVL1 was negatively correlated with FTO expression whereas expression of negative regulators of Wnt signaling APC, GSK3\u03b2, and AXIN2 was positively correlated with FTO expression in lung adenocarcinoma Fig. . These r6A RNA sequencing (m6A-seq) on H322 cells with or without FTO depletion. We showed that m6A peaks primarily occurred in a sequence context as GGAC (P\u2009=\u20093.5e10\u201341)  Fig. S and were1) Fig. S, which aays Fig. , which iion Fig. . MethylaRNA Fig. , FTO ovels Fig. S. Consistlls Fig. . These r6A regulation on c-Myc expression, we constructed a luciferase reporter gene with an integrated CDS sequence containing the WT or mutated m6A sites from the 3\u2032 end of MYC mRNA , but not in subunits smaller than 40S, 40S, and 60S   Table S. As expe) Table SA, B. Not) Table SC, D. Cor) Table S, S5E, la) Table S, S5F, ce) Table S, migrati) Table S, S5J of 48. Of note, the FTO depletion-enhanced these protein expressions were largely reduced by c-Myc depletion (Fig. We next subcutaneously injected H358 cells with or without FTO depletion or combined FTO and c-Myc depletion into athymic nude mice. We showed that FTO depletion-promoted tumor growth was blunted by c-Myc depletion Fig. . Immunohion Fig. . In addiion Fig. , furtherion Fig. . These r6A modification from mRNAs, plays instrumental roles in the regulation of many cellular functions7. However, how FTO is transcriptionally regulated, especially by oncogenic signaling, remains to be explored. We demonstrated here that FTO expression was downregulated in lung adenocarcinoma and positively correlated with overall survival. Depletion of FTO enhanced tumor cell proliferation, anchorage-independent growth, migration, invasion, and tumor formation in mice. Of special interest, we found that Wnt signaling, which is vital in lung adenocarcinoma tumorigenesis51, induced the binding of \u03b2-catenin/TCF/LEF to the TBEs of the FTO promoter region and suppressed FTO expression. Mechanistically, we showed that WNT signaling induced the binding of EZH2 to \u03b2-catenin, leading to an EZH2-dependent H3K27me3 increase at the FTO promoter region for inhibition of FTO expression. Downregulation of FTO expression enhanced m6A levels on mRNAs in a number of signaling pathways, including metabolic pathways, in which MYC is a master regulator of gene expression for glycolysis45. Enhanced m6A levels on MYC mRNA recruited the binding of YTHDF1 and enhanced MYC mRNA translation. FTO depletion and YTHDF1-dependent upregulation of c-Myc promoted tumor cell glycolysis, growth, migration, and invasion and accelerated tumor growth in mice and tumor metastasis to the lung (Fig. In addition to epigenetic regulation of gene expression, regulation of the epitranscriptome exerts another critical layer to regulate protein expression. FTO, a key demethylase that removes the mMYC gene transcription can be upregulated directly by \u03b2-catenin/TCF/LEF45. FTO downregulation can upregulate Wnt signaling by increasing m6A modification in FZD10 mRNA52. In addition, m6A-dependent and YTHDF2-dependent decreases in MYC transcript stability in leukemia cells were reported14. We showed that Wnt stimulation suppressed FTO expression, therefore inducing m6A-dependent and YTHDF1-dependent increases in MYC mRNA translation. Given that FTO globally regulates the expression of many genes, which can be signaling context-dependent and cancer type-dependent16, the differential and distinct regulation of m6A in mRNAs downstream of the FTO exhibit wide variation and may elicit distinct cellular activities. Our finding that FTO downregulation induced by Wnt/\u03b2-catenin signaling enhanced c-Myc expression through upregulation of m6A of MYC mRNA and subsequent YTHDF1 binding revealed a novel mechanism by which lung adenocarcinoma promotes glycolysis and growth. The discovery of the novel regulation of c-Myc expression may lead to an alternative approach for the therapeutic treatment of lung adenocarcinoma.Supplementary figure legendsSupplementary table 1Supplementary table 2Supplementary table 3Supplementary table 4Supplementary table 5Supplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5"}
+{"text": "Accumulating evidences supported that knock-down of DHCR24 is linked to the pathological risk factors of AD, suggesting a potential role of DHCR24 in AD pathogenesis. However, the molecular mechanism link between DHCR24 and tauopathy remains unknown. Here, in order to elucidate the relationship between DHCR24 and tauopathy, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some toxic sites. In present study, we found that DHCR24 knock-down significantly lead to the hyperphosphorylation of tau sites at Thr181, Ser199, Thr231, Ser262, Ser396. Moreover, DHCR24 knock-down also increase the accumulation of p62 protein, simultaneously decreased the ratio of LC3-II/LC3-I and the number of autophagosome compared to the control groups, suggesting the inhibition of autophagy activity. In contrast, DHCR24 knock-in obviously abolished the effect of DHCR24 knock-down on tau hyperphosphrylation and autophagy. In addition, to elucidate the association between DHCR24 and tauopathy, we further showed that the level of plasma membrane cholesterol, lipid raft-anchored protein caveolin-1, and concomitantly total I class PI3-K (p110\u03b1), phospho-Akt (Thr308 and Ser473) were significantly decreased, resulting in the disruption of lipid raft/caveola and inhibition of PI3-K/Akt signaling in silencing DHCR24 SH-SY5Y cells compared to control groups. At the same time, DHCR24 knock-down simultaneously decreased the level of phosphorylated GSK3\u03b2 at Ser9 (inactive form) and increased the level of phosphorylated mTOR at Ser2448 (active form), leading to overactivation of GSK3\u03b2 and mTOR signaling. On the contrary, DHCR24 knock-in largely increased the level of membrane cholesterol and caveolin-1, suggesting the enhancement of lipid raft/caveola. And synchronously DHCR24 knock-in also abolished the effect of DHCR24 knock-down on the inhibition of PI3-K/Akt signaling as well as the overactivation of GSK3\u03b2 and mTOR signaling. Collectively, our data strongly supported DHCR24 knock-down lead to tau hyperphosphorylation and the inhibition of autophagy by a lipid raft-dependent PI3-K/Akt-mediated GSK3\u03b2 and mTOR signaling. Taking together, our results firstly demonstrated that the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies. Previous studies found that there is a significant reduction in the expression of new gene in specific vulnerable brain regions in Alzheimer's disease (AD) patients, which was named selective AD indicator 1 (Seladin-1), or 3\u03b2-hydroxysterol-\u039424 reductase (DHCR24)  and mammalian target of rapamycin (mTOR) signaling, which are involved in the abnormal hyperphosphorylation of some tau sites in tauopathy. In our experiment, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some sites such as Thr181, Ser199, Thr231, Ser262, Ser256, Ser396, and autophagy, in order to elucidate the relationship between DHCR24 downregulation and tauopathy.DHCR24 is known as the key synthetase in cholesterol synthesis, which catalyzes the final step of cholesterol synthesis via the Bloch pathway, or converts lanosterol to cholesterol at the first step in the Kandutsch-Russell pathway , AKt (#4691), Phospho-Akt (T308) (#13038), Phospho-Akt (Ser473) (#4060), PI3 Kinase p110\u03b1 (#4249), GSK-3\u03b2 (#12456), Phospho-GSK-3\u03b2(Ser9) (#5558), mTOR (#2983), Phospho-mTOR (#2971), Tau (#46687), Phospho-Tau (Thr181) (#12885), Phospho-Tau (Ser199) (#29957), Phospho-Tau (Ser396) (#9632), LC3B (#3868), P62/SQSTM1 (#23214), GAPDH (#2118), secondary antibodies anti-rabbit IgG, HRP-linked antibody (#7074), Anti-rabbit IgG (H+L) (#5366), Anti-mouse IgG, HRP-linked antibody (#7076) were purchased from Cell Signaling Tech (USA), Primary antibodies against Caveolin-1 (ab2910), Phospho-Tau (Ser262) (ab131354) were purchased from British abcam Company, Primary antibodies against Phospho-Tau (Thr231) (4137) was purchased from China ABclonal Company. Primers were purchased from Sangon Biotech . Propidium Iodide (abs9105), Methyl-\u03b2-cyclodextrin (abs42021762), Filipin III (abs42018484) were purchased from China Absin Company.SH-SY5Y cells were obtained from Chinese Academy of Sciences  and kept in DMEM F12  with 10% fetal bovine serum . Following ATCC protocols, all cells were cultured in a 5% CO2 humidified incubator at 37\u00b0C.With regard to lentivirus transfection, lentivirus at multiplicity of infection (MIO) about five mediated DHCR24 shRNA, DHCR24 cDNA, and NC infected into the SH-SY5Y cells. At 48 h after infection, the medium was replaced with fresh complete growth medium containing the appropriate concentration of puromycin (6 \u03bcg/mL). At 3 to 4 days after infection, the selected cells were used for experiments. Note: ShRNA target sequence was as follow: (5'-GCATCATCCCTGCCAAGAAGT-3'); The empty vector sequence was as follow: (5'-TTCTCCGAACGTGTCACGT-3'), cDNA sequence was as follow: (5' -ATGGAGCCCGCCGTGTCGCTGGCCGTGTGCGCGCTGCTCTTCCTGCTGTGGGTGCGCCTGAAGGGGCTGGAGTTCGTGCTCATCCACCAGCGCTGGGTGTTCGTGTGCCTCTTCCTCCTGCCGCTCTCGCTTATCTTCGATATCTACTACTACGTGCGCGCCTGGGTGGTGTTCAAGCTCAGCAGCGCTCCGCGCCTGCACGAGCAGCGCGTGCGGGACATCCAGAAGCAGGTGCGGGAATGGAAGGAGCAGGGTAGCAAGACCTTCATGTGCACGGGGCGCCCTGGCTGGCTCACTGTCTCACTACGTGTCGGGAAGTACAAGAAGACACACAAAAACATCATGATCAACCTGATGGACATTCTGGAAGTGGACACCAAGAAACAGATTGTCCGTGTGGAGCCCTTGGTGACCATGGGCCAGGTGACTGCCCTGCTGACCTCCATTGGCTGGACTCTCCCCGTGTTGCCTGAGCTTGATGACCTCACAGTGGGGGGCTTGATCATGGGCACAGGCATCGAGTCATCATCCCACAAGTACGGCCTGTTCCAACACATCTGCACTGCTTACGAGCTGGTCCTGGCTGATGGCAGCTTTGTGCGATGCACTCCGTCCGAAAACTCAGACCTGTTCTATGCCGTACCCTGGTCCTGTGGGACGCTGGGTTTCCTGGTGGCCGCTGAGATCCGCATCATCCCTGCCAAGAAGTACGTCAAGCTGCGTTTCGAGCCAGTGCGGGGC CTGGAGGCTATCTGTGCCAAGTTCACCCACGAGTCCCAGCGGCAGGAGAACCACTTCGTGGAAGGGCTGCTCTACTCCCTGGATGAGGCTGTCATTATGACAGGGGTCATGACAGATGAGGCAGAGCCCAGCAAGCTGAATAGCATTGGCAATTACTACAAGCCGTGGTTCTTTAAGCATGTGGAGAACTATCTGAAGACAAACCGAGAGGGCCTGGAGTACATTCCCTTGAGACACTACTACCACCGCCACACGCGCAGCATCTTCTGGGAGCTCCAGGACATTATCCCCTTTGGCAACAACCCCATCTTCCGCTACCTCTTTGGCTGGATGGTGCCTCCCAAGATCTCCCTCCTGAAGCTGACCCAGGGTGAGACCCTGCGCAAGCTGTACGAGCAGCACCACGTGGTGCAGGACATGCTGGTGCCCATGAAGTGCCTGCAGCAGGCCCTGCACACCTTCCAAAACGACATCCACGTCTACCCCATCTGGCTGTGTCCGTTCATCCTGCCCAGCCAGCCAGGCCTAGTGCACCCCAAAGGAAATGAGGCAGAGCTCTACATCGACATTGGAGCATATGGGGAGCCGCGTGTGAAACACTTTGAAGCCAGGTCCTGCATGAGGCAGCTGGAGAAGTTTGTCCGCAGCGTGCATGGCTTCCAGATGCTGTATGCCGACTGCTACATGAACCGGGAGGAGTTCTGGGAGATGTTTGATGGCTCCTTGTACCACAAGCTGCGAGAGAAGCTGGGTTGCCAGGACGCCTTCCCCGAGGTGTACGACAAGATCTGCAAGGCCGCCAGGCACTGA-3').Total RNA was extracted using RNAiso Plus  according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript\u2122 RT Master Mix  with specific primers see . ReactioSH-SY5Y cells seeded in twelve-well-plate were incubated for 12-20 h, followed by washing three times with PBS, then fixed with 4% paraformaldehyde at room temperature for 30 min, followed by washing three times with PBS.0.5% Triton X-100 was adopted to permeate cells for 20 min. Cells were blocked with 5% bovine serum Albumin (BSA) in PBS at room temperature for 1 h, then incubated with the primary antibody at 4\u00b0C overnight followed by three times washing with PBS. Samples were incubated in secondary antibody in the dark for 1 h followed by stained with DAPI (0.5 \u03bcg/mL) for 5 min at room temperature. Finally, cells were observed and the fluorescent images were captured by a confocal microscope .After designated treatment, lysates were generated by placing the cells in SDS lysis buffer containing a cocktail of protease and phosphatase inhibitors. BCA was performed to determine total protein concentrations, which were normalized to 1 mg/mL for all samples. Samples were then prepared in sample buffer and heated to 100\u00b0C for 8 min. Equal amounts of lysates were fractionated using 8\u201312% sodium dodecyl sulfate polyacrylamide gels and were then electrotransferred onto nitrocellulose membranes. Gels were run at a constant voltage (80V) for 2\u20133 h for maximum separation. Wet transfer was performed for 120 min at constant current (250 mA) using polyvinylidenedifluoride membrane presoaked in methanol. The membrane was blocked in 5% milk in 0.2% TBST. The membrane was then washed three times in TBST for 10 min each. After overnight incubation at 4\u00b0C with the primary antibodies  the blots were washed and exposed for 1 h at room temperature to corresponding HRP-conjugated secondary antibodies. Chemiluminescent  detection was then used to detect the expression of each protein; GAPDH level served as internal loading controls.Cells were grown on coverglass bottom dishes. In order to allow a better visualization of intracellular cholesterol pools, cells were treated with 10 mM methyl-\u03b2-cyclodextrin  for 30 min at 37\u00b0C to remove cholesterol from the plasma membrane. Cells were then fixed for 10 min at RT with 4% paraformaldehyde in phosphate-buffered saline (PBS). Cells were incubated with a solution of filipin  for 30 min. Finally, after two washes, cells were counterstained with propidium iodide  for 5 min. Acquiring images with a confocal laser scanning microscope equipped with a 380 nm optically pumped semiconductor laser . Whole-cell cholesterol staining with filipin did not require M\u03b2CD treatment.SH-SY5Y cells were treated by stationary liquid which contained 3% glutaraldehyde and 0.22 mmol/L sucrose phosphate buffer for 4 h, then rinsed three times with 0.1 M phosphoric acid rinsing solution for 10\u201315 min, and fixed with 1% citric acid for 2 h. Next, stepwise ethanol dehydration, followed by epoxy resin embedding, overnight in a 30\u00b0C oven, then in a 60\u00b0C oven for 12 h. Using an ultra-thin slicer, 50-to 100-nm-thick slices were cut. Using uranium acetate and lead nitrate double staining, a transmission electron microscopy  was used to examine autophagosomes in SH-SY5Y cells.P-values were determined using one-way ANOVA. Significance was defined as P < 0.05.Statistical analysis was performed using SPSS statistical software. All data were expressed as mean \u00b1 SD from at least three independent experiments. To explore the influence of DHCR24 on tauopathy in Alzheimer's disease, a transient knock-down of DHCR24 by lentivirus-mediated shRNA (knock-down group) and knock-in of DHCR24 by lentivirus-mediated cDNA (knock-in group) in the SH-SY5Y cell line was performed. DHCR24 shRNA, DHCR24 cDNA were well-expressed in SH-SY5Y cells, as indicated by GFP (green) under fluorescence microscopy . TransduSo far, it is still not to elucidate the potential relationship between DHCR24 and tau hyperphosphorylation. Increasing data supported that the hyperphosphorylation of tau at several sites, such as Thr181, Ser199, Thr231, Ser262, Ser356, and Ser396, which are tightly associated with tauopathy in AD and other tauopathies  and western blot analysis. Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis, involving in the clearance of misfolded proteins and damaged organelles   and Phospho-Akt (Thr308) were also markedly lowered by DHCR24 knock-down in SH-SY5Y cells. In contrast, the DHCR24 knock-in obviously increased the expression level of total class I PI3-K and the Phospho-Akt (Ser473 and 308), leading to the enhancement of PI3-K/Akt signaling. Thus, we considered that the decrease of PI3-K expression induced by DHCR24 knock-down could directly inhibited the phosphorylation activation of Akt, leading to the inhibition of PI3-K/Akt signaling. In addition, previous study suggested that brain cholesterol deficiency induced by silencing DHCR24 was associated with reduce of membrane cholesterol and altered membrane cholesterol-rich lipid-raft composition including the dysfunction of caveolae in cholesterol-rich lipid-raft microdomains, resulting in the markedly Akt inactivation, which is partly consistent with our outcomes (Lu et al., /caveolae may lead to the alteration of raft-dependent functional protein kinase, such as PI3-K kinase (Noticeably, in our study, K kinase . TogetheIn present paper, we also further showed that the DHCR24 knock-down could induce overactivation of GSK3\u03b2 and mTOR kinase activity by the inhibition of PI3-K/Akt. Conversely, the DHCR24 knock-in could promote the activation of PI3-K/Akt signaling, and simultaneously inhibition of GSK3\u03b2 and mTOR signaling. Furthermore, GSK3\u03b2 was one of the first identified substrates of the heavily studied oncogenic kinase Akt, phosphorylation by which inhibits GSK3\u03b2 activity. Akt activation is highly dependent on the class I PI3-K, and in turn promte the GSK3\u03b2 at Ser9 phosphorylation and inhibition of GSK3\u03b2 activity (Buller et al., In addition, the alteration of GSK3\u03b2 and mTOR signaling plays a crucial role in tauopathy, which directly or indirectly participated in regulation of tau phosphorylation (Balaraman et al., Dysfunction of autophagy mechanism has been proposed to play a critical role in AD and other neurodegenerative disorders (Zare-Shahabadi et al., de novo synthetic ability and mainly rely on lipoprotein-conjugated cholesterol produced by Astrocytes (Dietschy and Turley, Noteworthily, Ledesma et al. revealed that the neuronal membrane cholesterol have a significant reduction, the loss of neuronal membrane cholesterol and anomalous raft microdomains, which contribute to excessive amyloidogenesis in AD patients (Abad-Rodriguez et al., In conclusion, in present study, our results further showed that DHCR24 could control the rate of cholesterol synthesis and cholesterol homeostasis by the Bloch pathway and/or K-R pathway. As a synthetase heavily involved in cholesterol synthesis, DHCR24 knock-down significantly lead to a reduction of cholesterol in plasma membrane and intracellular compartments, resulting in the cholesterol loss in SH-SY5Y cells. Furthermore, the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies, at least partly by membrane lipid raft-mediated signaling mechanism . Taken tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.HZ designed the study and wrote the manuscript. XB contributed to the experimental work about tau phosphorylation and autophagy. JW and MZ contributed to the transfection work of SH-SY5Y cells. YX, LD, and KY provided their experimental assistance and TEM work. JZ and JB contributed to analysis of data. YZ and GX provided their technical assistance. All authors have read the manuscript and provided the input.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z\u2019s ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription. Chromatin is a dynamic structure that has an important regulatory role in controlling the genomic DNA accessibility and various nuclear processes. One mechanism used by the cell to influence chromatin structure and function is through histone variant exchange, which confers the nucleosome with new chemical and physical properties. With the lack of details and insights into how histone variants influence chromatin structure and function, the molecular mechanism of variant-mediated gene expression is subject to much speculation.Drosophila, Tetrahymena and mouse   dimer and the (H3\u2013H4) tetramer and in the nucleosome surface around the acidic patch   while twle (NCP) ,15. An eic patch . The latThe DNA template containing twelve tandem repeats of 167-bp 601 Widom sequence was a kind gifts from Dr Craig Peterson. The DNA template containing twelve tandem repeats of 208-bp 601 Widom sequence was a kind gift from Dr Ed Luk. Large-scale plasmids were purified as previously described . To geneThe sequences for the 167-bp and 208-bp Widom sequence are listed as following with the 601 sequence underlined:CTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGACTAGAT167-bp DNA repeat: ATCCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.208-bp DNA repeat: ACTTATGTGATGGACCCTATACGCGGCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATGCATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.Modified 208-bp DNA repeat with NsiI site introduced: ACTTATGTGATGGACCCTATACGCGGCCGCCXenopus laevis histones H2A, H2B, H3 and H4 were expressed in BL21 (DE3) pLysS E. coli cells and purified as previously described . The H2A.Z.1 gene was sub-cloned into pET LIC expression vector. The H2A.Z mutant in the acidic patch includes residue substitution D97N, S98K, G106Q and Glycine insertion after I100. The H2A.Z L1 loop mutant was constructed with residues in H2A.Z replaced by those from the H2A L1 loop . These mutations were introduced using Q5 site-directed mutagenesis kit . The H2A.Z M6\u2013M7 cassette mutant was constructed by swapping residues G93\u2013G120 with the corresponding H2A sequence (residue N90-P118). The H2A.Z C-terminus mutant was constructed by replacing H2A.Z amino acid G123-V128 with the corresponding H2A sequence (amino acid T121-K130). The H2A.Z C-terminus-extended mutant was constructed by replacing H2A.Z sequence from amino acid G93-V128 with the corresponding sequence in H2A from amino acid N90-K130. These mutants were obtained by gene synthesis (Synbio Technologies). Both wild-type and mutant H2A.Z were expressed in BL21 (DE3) E. coli cells and purified using the same procedure as the canonical histones.escribed . Mouse Hin vitro using salt dialysis as previously described  of the chromatin fiber was screened using negative-stained EM.A 1:1 optimal ratio of octamer:DNA for nucleosome reconstitution and for array reconstitution was determined empirically through the electrophoretic mobility shift assay (EMSA). Induction of chromatin fiber formation and purification were done through the addition of Mgd method . BrieflyP\u00a0\u2264\u00a00.05.\u00a0Two-way Anova test and graphical representation was done using Prims 5 software.250 nM of 208 bp nucleosome was mixed with 45\u00a0U of restriction enzyme (HinfI or NsiI) in Cutsmart buffer (NEB)  in total volume of 45ul and incubated at 37\u00b0C. Samples were collected every 15 min (5 ul), quenched with 8ul of stop buffer  and incubated at 50\u00b0C for 20 min for deproteination. Samples were examined on an 8% Native-PAGE gel and stained with SYBR GOLD. Digestion results were analyzed using ImageJ software version 1.53e. Two-way Anova test was used to determine the level of significant difference at For the reactions mentioned in Figure P value.Fitting was done using the Solver add-in in Excel over 5 iterative cycles. The rate constants  under 8\u00b0C and 100% humidity. Aliquots of 3.5 \u03bcl of the nucleosome were applied to glow-discharged QUANTIFOIL grids (R1.2/1.3 \u2013 400 mesh), blotted for 4\u20135 s\u00a0and plunged into liquid ethane cooled by liquid nitrogen. Grids were stored in liquid nitrogen until they were imaged.2 per micrograph  in counting mode using EPU software, giving a pixel size of 1.12 \u00c5 at the specimen level. Defocus values range from 0.9 to 3 \u03bcm. Each movie was dose-fractionated to 78 frames with a dose rate of \u223c0.69 e/pixel/s. Total exposure time was 60 seconds, corresponding to a total dose of 33 e/ \u00c5crograph .2. For the H2A fiber, part of the dataset was collected with the same Titan Krios microscope using the condition mentioned above, and the rest of the dataset was collected using the Talos Arctica microscope operating at 200 keV with a Falcon 3EC direct electron detector at counting mode with a nominal magnification of 73 000\u00d7, giving a physical pixel size of 1.4 \u00c5/pixel. Each movie in the Arctica dataset was dose-fractionated to 78 frames with a dose rate of \u223c0.75 e/pixel/s. Total exposure time was 60 s, corresponding to a total dose of 30 e/ \u00c52 per micrograph. The micrographs from the Krios dataset were scaled to the same pixel size as the Arctica dataset, before the two datasets were merged . The histone core from mouse H2A.Z nucleosome structure (PDB: 1F66) and the 601 DNA from the NCP structure (PDB: 6FQ5) were combined to generate the initial template used for refinement. This template was manually fitted into the density map in UCSF Chimera . followeThe nominal resolution of the overall reconstructions of chromatin fibers is limited, due to the motions of individual nucleosome relative each other within the fiber, and thus it is intrinsically difficult to improve. However, the positions of nucleosome N3\u2013N10 in the multi-body-refined maps is sufficiently clear to fit the nucleosome crystal structures. The fiber models were built by rigid-body docking eight crystal structures of H2A.Z nucleosome (PDB ID: 1F66) or canonical nucleosome (PDB ID: 3LZ0) into the cryo-EM density map of the corresponding fibers using UCSF Chimera . The linIn Figure Xenopus histone H2A or mouse histone variant H2A.Z.1 were reconstituted using recombinant proteins , even though it appears faster than the H2A nucleosome control (P\u00a0= 0.015)  Figure . Concurr) Figure  is simil) Figure . In the ) Figure . For the) Figure . These ret\u00a0al. showed that variant histone H2A.Z facilitates the intramolecular folding of nucleosome arrays into 50S chromatin fibers in vitro, while simultaneously inhibiting the formation of a highly condensed structure  and \u03b3 (27.5 \u00ba) are larger than those of the H2A fiber (\u03b2 = 12\u00ba and \u03b3 = 9.7\u00ba). The average shift d between units is shorter for H2A.Z fiber (59.5 \u00c5) than for the H2A fiber (61 \u00c5). Using these parameters, we built two 30-nm fiber models, each containing 24 tandem repeats of 167-bp 601 Windom sequence    . Overall2+) , with a ) Figure , while t) Figure . Bendingin-vitro DNA accessibility assay. Several recent cryo-EM studies of histone variants such as CENP-A . Though the molecular details of these interactions and how their interplays contribute to chromatin higher-order folding are less clear, our model is generally consistent with an ic patch . The morLinker histones H1 are known as an important factor for stabilizing the higher-order chromatin structure  and for in vitro. Based on these results, we propose a model for H2A.Z-mediated transcription regulation where the function of H2A.Z is context-dependent  and the Protein Data Bank (https://www.rcsb.org/). EM maps are deposited at the Electron Microscopy Database for the canonical nucleosome (EMD-23632), H2A.Z nucleosome (EMD-23626), H2A fiber (EMD-23631), and H2A.Z fiber (EMD-23630) respectively. The protein coordinate of H2A.Z nucleosome is deposited at the Protein Data Bank (PDB ID: 7M1X).The data that support the findings of this study are openly available in the Electron Microscopy Database (gkab907_Supplemental_FileClick here for additional data file."}
+{"text": "Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses  Lagovirus genus within the Caliciviridae [Rabbit haemorrhagic disease virus (RHDV) is the causative agent of a highly infectious and fatal disease affecting domestic and wild rabbits ,2,3. It iviridae . The posiviridae ,7. Additiviridae ,9. The viviridae .Commercially available vaccines are still traditional inactivated forms of the original pathogens , which aIn the course of its evolution, RHDV has thus far diverged to form six recognised genotypes , all higEscherichia coli [Saccharomyces cerevisiae [Trichoplusia ni pupae as natural bioreactors [The VP60 capsid protein expressed in heterologous recombinant systems forms characteristic VLPs, which are protective in immunised rabbits. VP60-derived VLPs have been obtained in hia coli , Saccharrevisiae , plants revisiae ,27, and revisiae ,29,30 anrevisiae ,32,33,34reactors  to increSpodoptera frugiperda Sf21 cell line was cultured at 28 \u00b0C in TNM-FH culture media  supplemented with 10% heat-inactivated foetal bovine serum , gentamicin at 50 \u00b5g/mL ), and Antibiotic-Antimicotic 1\u00d7 ). Recombinant baculoviruses (rBacs) were generated using the TopBac expression cassette [\u00ae II Reagent (Thermo Fisher Scientific) and following the manufacturer\u2019s instructions.The cassette ,37 as doBaculovirus stocks were titered on Sf21 cells using the 6-well plate format. Subconfluent Sf21 cells were infected with 10-fold serial dilutions of recBac stocks for 1 h at 28 \u00b0C. Subsequently, inocula were removed and cells were overlayed with an insect cell media-agarose mix at 1% agarose final concentration  and further incubated for 3\u20135 days. Monolayers were stained with neutral red to facilitate plaque counting.The improved baculovirus vector system TopBac was used for the generation of recombinant baculoviruses. The construction of recombinant baculoviruses was carried out essentially as described in , with thRHDV GI.1 Isolate AST89 Genbank accession number Z29471MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVATTSVVTAENSSASIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYPGSNATNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNGPGIPAAGWVGFGAIWNSNSGAPNVTTVQAYELGFATGAPGNLQPTTNTSGSQTVAKSIYAVVTGTAQNPAGLFVMASGVISTPSANAITYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNATAGSANGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTGASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTANGFSYV*RHDV GI.2\u2014previously RHDVb/2 isolate Nav10/11 accession number KM878681MEGKARTASQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTFGWSSPRFAAIDHDRGNASYPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTVPTAGWVGFGGIWNSSNGAPFVTTVQAYELGFATGAPSNPQPTTTTSGAQIVAKSIYGVATGINQATAGLFVMASGVISTPNSSAITYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTVGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSELVDIRPVGPRPSTSTLVYNLGGTTNGFSYV*8 pfu (plaque-forming units)/mL were reached. Baculovirus stocks were stored at \u221280 \u00b0C containing 5% glycerol. The correct expression of VP60 proteins from recombinant baculoviruses was confirmed by detection of proteins using specific sera by Western blot analysis.The recombinant baculoviruses produced were subjected to two rounds of clonal selection by plaque formation, and after virus propagation, infective titres of approximately 10Trichoplusia ni  insects were reared following a previously described methodology [hodology . BrieflyThe crude protein extracts were obtained by homogenising the frozen biomass in the adequate protein extraction buffer. The resulting suspension was subjected to the downstream processing  to obtaiw/v) aqueous uranyl acetate. Micrographs were recorded with an EM 2000 Ex microscope . For the quantitation analysis of VLP stability, samples obtained from GI.2 (N11-) and GI.1 (AST) infections or from co-infections with the two viruses under different conditions were analysed at day 0 and 60 of storage at 4 \u00b0C. Samples were diluted to a protein concentration of 25 mg/mL to obtain an even distribution of the capsids on the electron microscopy grid and an easily countable number of capsids per image at the suitable magnification. Three grids were prepared per sample and images were taken in four different areas of each grid. We quantified the capsids with ImageJ tools in 15 pictures.Electron microscopy analyses were performed by conventional means. Briefly, purified VLPs (approximately 5 \u00b5L) were applied to glow-discharged carbon-coated grids for 2 min. Samples were negatively stained with 2%  was added. All washing steps were carried out in triplicate using PBS-Tween (0.05% PBS-Tw). For peroxidase-labelled antibodies, the Pierce\u2122 ECL Western Blotting Substrate system was used for detection (Thermo Fisher Scientific).2SO4. Absorbances were read at 450 nm using Varioskan Flash microplate reader and Skanit software.ELISA were carried out in flat bottomed 96-well plates . Plates were coated with 100, 50, or 10 ng of Mab (varying concentrations are indicated in the results section) in each well in PBS (1 mM a pH 7.3 with sodium azide 0.05% PBS-AS). Coating plates were incubated overnight at 4 \u00b0C. The next day the supernatant was removed, and plates were blocked with 1% BSA in PBS-AS for 1 h at 37 \u00b0C. Plates were washed 3 times with 200 \u00b5L PBS-Tw per well. Antibodies were added in volumes of 100 \u00b5L per well and incubated for 1 h at 37 \u00b0C followed by 3 washing steps as previously described. Secondary antibodies at the indicated dilutions were incubated for 1 h at 37 \u00b0C. Prior to revelation, plates were washed 3 times in PBS-Tw. For reaction development TMB  (Sigma) (100 \u00b5L per well) was added and incubated at room temperature in the dark until the reaction was stopped using 100 \u00b5L 2M HNew Zealand White rabbits were supplied by San Bernardo Farm . Prior to vaccination, sera were collected from the marginal ear vein of all animals to confirm the absence of RHDV antibodies.Two protection experiments were carried out. In general terms, rabbits were vaccinated with chimeric RHDV VLPs and in experiment 1; 7 days post vaccination they were challenged with RHDV GI.2 and in experiment 2; 7 days post vaccination they were challenged with classic RHDV GI.1 isolate RHDV AST89.Group A: Non-vaccinated controls.Group B: 20 \u00b5g chimeric VLPs.Group C: 40 \u00b5g chimeric VLPs.Group D: 5 \u00b5g RHDV G1 VLPs + 5 \u00b5g RHDVb VLPs.Experimental challenge 1: 30-day-old rabbits were divided into 4 groups (Groups A\u2013D), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Group E: Non-vaccinated controls.Group F: 40 \u00b5g chimeric VLPs.Experimental challenge 2: 2-month-old rabbits were divided into 2 groups (Groups E and F), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Rabbits were kept under observation throughout the experiment and housed in pairs in a biosecurity level 2 laboratory. L. europaeus GI.2 virus, common name RHDV-Gal08/13, with 10 LD50. Groups E and F were challenged 7 days post vaccination with RHDV isolate L. europaeus RHDV GI.1 RHDV-Ast89 (Genbank accession number Z49271). Rabbits were infected on day 7 post vaccination with 150 hemagglutination units/0.5 mL (as determined using O- and B-type human erythrocytes). The virus was administered subcutaneously in the cervical area as a dilution of clarified infected liver homogenates. Euthanasia of surviving rabbits was carried out by intravenous injection with Dolethal following the manufacturer\u2019s instructions .Two virus isolates were used in this study. Groups A\u2013D were challenged 7 days post vaccination with isolate Symptoms of RHDV infection, such as nervous, respiratory, and digestive signs, were monitored and recorded daily. Blood samples were collected on days 0 and 7 post vaccination, and from all surviving rabbits 7 days post challenge. Serum was separated after clotting by centrifugation and stored at \u221220 \u00b0C until analysis. After euthanasia, rabbits were necropsied, and gross lesions were recorded. Samples of liver were directly frozen at \u221280 \u00b0C for total RNA extraction using the GenElute Mammalian Total RNA Miniprep Kit , and RHDV GI.2 VP60 detection and quantification were obtained by specific RT-qPCR .n = 5) from each group.Rabbit serum RHDV antibody levels were tested by enzyme-linked immunosorbent assay (ELISA) using the commercially available INgezim RHDV kit  following the manufacturer\u2019s instructions. The absorbance was measured at 450 nm in a microplate reader  and data were analysed using the Skanit software Version 2.4.5 (Thermo Fisher Scientific). Negative and positive control sera were included in all plates. Results are expressed as mean optical densities \u00b1 SD . To determine if VLPs obtained after co-infecting with the two baculoviruses were formed by a single VP60 protein or were chimeras of the two VP60 proteins, we designed ELISA and dot blot-based sandwich analyses using monoclonal antibodies capable of recognising one or the two VP60 proteins. To determine the consequences of the co-infection conditions on the chimeric VLP stability, we compared the VLP stability after 2 months of storage at 4 \u00b0C. For this analysis, VLPs were produced using GI.1/GI.2-derived particles at baculovirus infection ratios of 1:1, 1:3, and 3:1. In parallel, we performed the same analysis using GI.2 (N11-)- and GI.1 (AST)-derived VLPs produced independently. After this storage period, we observed a dramatic degradation (>70%) of GI.2 (N11)-derived VLPs, indicative of their low stability . This ra5 viral RNA copies per microliter\u2014one rabbit in group B), indicating a solid protection conferred by the chimeric VLPs. The negative control groups showed 80% mortality after infection with RHDV GI.1 or RHDV GI.2 viruses and all animals were positive for virus genome detection. Viral RNA detection ranged between 6.03 \u00d7 105 and 5.81 \u00d7 106 copies per microliter for animals in group A.To analyse the protection conferred by the chimeric VLPs against the two predominant RHDV serotypes, we used a mixture of GI.2 (N11-) and GI.1 (AST89)-derived VLPs at a single previously determined protective dose and two doses of chimeric VLPs to immunise rabbits. As expected, the mixture of 5 \u00b5g of both VLPs induced protection against a lethal dose of RHDV GI.2 virus, showing no interference of the GI.1 (AST89)-derived VLP in this protection. Two doses of chimeric VLPs of 20 and 40 \u00b5g were also used to determine the protection ratios against RHDV GI.2, and only 40 \u00b5g was used to protect against an RHDV GI.1 virus. A high protection rate of 80% against RHDV GI.2 was achieved with the 20 \u00b5g dose of chimeric VLP, and a rate of 100% was achieved with a dose of 40 \u00b5g against both lethal viruses , demonstAntibodies generated against RHDV were analysed using a commercial ELISA from sera extracted from all rabbits on day 0 (pre-vaccination) and from surviving animals on day 14 post vaccination . As expected, all sera were negative for anti-RHDV antibodies on day 0. Seven days post challenge, the majority of surviving animals elicited an immune response as indicated by rising Ab levels . OverallDevelopment of efficacious recombinant subunit vaccines is essential, especially for causal agents for which a cell culture capable of supporting its propagation has not been identified. This is the case for RHDV, which is the causative agent of one of the most economically important diseases in rabbits worldwide. Commercially available vaccines are still traditional inactivated viruses collected from organs of artificially infected rabbits, which is unacceptable from both the safety and ethical points of view. The production of a new-generation vaccine against RHDV needs to meet two main requirements: first, cost-efficiency in production to make it commercially viable, and second, the vaccine has to induce a broad protection against RHDV GI.1 and RHDV GI.2 viruses as cross-protection is only partial at best ,40.Caliciviridae. The productivity of this protein is generally good in most studied systems, but the development of a subunit vaccine based on the VLP that this protein forms is commercially difficult for the production and regulatory-associated costs, considering the limited number of vaccine doses applied in the domestic rabbit population worldwide. In addition, any vaccine against this virus has to be dual because of the potential of two circulating virus serotypes today, RHDV GI.1 and RHDV GI.2. In the present work, we attempted to produce RHDV VLPs in one of the most cost-efficient methods developed to date based on the use of Trichoplusia ni insect chrysalises (CrisBio technology), and also to develop a single production process (upstream and downstream) of a chimeric VLP-based vaccine containing the critical antigens from both predominant RHDVs circulating today.Virus-like particles have received considerable attention due to their potential application in human and veterinary vaccines against infectious diseases. VLPs are highly immunogenic and are extremely safe. In the case of RHDV, the capsid protein forms characteristic VLPs when expressed in different systems as for the other members of the RHDV has been previously described as a versatile platform for foreign B-cell epitope display, inducing protective humoral immune responses . It has In the absence of a widely available tissue culture system that can sustain replication of human noroviruses, VLPs have been used as a surrogate to study the capsid\u2019s structural features and as immunogens to elicit protective humoral responses . The mosTrichoplusia ni larvae [Trichoplusia ni Lepidoptera is highly susceptible to AcMNPV-derived vectors, and therefore, any common baculovirus can be used for industrial or experimental production. This technology is also linearly scalable, facilitating their implementation for large quantities of vaccine doses in a record time.RHDV GI.1 VLPs have been previously produced in i larvae  and pupai larvae . Recentli larvae . The proIn the present work, we also used the TopBac expression cassette to generate the baculovirus vectors ,37. The In the present work, we demonstrated the feasibility of producing a vaccine against RHDV that incorporates two different capsid proteins derived from two virus subtypes and confers broad protection in immunised rabbits. The chimeric RHDV VLPs were produced in a single upstream and downstream production process, significatively reducing the complexity of a binary vaccine. In addition, the combination of TopBac baculovirus vector and CrisBio technology allows for a cost-efficient production required for a vaccine directed to domestic rabbits to make it commercially viable."}
+{"text": "The authors wish to introduce the following corrections to their article .OLLAS::RPA-1 is incorrect. The correct sequence is:1) The DNA sequence for the TTCCCCAATTTTTATGTATCTGTTTCAGATAGTGAAAGATGTCCGGATTCGCCAACGAGCTCGGACCACGTCTCATGGGAAAGGCGGCAATTCACATCAATCACGATGTCTTCAATAArpa-1 was indicated to be placed on chromosome I, when its correct position is on chromosome II.2) In some places in the list of Strains The published article has been updated. These changes do not affect the results, discussion and conclusions presented in the article."}
+{"text": "What is more, interfering with this process is an innovative strategy to overcome the chemo\u2010resistance of GBM cells.Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi\u2010drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo\u2010resistance. We tried to explore the mechanism of GBM chemo\u2010resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the  The progression\u2010free survival of patients with this condition after diagnosis is 6.2\u20137.5\u00a0months; the median survival is 14.6\u201316.7\u00a0monthsIn the studies of the chemo\u2010resistance, mitochondria are under spotlight for a long time. Mitochondria are involved in vital processes such as energy synthesis, regulation of calcium ion, ROS generation and apoptosis executionAs we all know, TMZ kills tumour cells by inducing DNA damage, and the survival cells are always with upregulated DNA damage response or multiple stress responses. Although these responses vary, energy support is essential.22.1TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N\u2010Acetyl\u2010L\u2010cysteine (NAC) were purchased from Sigma\u2010Aldrich. MitoTracker\u2122 Red FM (M22425), Hoechst 33342 (H1399) and anti\u2010Ubiquitin WB Antibody (13\u20131600) were purchased from Invitrogen  . Anti\u2010phospho\u2010Ubiquitin (Ser65) (ABS1513\u2010I) was purchased from Merck KGaA company (1:1000). Anti\u2010P53 (21891\u20131\u2010AP), anti\u2010PINK1 (23274\u20131\u2010AP), anti\u2010Parkin (14060\u20131\u2010AP), anti\u2010Drp1 (12957\u20131\u2010AP), anti\u2010Opa1 (27733\u20131\u2010AP), anti\u2010Mfn1 (13798\u20131\u2010AP), anti\u2010Mfn2 (12186\u20131\u2010AP), anti\u2010Caspase\u20109 (10380\u20131\u2010AP), anti\u2010BAX (50599\u20132\u2010Ig), anti\u2010Caspase\u20103 (19677\u20131\u2010AP), anti\u2010VDAC1 (10866\u20131\u2010AP), anti\u2010Lamin B (12987\u20131\u2010AP), anti\u2010Cytochrome c (10993\u20131\u2010AP), and anti\u2010\u03b2\u2010actin (60008\u20131\u2010Ig) were purchased from ProteinTech Group, Inc.,  . Anti\u2010\u03b3\u2010H2A.X (ab81299) was purchased from Abcam (1:1000). Anti\u2010AMPK\u03b1 (2532) and p\u2010AMPK\u03b1 (50081) were purchased from Cell Signaling Technology, Inc.  (1:1000). Anti\u2010phospho\u2010DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti\u2010phospho\u2010DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. .2.22\u00a0atmosphere.Human glioblastoma cell lines  were obtained from the Chinese Academy of Medical Sciences and cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, penicillin (50\u00a0U/ml) and streptomycin (50\u00a0\u00b5g/ml) purchased from Life Technologies and incubated at 37\u00b0C in a 5% CO2.36 U87MG cells were suspended in 100\u00a0\u00b5l of phosphate buffer saline (PBS) (Beyotime Institute of Biotechnology) and injected subcutaneously into the right armpit of the nude mice. The animals were raised in a specific pathogen\u2010free (SPF) environment, and tumour volume was observed regularly. One week later, when the tumour volume reached 50\u00a0mm3, the nude mice were randomly divided into four groups, with eight mice in each group. The treatment conditions for each group were as follows: control group: 200\u00a0\u00b5l of 5% Carboxymethyl Cellulose (CMC)\u2014daily gavage; TMZ group: 40\u00a0mg of TMZ suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage; WY14643\u00a0group: 10\u00a0mg of WY14643\u00a0suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage; TMZ+WY14641\u00a0group: TMZ (40\u00a0mg) + WY14643 (10\u00a0mg) suspended in 200\u00a0\u00b5l of 5% CMC\u2014daily gavage. The status of the nude mice was observed daily. Mice were weighed and the size of each tumour was measured every 2\u00a0days. The experiment was terminated when the longest diameter of the tumour reached 2\u00a0cm or signs of ulceration appeared. Upon termination of the experiment, the nude mice were sacrificed, and the tumour was stripped, photographed and weighed. The tumour tissue was used for the following experimental tests: (1) evaluating the ATP content of tissue cells; (2) the total protein was extracted from the tumour tissue for Western blotting (WB); (3) part of the tumour tissue was stored in 2.5% dialdehyde and analysed by transmission electron microscopy; and (4) part of the tumour tissue was stored in 4% paraformaldehyde for immunohistochemical analysis.The experimental protocol was approved by the local ethics committee (20200921\u20131). Nude mice were adapted to the experimental environment for a week prior to experimentation. Then, 5\u00a0\u00d7\u00a0102.4Cells were lysed in Prusiner's buffer , EDTA (5\u00a0mM), Triton \u00d7\u2010100 (0.5%), and deoxycholate and protease inhibitor cocktail). The homogenates obtained were then briefly sonicated. Aliquots of 10\u00b5g of total protein were then loaded onto 8%\u201316% SDS\u2010PAGE gels. After migration, proteins were wet\u2010transferred to PVDF membranes and immunoblotted using certain antibodies. Immunological complexes were detected with either anti\u2010rabbit or anti\u2010mouse IgG\u2010coupled peroxidase antibodies  by the electrochemiluminescence detection method (Roche Diagnostics S.A.S).2.5Cells were grown on coverslips in 24\u2010well plates and treated with different agents. After staining with MitoTracker for 30\u00a0min, the coverslips were rinsed with PBS and fixed with 4% (w/v) paraformaldehyde for 25\u00a0min at room temperature followed by a PBS rinse. Permeabilization with 0.1% (v/v) Triton \u00d7\u2010100 for 7\u00a0min was followed by blockade with 10% horse serum for another 30\u00a0min. Following incubation with primary antibodies (1:100) at 4\u00b0C for 12\u00a0h, the coverslips were incubated with fluorescent secondary antibodies (1:200) for 30\u00a0min, rinsed with cold PBS and stained with Hoechst 33342 in accordance with the manufacturer's instructions for 7min before a final PBS rinse. A Revolve Hybrid Microscope (Discover ECHO) was used for image capture. Images were further processed by ImageJ Software version 1.52\u00a0s  to assess the intensity of immunofluorescence (IF), as well as the mitochondrial parameters. For the evaluation of mitochondrial fusion, we evaluated three images in each treatment group from three independent experiments using ImageJ Software. Two parameters were used to assess the level of mitochondrial fusion: the mean size of the mitochondrial network (MS) and the mean length of mitochondria (ML).2.65 cells/well. Cells were then incubated overnight at 37\u00b0C, and the medium was replaced with fresh complete medium. After 24\u00a0h, the culture medium was collected and proteins were extracted by sonication. Extracted proteins were then quantified with a Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Then, we measured the concentrations of glucose and lactate with glucose (RsBio) and lactate assay kits (Jiancheng Bio), respectively. The glucose consumption in each experimental group was calculated as follows: Glucose consumption\u00a0=\u00a0glucose concentration (fresh complete medium)\u2013glucose concentration .Cells were seeded in 6\u2010well plates at a density of 3\u00a0\u00d7\u00a0102.75 cells/well. Following overnight incubation at 37\u00b0C, the medium was replaced with fresh culture medium. After 6 or 24\u00a0h, in accordance with the manufacturer's instructions, cells were washed with PBS, and then, their ATP levels were determined using an ATP Assay Kit (Beyotime Institute of Biotechnology).Cells were seeded in 6\u2010well plates at a density of 5\u00a0\u00d7\u00a0102.8\u00ae Xtra\u2010Oxygen Consumption Assay (Luxcel Biosciences Cork) using a CLARIOstar microplate reader (BMG Labtech). In brief, cells were plated at a density of 8\u00a0\u00d7\u00a0104 cells/well in 96\u2010well plates  and allowed to adhere overnight at 37\u00b0C using a plate block heater. The culture medium was removed from all wells and replaced with 160\u00a0\u03bcl of pre\u2010warmed reaction mixture, including 10\u00a0\u03bcl of reconstituted MitoXpress\u00ae Xtra reagent, 150\u00a0\u03bcl of fresh culture media with TMZ  in each well. The wells were sealed by adding two drops of pre\u2010warmed HS Mineral Oil, and the plate was immediately analysed in a microplate reader.Cellular oxygen consumption rate (OCR) was measured by the MitoXpress2.94 cells per well. After exposure to TMZ and/or Mdivi1 or WY14643, for 24\u00a0h, the cells in each well were incubated with MTT solution (0.5\u00a0mg/ml) dissolved in PBS for 4\u00a0h at 37\u00b0C. Then, 150\u00a0\u00b5l of DMSO was added to each well. The absorbance was measured at 570\u00a0nm using a CLARIOstar microplate reader (BMG Labtech). Cell viability was then calculated as follows: cell viability\u00a0=\u00a0absorbance of experimental group/absorbance of control group\u00a0\u00d7\u00a0100%.Cells were seeded in 96\u2010well plates with 100\u00b5l of complete DMEM medium at a density of 1\u00a0\u00d7\u00a0102.10RNA was extracted using a RNeasy kit (Qiagen) in accordance with the manufacturer's protocol. Quantitative real\u2010time reverse transcription\u2010PCR (qRT\u2010PCR) was then performed using a two\u2010step method. Data analysis was based on the delt\u2010delt\u2010Ct method using \u03b2\u2010Actin as a normalization control. PCR was conducted in a QuantStudio5 real\u2010time PCR system (Thermo Fisher) and analysed using QuantStudio Design & Analysis software v1.3.1 (Thermo Fisher).In order to evaluate the mtDNA copy number, we extracted DNA from cells in each group and determined the copy number of the mtDNA gene ND1, as normalized to the 18S gene. In order to evaluate the extent of DNA damage, we selected two fragments of mtDNA (79bp and 230bp in length) as target templates for a qPCR array using the 18S gene as an internal reference. The extent of mtDNA damage was parallel to the value of 79bp/230bp. The primers used for the real\u2010time PCR are listed in Table\u00a02.11The Mitochondria and Nuclear Extraction Kit for Cells (Beyotime Biotechnology) was used to isolate nuclear and mitochondrial fractions from each experimental sample. In each case, we followed the manufacturer's guidelines.2.12A plasmid that overexpressed TP53, along with a negative control (NC) plasmid, were constructed by GenePharma Co., Ltd. Cells were transfected with the plasmids using the Thermo Scientific\u2122 TurboFect\u2122 Transfection Reagent in accordance with the manufacturer's protocol. In brief, cells were plated in 6\u2010well plates and transfected the next day with 4\u00a0\u03bcg of plasmid using 6\u00a0\u03bcl of transfection reagent. Cells were harvested 24\u00a0h after transfection, and whole\u2010cell lysates were isolated for Western blotting. The sequence of TP53 is as follows: ATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA.2.13Tumour tissues were fixed in 4% paraformaldehyde for 24\u00a0h and embedded in paraffin. Paraffin sections were cut into sections (4\u00a0\u03bcm thick) and then deparaffinized and rehydrated prior to antigen retrieval. Sections were blocked with 10% bovine serum albumin in TBS\u2010Tween 20 (Sigma\u2010Aldrich) for 1\u00a0h at room temperature. Sections were then incubated overnight at 4\u2103 with primary antibodies against P53 (21891\u20131\u2010AP) and PINK1 (23274\u20131\u2010AP). The following morning, sections were incubated with secondary antibodies for 30\u00a0min at 37\u2103. Then, slides were incubated with diaminobenzidine for 5\u00a0min, and then counterstained with Gill's haematoxylin for 30\u00a0s. Images were captured on a microscope (Discover ECHO Company).2.14Tumour tissues from the different treatment groups were fixed in 2.5% glutaraldehyde and then embedded in an embedding agent. Next, specimens were sliced into sections (50\u201370\u00a0nm in thickness). The sections were then dyed with uranyl acetate and citrate dye solution. The TEM system (Thermo Fisher Scientific Company) operated at an acceleration voltage of 80\u00a0kV and an electron tomography voltage of 120\u00a0kV. Specimens were magnified by \u00d7150,000, and images were acquired. Three tumour specimens in each treatment group were randomly selected for TEM examination. And each specimen outputted a clear picture of which the mean length of mitochondria was measured directly by the software of TEM. Then, the data were analysed.2.15\u00ae easyCyte flow cytometer (Merck KGaA).Apoptosis assays were performed using an Apoptosis Detection Kit (BD Biosciences). Cells from different treatment groups were trypsinized with 0.25% trypsin, and assays were conducted in accordance with the manufacturer's protocol. In total, 10000 cells were counted in each treatment group using a Guava\u00ae easyCyte flow cytometer (Merck KGaA).For the analysis of mitochondrial mass, cells were grown in 6\u2010well plates and treated with the different agents. Cells were trypsinized with 0.25% trypsin and suspended cells were stained with MitoTracker\u2122 Red FM (Invitrogen) (1:10000) for 30\u00a0min at 37\u2103. Cells were then rinsed three times with PBS, and 10,000 cells were counted for each treatment group using a Guava\u00ae easyCyte flow cytometer (Merck KGaA).The production of ROS was measured by a ROS Assay Kit (Beyotime Biotechnology). In brief, cells were collected and resuspended in DMEM medium after treatment and then incubated with DCFH\u2010DA for 30\u00a0min at 37\u00b0C. Cells were then rinsed three times in PBS, and 10,000 cells were for each treatment group using a Guava2.16U87 cells were seeded at a density of 8000 cells/well and treated for 24\u00a0h with the respective treatments . On the day of the assay, cell culture medium was removed and replaced by assay medium supplemented with agents according to the protocol and the Seahorse XFe96 analyzer was used. In the test, basal cellular oxygen consumption was determined, followed by injection of the ATP synthase inhibitor oligomycin (5\u00a0\u00b5M), the uncoupler fluoro\u2010carbonyl cyanide phenylhydrazone (FCCP) (2\u00a0\u00b5M) and the complex I inhibitor rotenone (0.5\u00a0\u00b5M) combined with the complex III inhibitor antimycin A (0.5\u00a0\u00b5M). The oxygen consumption rate was detected, and each measurement was normalized to cell content.2.17t test. Analysis of variance (ANOVA) was also used to conduct multiple comparisons using GraphPad Prism 7.00 . Dunnett's post hoc multiple comparisons test was used after ANOVA. Data are presented as means\u00a0\u00b1\u00a0standard deviation, and p\u00a0<\u00a00.05 was considered to indicate a statistically significant difference.Data from three independent experiments were collected and analysed using SPSS 22.0 (IBM Corp.) and the Student's 33.1As an alkylating agent, TMZ can induce damage to nuclear DNA and mitochondrial DNA.In order to identify the mechanisms underlying the increased level of oxidative phosphorylation, we first detected the mass of mitochondria in the two cell lines, using three methods: (1) labelling mitochondria with specific fluorescent probes Figure\u00a0A, 2) by by2B) an3.2The mass of mitochondria within a cell is precisely controlled by mitophagy.3.3In order to further explore the mechanism by which mitophagy was downregulated, we focused on how the expression of PINK1 was regulated. We also investigated the elevated expression of intracellular nuclear transcription factor (NTF) in tumour cells under TMZ treatment. First, we found that TMZ treatment\u2010induced cellular DNA damage responses in both SHG44 and U87 cells; these responses involved high expression of \u03b3\u2010H2A.X and the increased aggregation of the protein in the nucleus  establishing a cell model overexpressing TP53 by plasmid transfection; under this condition, we observed that PINK1 expression was downregulated in cells overexpressing TP53, and that the expression of Ser65 phosphorylated ubiquitin was correspondingly downregulated  after TMZ treatment, the tumour cells showed reduced levels of ATP; thereafter, however, the levels of ATP increased. The curve created by plotting ATP concentration with time showed two troughs; one at 10\u00a0min and the other at 6\u00a0h Figure\u00a0A. FurtheWhat is more, to confirm that the AMPK regulated the function of mitochondria, we performed the mito stress test by using the seahorse analyzer and revealed that: 1.TMZ elevated the OXPHOS potential of mitochondria while repressing the activity of AMPK impaired the effect Figure D.3.6In order to verify that the mobilization of mitochondrial dynamics was involved in the metabolic reprogramming of tumour cells under chemotherapy stress and to explore whether interfering with mitochondrial dynamics could sensitize GBM to TMZ treatment, we used two different agents: WY14643 to promote mitochondrial fissionFinally, we found that WY14643 promoted mitochondrial fission and increased the efficacy of TMZ chemotherapy. This treatment also enhanced the efficacy of TMZ to induce apoptosis of SHG44 and U87 cells . Renxuan Huang: Software ; Visualization . Kunmeng Yang: Data curation . Yichun He: Writing \u2013 review & editing . Delu Dong: Supervision . Yufei Gao: Conceptualization (lead); Funding acquisition (lead).Fig S1Click here for additional data file."}
+{"text": "Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of  \u2022Vein-specific enhancers can contain essential ETS motifs.\u2022VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.\u2022VEGFA stimulation cannot induce vein-specific enhancer activity. The vascular system is subdivided into arteries, veins, lymphatics and capillaries, each comprised of genetically distinct ECs expressing specific fate-determining genes . The essE-26 transformation-specific) transcription factor family  identified a group of ETS binding motifs required for arterial and angiogenic activity  signalling may act via ETS transcription factors to enable specific activation of arterial and angiogenic genes. VEGFA signalling influences many processes during early vascular growth, and plays essential roles in vasculogenesis, arterial specification and angiogenesis . ETS traactivity . ERG binactivity . A similactivity , whilst activity .Despite their hypothesised role in arterial and angiogenic-specific patterns of gene expression, binding motifs for ETS factors are also a common feature of many pan-endothelial expressed gene promoters and enhancers . ETS1, Ein vivo. These results indicate that within the endothelium, VEGFA-stimulated ETS factor binding is a shared feature at enhancers associated with multiple different patterns of gene expression, and suggests that additional transcription factors may be primarily responsible for directing arterial, angiogenic and venous-specific gene expression patterns downstream of different growth factor signalling inputs.In this paper, we undertake a detailed analysis of two recently characterized vein-enriched gene enhancers. We demonstrate that, similar to arterial and angiogenic enhancers, ETS factor binding at these venous enhancers is necessary for enhancer activation and vein-specific patterns of reporter gene expression, and that this binding is also enriched by VEGFA signalling. However, unlike arterial and angiogenic enhancers, these venous enhancers cannot be directly activated by over-expression of VEGFA 22.1Ephb4 and Coup-TFII (Nr2f2) gene loci (Coup-TFII. Neither enhancer was active in the mature microvasculature (We have recently identified enhancers within the venous-enriched ene loci . Both enA/T)  were able to compete for binding of either ETS1-DBD or ERG, or both proteins   and Fig.A/T) A. Of theCGGAAG/A . We perfCGGAAG/A B\u2013C and fCGGAAG/A D\u2013E, bothproteins C while Eproteins E. The spproteins C and E. EPHB4 and COUP-TFII/NR2F2 were significantly enriched in these HUVECs . Similar to the venous enhancers investigated, ERG binding around these arterial-, angiogenic- and pan-endothelial-expressed enhancers was seen in both HUVEC and HUAEC cells, suggesting that binding of ERG to specific enhancer regions was not routinely restricted to the EC subtypes in which the associated genes are preferentially active . Taken together, these results demonstrate that functional ETS binding motifs can be present within vein EC-specific enhancers, and show that the ability to bind ERG and other ETS factors is not restricted to enhancers that are active in arterial and angiogenic ECs.Since the binding of ETS factors to vascular enhancers has been previously associated with arterial-specific and angiogenic-specific enhancers, we next investigate whether ETS factor binding was also a feature at venous-specific enhancers . We found significant binding for ERG, FLI1 and ETS1 at both Ephb4-2 and CoupTFII-965 venous enhancers . EMSA analysis confirmed that the mutated ETS binding motifs could not bind ETS proteins   exclusively to the venous endothelium .In agreement with previous  studies , our resproteins C and E. on (hpf)  and Fig.on (hpf) A\u2013B. Howeon (hpf) A\u2013B. We sothelium  and Fig.othelium C\u2013D, the The loss of Ephb4-2 and CoupTFII-965 vein enhancer activity after ETS motif mutation observed here is similar to the loss of activity seen when ETS motifs are mutated in the arterial and angiogenic Dll4in3 enhancer , in othe2.3erg and fli1. There are multiple ETS factors expressed in the developing zebrafish vasculature  zebrafish was significantly reduced after morpholino knockdown of erg and fli1, with the strength of Ephb4-2:GFP transgene activity inversely correlated with the levels of erg/fli1 MO . Conversely, we saw a reduction in the expression of endogenous ephb4 and stab1l, a zebrafish venous marker, when assessed by whole mount in situ hybridization analysis . Similar reductions in the expression of some venous-enriched genes was also observed after ERG depletion in HUVECs  transgenic zebrafish, although this was not as marked . As previously reported, erg/fli1 knockdown also resulted in reduced activity of the arterial/angiogenic Dll4in3 enhancer in transgenic zebrafish  and tg(CoupTFII-965:GFP) zebrafish lines compared to the arterial (tg(Dll4in3:GFP) zebrafish line. At the lowest concentration of inhibitor, Dll4in3:GFP activity was more notably reduced than either venous enhancer, while higher SU5416 doses significantly reduced activity of all enhancers  results in EC apoptosis, lower levels of VEGF inhibition result in reduced arterial and venous marker gene expression, although the reduction in venous genes was less pronounced and sometimes compensated by expansion of vein gene activity into the dorsal aorta . We therAlthough Dll4in3:GFP was more sensitive to inhibition of VEGFA signalling than either the venous Ephb4-2:GFP or the CoupTFII-965:GFP, our previous results show that changes in ETS factor binding after VEGFA stimulation can be seen at both venous and arterial enhancers. Consequently, it is unlikely that changes in ETS factor occupancy at enhancers can explain the differences between venous and arterial enhancer responses to VEGFA inhibition. An alternative explanation may be that additional transcription factors  may be instead responsible for allowing arterial and angiogenic enhancers a greater sensitivity to VEGFA signalling.2.6in vivo. We first used an established model of VEGFA-stimulated blood vessel growth in mice, in which an adenovirus expressing VEGFA164 (Ad-VEGFA164) is injected intradermally into the ears of adult mice . In both enhancers, angiogenic expression was lost by 60 days after injection, a time-point known to be independent of VEGFA signalling.We next examined whether VEGFA over-expression, and subsequent increased ERG occupancy, is alone sufficient to initiate activity of venous-, arterial- and angiogenic-specific enhancers ult mice . This reult mice . Vessel ult mice . To deteenic ECs A. Ad-VEG164 injection can specifically re-activate the Dll4in3 and HLX-3 enhancers in their native EC sub-types. However, they cannot determine whether this occurs via a VEGFA-mediated increase in ERG binding, VEGFA-mediated increase in other transcription factors binding to these enhancers or VEGFA-mediated removal of repressive factor binding. If VEGFA-mediated activation of these enhancers occurs primarily via changes to ETS factors, we would expect a similar reactivation of venous enhancers. We therefore next determined if Ad-VEGFA164 injection was able to directly activate the Ephb4-2:lacZ and CoupTFII-965:lacZ transgenes. Unlike with the arterial and angiogenic enhancers, we observed no endothelial transgene activity at any time point in either Ephb4-2:lacZ or the CoupTFII-965:lacZ Ad-VEGFA164 injected ears, although occasional ectopic expression could be detected  embryos (following protocol and concentration from tg(Ephb4-2:GFP) embryos . This did not change significantly when we increased the amount of vegfaa injected . This result therefore further indicates that increased VEGFA signalling does not increase Ephb4-2 enhancer activity. Additionally, at 50\u00a0\u200bpg vegfaa121 and vegfaa165 we saw no clear expansion of Ephb4-2:GFP expression beyond the venous endothelial expression pattern observed in control embryos (vegfaa121 and vegfaa165 mRNA into 1-cell stage tg(Dll4in3:GFP) embryos resulted in slightly increased GFP intensity and expansion into the caudal vein plexus, as previously described by vegfaa levels . Taken together, these results suggest that the failure of Ad-VEGFA164 to activate venous gene enhancers in the mouse ear is unlikely to be a result simply of developmental context. Furthermore, the Ephb4-2:lacZ transgenes can be reactivated in injured neonatal hearts , suggesting that absence of normal activity does not affect enhancer reactivation. Taken together, these results indicate that VEGFA stimulation is not sufficient to activate transcription from the Ephb4-2 venous EC-specific enhancer, despite its reliance on VEGFA-augmented ETS transcription factors.Although developmental arterial and angiogenic enhancers were reactivated in the adult mouse ear by Ad-VEGFA164 A and B,  reduced . Furthern plexus . To dete3The role of ETS transcription factors in the regulatory hierarchy of endothelial cells has been unclear. The specification and maintenance of the endothelial cell lineage requires the shared activation and repression of many lineage-defining genes. However, differential gene expression within specific sub-populations of endothelial cells is also essential for vascular function. Consequently, spatial and temporal control of endothelial gene expression must involve multiple layers of regulation. While analysis of arterial and angiogenic-specific enhancers has supported a proposal that ETS factors play a specific role in the activation of arterial and angiogenic genes downstream of VEGFA signalling, ETS factors have also been implicated in the more general activation of genes, and their cognate regulatory elements, involved in endothelial identity and maintenance. However, the analysis of the precise roles played by ETS factors in the vasculature has been complicated by the abundance of different ETS factors in the endothelium coupled with extensive redundancy between different ETS family members. Parsing their two potential functions is further challenged by the multiple roles played by VEGFA signalling in the vasculature. Recent analysis in zebrafish concluded that low levels of VEGFA signalling promotes general endothelial identity and survival, while higher levels of VEGF signalling primarily promotes arterial specification . ConsequIn this paper, we have clearly demonstrated that vein-specific gene enhancers can be reliant on ETS factors for activation in venous ECs. This is similar to that previously reported in arterial-specific and angiogenic-specific enhancers, even though expression of venous genes is not induced to the same extent by high VEGFA signalling. Further, we have shown that VEGFA signalling can also increases ETS factor binding at venous enhancers, indicating that selective arterial and angiogenic gene activation is unlikely to be achieved through this mechanism. Because our analysis was predominantly restricted to two venous enhancers, the conclusions may not equally apply to all venous-expressed genes. Of note, Notch1 expression in arterial ECs , Tg(CoupTFII-965:lacZ) Tg(Dll4in3:lacZ) and Tg(HLX-3:lacZ) were generated as previously described  containing 5\u00a0\u200bmM potassium ferrocyanide, 5\u00a0\u200bmM ferricyanide, 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 2\u00a0\u200bmM MgCl2 and 1 X PBS. After staining, embryos were rinsed through a series of 1 X PBS washes, then fixed overnight in 4% paraformaldehyde at 4\u00a0\u200b\u00b0C. All embryos were imaged using a Leica M165C stereo microscope equipped with a ProGres CF Scan camera and CapturePro software (Jenoptik). In instances that images have been altered to improve quality and colour balance, each image within a set have been altered using the same parameters. This occasionally included to selective depletion of the yellow or red colour channel, in order to counteract issues from the X-gal stain solution (which is orange). All samples are stored in 4% PFA indefinitely and slowly become less orange. Consequently, samples stained more recently have a greater yellow/orange hue. An example of this alteration can be seen in  and tg(CoupTFII-965:GFP) zebrafish lines were generated in  at 28.5\u00a0\u200b\u00b0C. To image, all embryos were dechorionated and anesthetized with 0.01% tricaine mesylate. For analysis of F0 transgenic zebrafish, single embryos were transferred into a flat bottom 96-well plate, and GFP reporter gene expression screened with a Zeiss LSM 710 confocal microscope at 46\u201350 hpf. Whole zebrafish were imaged using the tile scan command, combined with Z-stack collection under a confocal microscope Zeiss LSM 710\u00a0\u200bMP (Carl Zeiss) at 488\u00a0\u200bnm excitation and 509\u00a0\u200bnm emission (EGFP).All animal procedures comply with all relevant ethical regulations, were approved by Clinical Medicine Local Ethical Review Committee, University of Oxford and licensed by the UK Home Office. Stable rated in . F0 mosarated in . EmbryosFor pharmacological inhibition of VEGF signalling, embryos were manually dechorionated and 0.625\u00a0\u200b\u03bcM, 1.25\u00a0\u200b\u03bcM, 2.5\u00a0\u200b\u03bcM and 5\u00a0\u200b\u03bcM of SU5416 (Stratech Scientific Ltd.) added at approximately 5ss as described in . Control4.3Ephb4-2mutETS and CoupTFIImutETS enhancer sequences were initially generated as custom-made, double-stranded linear DNA fragments . DNA fragments were cloned into the pCR8 vector using the pCR8/GW/TOPO TA Cloning Kit  following manufacturer\u2019s instructions. Once cloning was confirmed, each enhancer was transferred from the pCR8/GW/enhancer entry vector to a suitable destination vector using Gateway LR Clonase II Enzyme mix  following manufacturer\u2019s instructions. For mouse transgenesis, the enhancer was cloned into the hsp68-LacZ-Gateway vector (provided by N. Ahituv). For zebrafish transgenesis, the enhancer was cloned into the E1b-GFP-Tol2 vector (provided by N. Ahituv).4.3.1AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTTTCCTGAGGCTGTTTCCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTTCCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTTCCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTTCCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGGAAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.4.3.2agCTGAGGCTGTTagCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTagCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTagCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTagCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGAGAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTT4.3.3GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAGGAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACATCCTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAGGAAATGATCGAGATGCCTTGTCAGATCTTC.4.3.4GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAtcAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACagCTTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAtcAAATGATCGAGATGCCTTGTCAGATCTTC.4.4Mouse and human sequences of putative enhancers were aligned using ClustalW . Binding4.5italic underlined nucleotides modified (GGA to TCA or TCC to TGA) in mutant version:Proteins were made using the TNT Quick Coupled Transcription/Translation system as described in the manufacturer\u2019s directions. The truncated ETS1 DNA binding domain (ETS-DBD) and full length ERG were in the pCITE2 plasmid, and transcribed using T7 polymerase. To make each probe, double stranded oligonucleotides with CTAG 5\u2032 overhangs were labelled with 32P-dCTP using Klenow (Promega) to fill in overhanging 5\u2032 ends, and purified on a non-denaturing polyacrylamide-TBE gel. 20\u00a0\u200b\u03bcl binding reactions consisted of 2\u20133\u00a0\u200b\u03bcl protein or lysate control, 2\u00a0\u200b\u03bcl 10X binding buffer  and 0.5\u00a0\u200b\u03bcg poly dI-dC. For competitor lanes, a 100-fold excess of competitor DNA was added in a volume of 1\u00a0\u200b\u03bcl. Binding reactions were incubated at room temperature for 10\u00a0\u200bmin before the addition of radiolabelled probe, after which they were incubated an additional 20\u201330\u00a0\u200bmin. Gels were electrophoresed on a 10% non-denaturing polyacrylamide gel. Sequences of the probes and competitor regions are listed below, with tccccgccg;ETS control probe  CTAGtaaatcctgaggctg.Ephb4-2 ETS-b CTAGgttgtggctctttcctgtctggc.Ephb4-2 ETS-c CTAGgaggctgttggatggctggga.Ephb4-2 ETS-d CTAGggccctcgtcctctcatttg.Ephb4-2 ETS-e CTAGagggccctggatggttgg.Ephb4-2 ETS-f CTAGgtggggcacgggatgagagaggtgc.Ephb4-2 ETS-g CTAGgatggtttcctggcgcaagta.Ephb4-2 ETS-h CTAGcacttatcttcctcacagcccEphb4-2 ETS-I CTAGcctgccctggaaacagcc.Ephb4-2 ETS-j CTAGagccagggcaggaaagctgaagataa.Coup-965 ETS-a CTAGacaaatggatcctctgag.Coup-965 ETS-b CTAGgctgaagataaggaattgccagaga.Coup-965 ETS-c CTAGagctgttgggaagtcactat.Coup-965 ETS-d CTAGcggaccaataaaggaaatgaagcaacatc.Coup-965 ETS-e CTAGaaggttcctggccaaag.Coup-965 ETS-f CTAGaagcaacaggaaatgatcgagatc.Coup-965 ETS-g CTAGcaaaaccatccaggcctg.Coup-965 ETS-h CTAGgtcactatttt4.6165 (PeproTech) at 25\u00a0\u200bng/ml for 1.5\u00a0\u200bh. Cells were then trypsinised and the cell pellet collected.For VEGF stimulation experiments in cells, human umbilical vein pooled endothelial cells  were grown in Endothelial Cell Growth Medium 2 with BulletKit (PromoCell). Media was changed every 48\u00a0\u200bh. Four 80% confluent 15\u00a0\u200bcm dishes per condition were serum starved in 0.5% Foetal Bovine Serum (Gibco) overnight before being stimulated with VEGFAChromatin immunoprecipitation was carried out as previously described . BrieflyImmunoprecipitated DNA was analyzed by qPCR using TaqMan Custom Gene Expression Assay Probes (ThermoFischer) designed against 100bp regions of the Ephb4-2 enhancer, the CoupTFII-965 enhancer or a gene dessert region of Chromosome 7 as a negative control.4.6.1ACCCCTGCCCTTCCTTGCTGTTCTGCCTGGGTCCTGCGCCCGGGTTGGGGGGGGTGGGCCGGTCACCGAGGGCAGGAAACAGCCGGCTTCACTGGAGCCAGGCAGACCAG.4.6.2AGCGGCTGTATATTGCTGAAAGAGCATCATTCACATTCAGGCAGAGACAAAAGGTGGAAATGAAGTAACATCCTGGCTGAAGAAGGCCTCACGACAGAATA.4.6.3CCTCAGCCTCCCAAGTAGCTGGGATTACAGGTGTGTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGAACTCAGG TGATCTA.Each ChIP was performed on at least three biological replicates, with three technical replicates for each. Statistical analysis was performed in StepOne plus software, Microsoft Excel. Input was taken as the supernatant from the non-antibody control condition. Results are expressed as the mean of the % input defined as 100\u2217(2^(adjusted Input ct \u2013 ct IP)) across all replicates. Significant differences were calculated using ANOVA f test with p values subsequently derived from Tukey HSD test, accounting for multiple comparison correction. Graphs were produced using R statistical package.4.7https://ncbi.nlm.nih.gov/geo/) under accession GSE93030. The four ChIP-seq datasets used have accession numbers GSM2442775 to GSM2442778. Data consisted of ChIP-seq Model-based Analysis of ChIP-Seq (MACS) (https://CRAN.R-project.org/package = data.table and https://CRAN.R-project.org/package=ggpubr). GRCh37 ROIs: HLX-3 chr1:221,049,659\u2013221,050,776, Dll4in3 chr15:41,222,807\u201341,223,778, Ephb4-2 chr7:100,426,194\u2013100,427,393, and CoupTFII-965 chr15:95,908,708\u201395,909,301.ChIP-seq analysis was conducted on the published and publicly available data from q (MACS)  regions q (MACS) . MACS pe4.8Antisense MOs were ordered from GebeTools LLC and dissolved in water before injected into 1\u20132\u00a0\u200bcell stage zebrafish embryos as previously described . Sequencfli1 MO (3 \u2013 6\u00a0\u200bng) TTTCCGCAATTTTCAGTGGAGCCCG (GGAGCCCG .erg MO (3 \u2013 6\u00a0\u200bng) CAGACGCCGTCATCTGCACGCTCAG .4.9in situ hybridization ephb4, efnb2 and stab1l probes were generated as custom-made, double-stranded linear DNA fragments , cloned into the pCR2 vector using the TOPO/TA cloning kit (Invitrogen 450641) and transcribed using SP6 and T7. The sequences are provided below. dll4 probe was kindly provided by R. Patient, University of Oxford, Oxford. Whole-mount in situ hybridization was conducted as previously described . Embryos were washed through a dilution series of 2 x SSC followed by 0.2 x SSC at 65\u00a0\u200b\u00b0C and thereafter taken through room temperature dilution washes of 100% MABT . Nonspecific sites were blocked with MAB block (MABT with 2% Boehringer block reagent) and the embryos incubated for 15\u00a0\u200bh with 1:4000 antiDIG antibody (Roche) at 4\u00a0\u200b\u00b0C, before washing in MABT. Prior to staining, embryos were washed in AP buffer and the in situ signal developed at room temperature with BM Purple (Sigma-Aldrich). Staining was stopped as appropriate, and embryos were bleached in 3% H2O2/0.5% KOH until pigmentation disappeared, then re-fixed in 4% PFA for 20\u00a0\u200bmin and washed 4 times with PBST. Embryos were transferred to 80% glycerol for imaging.For zebrafish whole-mount escribed . Brieflyin situ/condition. Experimental blinding was not used as phenotypes of control and treated were easily detectable due to morphological defects.Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern. Numbers of zebrafish embryos was no less than 30/4.9.1TCTCAGCTCTGGACAAGCTGATCCGCAACCCGGCCTCACTCAAAATCACAGCGCAGGAGGGGGCGGGCCCCTCTCACCCTCTGCTGGACCAGCGGTCTCCACTCACGCCCTCATCCTGCGGGACAGTGGGTGACTGGCTGCGGGCCATCAAGATGGAGCGCTACGAGGAGACATTTCTGCAGGCGGGATACACGTCCATGCAGCTCGTCACCCACATCAACACGGAGGATCTGCTGCGTTTGGGAATAACTTTAGCAGGTCACCAGAAGAAGATTCTCTCCAGCATTGAGGCTCTCGGGATTCAAAACAAAGCACCAGGGAATGTGCTGTACTGA.4.9.2AAAACCAAGTCGATGAAAATCATCATGAAGGTTGGACAAAACCCCTCTGATCCCATTTCCCCCAAAGACTACCCTACCAGTTACCCTCCCAAACACCCTGACTTAGGGGGCAAGGACAGCAAATCGAATGAAGTACTTAAGCCAGATGCATCTCCTCATGGGGAAGATAAGGGAGATGGAAATAAATCCTCATCAGTCATTGGCTCAGAGGTGGCCCTGTTTGCCTGCATCGCCTCAGCAAGCGTCATCGTCATCATCATAATCATCATGCTAGTTTTCCTTCTCCTGAAGTATCGACGA.4.9.3GGATTCAGCAGCTACAGACACACCCAACCTCATCGACTAGCACAGACAGCAGCGTTAAACTCTCCCTTCATTTACCTGCAATCAGCTGACCGCTCTTAAAATAAAGGTTCTGTATTGGCATTGATGGTTCCGCGAAGAATCTTTATAAGCCATAACATCTTTCCATTTCCATGAGGTGTAAAAAGACTCTTTAGAATATTAAAATGTTACTTCATAAACATTTGATGTGTTTGATTGCAGATACTTCAGAGTGTTTAACTTCCACCCATTTATTTCTGCGTTTCACACATATTTTTGACTAAAAATGTTCTTTACATTAAGAAAAAATGGTGTACTCACCCTCAAGTAGGTCCAAACCTTCACGAGTTTCTTTTTTTGCCTTCTGTTGAACACAAACAAGAAAATATTTTGATAATGCGTTAAGCAAGGGGCCATTAGCTGTTTTGATCCAACTTTTTTCATGCGCATTTTAAATTATCGCATGTAAAAAAGCTTAATGGAAACCCAAGATGTGCTTAATTTGTCAAAATCTGCAT.4.10164 injections were performed on nude mice as described in (164 (provided by Lilly) diluted 1:30 in sterile 3% glycerol/PBS. At the required time-point after injection, ears were harvested and skin removed from the dorsal side. Ears were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 20\u00a0\u200bmin at 4\u00a0\u200b\u00b0C, washed twice in PBS then X-gal stained overnight at room temperature. Ears were then placed in 4% paraformaldehyde for storage.Intradermal Ad-VEGFAribed in . Briefly164 injected ears, ears were harvested and X-gal stained as described above, then dehydrated through a series of ethanol washes, cleared by xylene and paraffin wax-embedded. 5 or 6-\u03bcm sections were prepared and de-waxed. For imaging of X-gal staining, slides were counterstained with nuclear fast red (Electron Microscopy Sciences).For histological analysis of Ad-VEGFA4.11vegfaa121 and pCS2+vegfaa165 plasmids were kindly provided by S. Sumanas, Cincinnati Children\u2019s Hospital Medical Center, Ohio, USA. vegfaa121 and vegfaa165 mRNA was synthesised in vitro using the mMessage mMachine SP6 transcription kit (ThermoFisher Scientific) and injected into 1\u00a0\u200bcell stage zebrafish embryos at a final concentration of 50\u00a0\u200bpg of mRNA per embryo. Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern.pCS2+No competing interests declared."}
+{"text": "The rat progression elevated gene-3 (PEG-3) promoter displays cancer-selective expression, whereas the rat growth arrest and DNA damage inducible gene-34 (GADD34) promoter lacks cancer specificity. PEG-3 and GADD34 minimal promoters display strong sequence homology except for two single point mutations. Since mutations are prevalent in many gene promoters resulting in significant alterations in promoter specificity and activity, we have explored the relevance of these two nucleotide alterations in determining cancer-selective gene expression. We demonstrate that these two point mutations are required to transform a non-cancer-specific promoter (pGADD) into a cancer-selective promoter (pGAPE). Additionally, we found GATA2 transcription factor binding sites in the GAPE-Prom, which regulates pGAPE activity selectively in cancer cells. This newly created pGAPE has all the necessary elements making it an appropriate genetic tool to noninvasively deliver imaging agents to follow tumor growth and progression to metastasis and for generating conditionally replicating adenoviruses that can express and deliver their payload exclusively in cancer.Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at \u2212260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells  vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells. Defining the steps necessary to convert a normal cell into a cancerous one has been aided by identification and interrogation of defined genetic elements that regulate these processes ,2,3. Usipeg-3 and gadd34 genes share 73% nucleotide and 59% amino acid sequence homology [peg-3 and the murine gadd34 genes [The N-terminal domain first 415 aa) of rat PEG-3 is identical to the rat GADD34 protein  aa of ra,8. The p34 genes ,11,12,1334 genes . In cont34 genes . These speg-3 and gadd34 genes is chiefly transcriptional [Previous studies indicate that regulation of both the iptional . The fuliptional ,16. Promiptional ,15,16. Siptional . Furtheriptional ,15,16.Mutations are common events in gene promoters, which can result in dramatic changes in promoter specificity and activity ,19,20,21To determine why the cancer-specific min-PEG-Prom (pPEG) is functionally different than the pGADD, which shares almost identical sequence similarity, we compared the sequence of the promoter regions of the pPEG  with the pGADD , which resulted in the identification of only two base pairs that were different . Using sUsing the luciferase assay, we compared the activity of the pGADD-, pPEG- and pGAPE-Luc in cancer vs. normal  human cells. As shown in Previous studies have confirmed that when pPEG-Luc or pPEG-HSV-Tk were complexed with in vivo jetPEI a linear polyethylenimine (l-PEI), intravenous injection permitted imaging of both primary tumors and metastases . We initWe used in vivo jetPEI for delivery and imaging of the different promoters ,27. JetPAn important question was whether a single mutation in the pGADD was sufficient, or if both mutations were necessary, for pGAPE to display cancer-specific activity. To accomplish this goal, we generated a pGADD with a single mutation at bp \u2212260  and a second pGADD mutation at bp +159 . Using the same in vitro Luc-assay-based approach (described above), we analyzed the activity of pGADD, pPEG, pGAPE, pGADD1-1 and pGADD2-2 in the same series of human cancer, human immortalized normal cell lines, and primary normal cells shown in pPEG and pGAPE show tumor-specific in vivo imaging capabilities as well as enhanced tumor-specific expression in vitro, whereas the full-length GADD promoter, pT-GADD, does not show tumor specificity compared to normal cells. The same is true with the single mutation modification of bp \u2212260 (pGADD1-1), which lacks cancer specificity, whereas the change of bp +159 (pGADD2-2), shows cancer selectivity when engineered in pGADD (the minimum GADD34-Prom). To check the transcription factors involved in this cancer-selective activity, we analyzed the pGADD with and without mutations with the TFBIND software , which pChromatin immunoprecipitation (ChIP) assays confirmed an interaction between GATA2 and pGAPE in DU-145 and RWPE-1 cells A. DU-145We next determined whether changing GATA2 expression could modulate promoter binding to GATA2. RWPE-1 and DU-145 were transfected with GATA2 OE plasmid or shGATA-2 (inhibitory) plasmid for 48 h and then processed for ChIP assays. We predicted and confirmed that, following overexpression of GATA2, the binding to pGAPE/pPEG increased as compared to non-transfected cells, and conversely, GATA2 inhibition reduced binding to the pGAPE/pPEG. However, binding to the pGADD was not altered in either of these cell lines under these conditions C. CollecTo determine if a single mutation or both mutations (bp \u2212260 or bp +159) in the full-length GADD34-Prom  could confer cancer selectivity, we developed total pT-GADD constructs with a single mutation at G-A, pT-GADD1-1-Luc or C-T, pT-GADD2-2-Luc, and with both the mutations, pT-GADD2-Luc . The CTV induces profound anticancer activity in vitro and in vivo in diverse cancer indications [Tissue- and cancer-selective/specific promoters also provide functional tools to potentially regulate virus replication in a conditional manner, including adenovirus, herpesvirus, human immunodeficiency virus, simian immunodeficiency virus, and others ,32,59,60-7/IL-24 , under cications ,30,31,32In summary, we document that two-point mutations are critical to convert a non-cancer-specific pGADD into a cancer-selective pGAPE. A single mutational change at bp +159 of pGADD to that of the pPEG also promotes cancer selectivity both in vitro and in vivo. Additionally, we define a new transcription factor, GATA2, to which binding to the GAPE-Prom is indispensable for cancer-selective expression and activity. This newly engineered pGAPE embodies all the necessary features that make it an ideal tool to regulate transgene expression in a cancer-selective manner to both deliver imaging agents noninvasively to follow tumor promotion and metastasis and create CRADs expressing replicative functions uniquely in cancers.2 at 37 \u00b0C. Cells were validated for authenticity in prior studies [Immortalized human prostate epithelial (RWPE-1) and prostate cancer  cells, human immortalized pancreatic mesenchymal (LT-2) and pancreatic cancer  cells, early passage primary human mammary epithelial (HMEC) and breast cancer (MDA-MB-231 and SUM159) cells, H-TERT immortalized primary human fetal astrocytes (IM-PHFA) and neuroblastoma  cells were cultured as described previously ,64,65. S studies ,64,65 an studies ,64.\u00ae Plus Enzymatic Chromatin IP Kit (Magnetic Beads) was purchased from Cell signaling technology. Dual Luciferase Reporter Assay Kit was obtained from Promega  and used in the study. Lipofectamine 2000  and Invivo JetPEI  and transfection reagents, D-Luciferin Potassium Salt Bioluminescent Substrate , Agilent Quickchange II site directed mutagenesis kit  were used in the study.Antibodies specific for: GATA2, Control Rabbit IgG , horseradish peroxidase (HRP)-conjugated secondary antibodies , and \u03b2-actin  were used in this study. SimpleChIPRat progression-elevated gene-3 (PEG-3) was isolated from H5ts125-transformed rat embryo cancer cells by subtraction hybridization and is not expressed in normal rodent or human cells . The minpGADD34-FCGGGGTACCGAAAGAGAAAGAGAATGGGACAGCApGADD34-RCGGAAGATCT GGTCCGGTTCGGTTTGCCAAAAGCGGTCpGADD-FCGGGGTACCGAAAGAGAAAGAGAATGGGACGGCApGADD-RCGGAAGATCTGGTCCGGTTCGGTTTGCCAAAAGCGATCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCSequences of the full-length GADD promoters with mutations are described in the The prostate cancer, pancreatic cancer, breast cancer, and neuroblastoma cell lines and primary or immortalized normal cells were plated in 24-well plates (BD Biosciences) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s protocol. The indicated cells were transfected with Luc reporter constructs pPEG-Luc, pGADD-Luc, pGAPE-Luc or a empty vector (control). Luminescence was normalized for transfection efficiency by co-transfection with a vector expressing Renilla luciferase (Luc). After 48 h of transfection, the expression level of the Luc reporter was measured by the Dual Luciferase Reporter Assay Kit (Promega) ,16.Western blotting analysis was performed as described previously ,11,64. Rwt/vol) glucose. The glucose solutions of DNA and l-PEI polymer were then mixed together to give an N:P ratio (the number of nitrogen residues of in vivo jetPEI per number of phosphate groups of DNA) of 6:1 in a total volume of 400 \u03bcL. The DNA-PEI polyplex was injected intravenously as two 200 \u03bcL injections with a 5 min interval.Low-molecular-weight l-PEI\u2013based cationic polymer, in vivo jetPEI (Polyplus Transfection), was used for gene delivery ,27. The In vivo BLI was conducted at 24 and 48 h after the systemic delivery of reporter genes ,27. All 3 in 1% SDS. Primer pairs are listed below.ChIP assays were performed as described previously . BrieflyThe following primers were used in the ChIP assay:GADD F- GAA AGA GAA AGA GAA TGG GAC GGADD R- GTC CGG TTC GGT TTG CCA AAA GCG GGAPE F- GAA AGA GAA AGA GAA TGG GAC AGAPE R- GTC CGG TTC GGT TTG CCA AAA GCG AGAD1-1 F- GAA AGA GAA AGA GAA TGG GAC AGADD1-1 R- GTC CGG TTC GGT TTG CCA AAA GCG GGADD2-2 F- GAA AGA GAA AGA GAA TGG GAC GGADD2-2 R- GTC CGG TTC GGT TTG CCA AAA GCG AStatistical analysis was performed using SPSS 22.0 software , and statistical graphs were generated using GraphPad Prism . The generalized odds ratios (ORs) and 95% confidence interval (CI) of the promoters were calculated. The multiple comparisons were performed using false discovery rate (FDR) correction. False discovery rate (FDR) correction was used to analyze the promoter activity among the different promoters."}
+{"text": "The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1\u2019s molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes , and metastasis . Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration  Metastasis is a multi-step process that occurs when cells disseminate from the primary neoplasm and eventually colonize distant organs. Successful completion of this complex cascade is associated with nearly all cancer-related morbidities and mortalities. Despite its causative role in cancer-specific mortality and morbidity, a complete understanding of the process and its mechanisms remain elusive. Metastasis is regulated by three types of genes: metastasis-promoting, -suppressing, and -efficiency modifying . The proBRMS1 was discovered in the year 2000 and its re-expression in multiple cell lines significantly decreases lung metastases in syngeneic and xenograft models \u20138. In adBRMS1 is a known member of Sin3 histone deacetylase (HDAC) transcriptional regulatory complexes in multiple eukaryotic cell types \u201313. It iIn order to translate BRMS1 into clinical practice, additional structural and interactome data are necessary. This study leverages previously described features of BRMS1 to clarify its molecular functions within a cell. Previous attempts to define BRMS1 structure-function have been met with uneven success. To date no one has successfully crystallized nor determined structure for full-length, wild-type BRMS1. Computer algorithms and NMR studies have identified structural information for BRMS1 domains . BRMS1 hin vivo studies demonstrating that alteration of the domain impacts overall metastatic burden [The C-terminus of the BRMS1 protein is of particular interest as it has been shown to play a critical role in metastatic suppression, due to c burden . Of notec burden . Due to The BRMS1 primers listed in 7) were cultured into a 15 cm tissue culture plates for 24 hours then Halo-BRMS1 constructs were transfected using Lipofectamine LTX (Thermo Fisher Scientific). After 48 hours cells were harvested and washed twice with ice-cold PBS. Cells were then resuspended in mammalian cell lysis buffer (Promega) (50 mM Tris\u00b7HCl (pH 7.5), 150 mM NaCl, 1% Triton\u00ae X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 \u03bcM phenanthroline, 1 mM PMSF, 10 \u03bcM bestatin, 5 \u03bcM pepstatin A, and 20 \u03bcM leupeptin) followed by centrifugation at 21,000 \u00d7 g for 10 min at 4\u00b0C. To remove insoluble materials, cell extracts were diluted with 700 \u03bcl of TBS  and centrifuged at 21,000 \u00d7 g for 10 min at 4\u00b0C. Next, cell extracts were incubated overnight at 4\u00b0C with magnetic beads . Before elution, magnetic beads were washed four times with wash buffer . Proteins bound to magnetic beads were eluted for 2 hours at room temperature using elution buffer containing 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 0.005 mM DTT, and 2 Units AcTEV\u2122 Protease (Thermo Fisher Scientific). The eluate was further purified by passing through a Micro Bio-Spin column  to remove residual beads prior to proteomic analyses.HEK293T cells . This application utilizes the average spectral counts of each bait across all baits to calculate the TopS score, which indicates a likelihood ratio of binding.Topological Scoring was completed for all proteins as previously described , 28. BriBRMS1 protein structure was predicted based upon its amino acid sequence retrieved from NCBI using I-TASSER , 30. BriThe primary sequence of BRMS1 was mutated at S237 to either Aspartic Acid (D) or Alanine (A). These mutants were subject to multiple predictive software applications. MuPro  utilizesThree biological replicates were performed for each bait protein . The distributed normalized spectral abundance factor (dNSAF)  was usedTo spatially map all significant 1175 proteins, we first applied a T-Distributed Stochastic Neighbor Embedding (t-SNE), a nonlinear visualization of the data followed by a k-means clustering on the two vectors generated from the t-SNE . The numTo compare the overall composition of SIN3A associated proteins between metastatic and normal breast cells, co-IP of SIN3A  from thIn order to determine the biological enrichment of differentially expressed proteins in the mutants, we subjected 1175 proteins to Reactome pathway analysis. Enrichment was completed within the R environment and several packages such as clusterProfiler, ReactomePA, DOSE and enrichplot were used.http://www.ingenuity.com, Release date: December 2016). Visualization for overlapping significant proteins was completed in Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). Heatmaps were generated within ClustVis (https://biit.cs.ut.ee/clustvis/).Significant proteins were subjected to multiple analyses. Disease enrichment was completed within Ingenuity Pathway Analysis  cell culture medium (Thermo Fisher Scientific). MDA-MB-231 cells were cultured in 5% FBS, 0.5 mM NEAA 0 + DMEM/Sodium Bicarbonate (2.438 g/L) cell culture medium (Thermo Fisher Scientific). In both 293T and MDA-MB-231s, overexpression cells were first transduced with pENTR followed by pLENTI expression within 293FT cells according to manufacturer\u2019s instructions (Invitrogen). Clones were selected with blasticidin. Each construct contained a single or 3x-Flag epitope tag. Expression was quantified using validated BRMS1 1a5.7 antibody (as described previously ), FLAG-M5\u2019- GATCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGAAGGCTAGGGCAGCTGTGTCCCCTCAGAAGAGAAAATCGGATGGACCTTGATGATGAC -3\u2019, R: 5\u2019 -TCGAGTCATCATCAAGGTCCATCCGATTTTCTCTTCTGAGGGGACACAGCTGCCCTAGCCTTCTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCATG -3\u2019.F: Predicted amino acid sequences for each construct are:WTBRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVSPQKRKSDGP1-229BRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIS237ADYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVAPQKRKSDGPS237DDYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVDPQKRKSDGP230-246BRMS1MDYKDHDGDYKDHDIDYKDDDDKKARAAVSPQKRKSDGP293T cells were grown to 90% confluency, collected and RNA extracted an RNA Isolation kit (Zymo Research). Following RNA extraction cDNA completed with the iScript cDNA synthesis kit (Bio Rad). qRT-PCR was completed with a SYBER Green assay, and cDNA loaded at a concentration of 50 ng/uL. The following primers were utilized for analysis:https://imagej.nih.gov/ij/) was utilized for analysis, followed by statistical analysis in R (\u2018FAS\u2019 package), in which a Kruskal Wallis test followed by a Dunn\u2019s adjustment was completed (https://www.R-project.org/).Cells were cultured at a seeding density of 750,000 cells/well in 5% FBS, 0.5 mM NEAA + DMEM/Sodium Bicarbonate (2.438 g/L) media in 6-well plates (Thermo Fisher). After 48 hours cells were scratched, washed, and media replaced with serum-free DMEM/Sodium Bicarbonate (2.438 g/L) (Thermo Fisher). Cells were imaged at t = 18 to 24 hrs. Image J  MDA-MB-231 cells were transduced with the construct as described above. For each construct, 3 clones were selected. 2x105 cells suspended in 100 uL of Hanks Balanced Salt Solution  were intravenously injected within the tail vein of three week-aged athymic mice (Harlan HSD athymic nude-Foxn1nu), which were restrained in a tube to facilitate tail manipulation. The lung metastases grew for 6 weeks or until the mouse was moribund and required euthanasia. Lungs were removed and fixed in a mixture of Bouin\u2019s fixative and neutral buffered formalin (1:5 v/v), and macroscopic lung metastases were counted. This study was completed in 30 mice per construct, and 3 clones per construct. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center (#2014\u20132208). These experiments were performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, USDA regulations , the Federal Animal Welfare Act (7 USC 2131 et. Seq.), and the Guide for the Care and Use of Laboratory Animals, that were followed in attempts to alleviate animal suffering. A Kruskal Wallis test followed by a Dunn\u2019s adjustment was completed as per above for statistical analysis.To measure the successful colonization of the lungs by metastases for each construct  [https://kmplot.com/analysis/) [The UALCAN web resource was utilized to assess the expression of BRMS1, BRMS1L, HDAC1, SIN3A, and SUDS3 in tumor compared to normal expression (ex.html) . All samalysis/) . PatientR packages Gplots, GGplots2, and RColor Brewer were utilized for image analysis of data.S237D ; BRMS1S237A ; BRMS11-229 ; and BRMS1230-246   . Regardl methods . These dGiven the lack of crystal structure for BRMS1 , we used the I-Tasser structure prediction method to predict the three-dimensional structure of BRMS1 using its amino acid sequence . The algin silico prediction methods, when combined with our previous findings that S237 near the C-terminus can be phosphorylated compelled us to posit that the phosphorylation site could regulate protein binding. To test this hypothesis, mass spectrometry was completed (WT), BRMS11-229, S237A, S237D, or BRMS1230-246 , applied to Z-statistics obtained from the QSPEC analysis [Mass spectrometry identified 1175 proteins whose binding was significantly (QSPEC Z-Score \u00a3-2) altered in at least one mutant compared to BRMS1analysis . This an1-229, and BRMS1230-246 cluster the most similarly. This finding may suggest that in order for BRMS1WT to interact with certain proteins it must include both phosphorylation at S237 as well as additional factors near the N-terminus that BRMS1230-246 lacks. That being said, BRMS1WT and S237D have little overlap, sharing only HDAC1, HDAC2, and SIN3A  was employed . This mend SIN3A . This coTo further examine Sin3/HDAC complex binding with BRMS1 we utilized recently published crosslinking data of complex members . BRMS1 wTo examine the interaction between SIN3A and BRMS1 further, we utilized SIN3A-endogenously expressing cells. SIN3A was co-immunoprecipitated from the nuclear lysates of immortalized, but otherwise normal, MCF-10A breast cell line, as well as within the metastatic MDA-MB-231 breast cancer cell line. These precipitates were then subjected to mass spectrometry through MALDI-TOF analysis. Interactions with SIN3A were found to be context-dependent, i.e., normal breast cell line interactions differ from metastatic cancer cell interactions, though several of these interactions have been previously published and validated in many cell lines and systems , suggest\u22125) with SIN3B  versus C-terminal (BRMS1230-246) binding were observed. Many of the significant pathways represented in the analysis were not represented in BRMS11-229, e.g., mRNA processing, rRNA processing, and immune responses (230-246), emphasizing the importance of the C-terminus in overall protein function and regulation of protein interactions. Importantly, many of the biological processes are tied to cancer-associated pathologies  and further study into the BRMS1 C-terminus role in these pathways could identify the molecular role of BRMS1 in metastasis. To identify binding partners distinctly affected by particular domains of BRMS1, all protein interaction changes for each mutant were considered. To ensure that putative interacting proteins were truly associated with BRMS1, an additional criterion was added, i.e., the interactor must have a high TopS value in BRMS1WT, to demonstrate an increased likelihood of binding ratio . Several of the enriched pathologies are often associated with neoplasia, including cell cycle dysregulation, cell death and survival, RNA damage repair, embryonic development, immunological disease, and infectious disease. The latter findings compliment previously published results in which re-expression of BRMS1 in metastatic MDA-MB-435 cells were highly enriched for upregulated immune response genes [To further characterize how changes in interactor binding may play a role in the development of certain pathologies, disease enrichment within Ingenuity Pathway Analysis (IPA) was completed , Src, and Fascin \u201371. ThesWithin these two enriched disease pathways, several proteins overlap, including BReast CAncer gene 1 (BRCA1), mechanistic target of rapamycin (mTOR), and Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) . SeveralWT and its mutants , we examined how BRMS1 interacting partners  as well Firstly, we compared the expression of the Sin3/HDAC members from normal to breast tumor samples. BRMS1, HDAC1 and SUDS3 significantly increase expression within the tumor samples, while BRMS1L has significantly decreased expression . SIN3A iTo address this speculation, we also looked for an association between one of the Sin3/HDAC members and BRMS1 expression. To do this we first separated patients by expression of each Sin3/HDAC BRMS1-interacting member by expression greater than or less than the median expression of that particular member. Within those subsets, BRMS1 expression (high vs low) was examined for patient overall survival . When loin silico prediction methods, that the C-terminus functions in protein binding, and demonstrated through mass spectrometry that the C-terminus can impact BRMS1 associations with Sin3/HDAC complexes as well as proteins outside of the complexes. We model this hypothesis in This study investigates the role of BRMS1 C-terminus in protein function, with an emphasis on the function of the S237 phosphorylation site. We hypothesized, based upon It is interesting to speculate that once expression of either BRMS1L or SIN3A is lost, it appears that BRMS1 will then function to suppress metastasis and increase survival. Our data begin to define these interactions, in particular with SIN3A, but further studies are required in order to directly define those relationships, including whether the interactions are based on phosphorylation-status to regulate metastasis. The C-terminus and phosphorylation status appear to be vital to regulating the metastasis promoting miRNA miR-10b and corresponding reductions in motility and metastasis. The data also identify specific functionalities of protein complexes previously associated with BRMS1 and metastasis.This combination of results refine the molecular impact of BRMS1 on regulating metastasis and the potential development of therapeutics based upon BRMS1.S1 FigExpression of BRMS1WT, BRMS11-229, and phospho-mutants compared to parental and vector control 293T cells. (B) qPCR quantification of BRMS1230-246 expression in 293T cells. (C) Expression of BRMS1WT and phospho-mutants compared to vector control MDA-MB-231 cells. (D). Expression of BRMS11-229 within MDA-MB-231 cells. (E) qPCR quantification of BRMS1230-246 expression in MDA-MB-231 cells.(TIF)Click here for additional data file.S2 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those greater than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S3 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those less than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Data(DOCX)Click here for additional data file."}
+{"text": "EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. Methods: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae\u2013EC adhesion. Results: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. Conclusions: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.Introduction: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 ( Rickettsia (R.), including R. rickettsii [R. parkeri [R. parkeri rickettsiosis [R. conorii, the causative agent of Mediterranean spotted fever [R. australis, which is a part of the transitional group (TRG) [R infections are frequently associated with severe morbidity and mortality [R infections, it only stops bacteria from reproducing, but does not kill the rickettsiae. A fatality rate as high as 32% has been reported in hospitalized patients with Mediterranean spotted fever [Rickettsioses are devastating human infections . These ackettsii ,3 and R. parkeri ,5,6 thatttsiosis , respected fever ; and R. up (TRG)  and causup (TRG) . A licenup (TRG) ,2,9. Typup (TRG) ,12. Untrortality ,14,15. Aed fever ,17. Comped fever ,20,21,22EPAC1, not EPAC2, is the dominant isoform [EPAC1 protected mice from fatal rickettsioses [EPAC1 suppressed rickettsial adherence to and invasion into ECs [cAMP is one of the most common and universal second messengers. cAMP regulates a myriad of important biological processes under physiological and pathological conditions. Therefore, current pharmaceutical medications target the cAMP signaling pathway more than any other . cAMP tr isoform ,39,40. Wttsioses . Our ex into ECs . HoweverANXA2), a well-characterized plasminogen and plasminogen activator receptor [Staphylococcus aureus adhesion to EC surfaces [EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation [EPAC1-ANXA2 signaling pathway is involved in the regulation of rickettsial adhesion to ECs.The mechanism(s) underlying bacterial adherence to vascular endothelial cells (ECs) under shear stress from flowing blood is critical to our understanding the initial stages of the pathogenesis of endovascular bacterial infections. The constitutively expressed native proteins on and/or in the mammalian host plasma membrane play crucial functional role(s) during the initial stage of infection, just prior to EC activation. Recently we reported that endothelial surface annexin A2  provides evidence to support that EPAC1 governs rickettsial adhesion to EC surfaces in association with regulation of ANXA2 Y23 phosphorylation.In the present study, an established, anatomically based, in vivo quantitative system that measures bacterial adhesion to ECs  was combEPAC1 plays a critical regulatory role in the early stage of rickettsial invasion into nonphagocytic host cells [EPAC1-knock out (KO) mouse model [EPAC1 during rickettsial adhesion. Plaque assays reveal that the number of viable rickettsiae in circulating blood was higher in EPAC1-KO mice (n = 11) than wild-type (WT) mice (n = 10) at 30 min and 1 hr post-infection (p.i.) when the bacteria were given by the intravenous route , we found that rickettsiae mediate adhesion to the surface of ECs via host ANXA2 . CompareANXA2 .p < 0.01). To examine whether EPAC1 contributes to reOmp and BMEC binding, we compared the adhesion forces generated in WT versus EPAC1-KO BMECs; reduced adhesion forces between a reOmpB and a EPAC1-KO BMEC were recorded (p < 0.01)  microscopy [EPAC1-KO BMECs at 15 min p.i. with 10 MOI rickettsiae  . Furthercroscopy . All ECskettsiae .EPAC1 specific activator named I942 that competitively binds the cyclic nucleotide-binding domain of EPAC1 [p < 0.05) (ANXA2 in ECs diminished reOmpB binding forces to the ANXA2-KO BMEC surface (p < 0.01) , independent of its concentration in the culture medium  . Depleti < 0.01) , corroboiving EC . Interese medium .EPAC1-ANXA2 pathway is involved in the interaction of OmpB with ECs during the initial stages of bacterial binding.Collectively, these data suggest that the EPAC1 regulates endothelial luminal surface ANXA2 expression by modulating the phosphorylation of Y23 [EPAC1-targeted site in the N-terminus of ANXA2 that leads to regulation of rickettsial adhesion, we transfected ANXA2-KO BMECs with mouse WT ANXA2 construct, phosphodefective ANXA2 mutant Y23F, or phosphomimic ANXA2 mutant Y23E, respectively, which were generated by site-directed mutagenesis using their respective mutant oligonucleotides [ANXA2 in these constructs-transfected ANXA2-KO BMECs were detected, respectively (Our previous study suggested that n of Y23 . To idenleotides . Expressectively .p = 5.36 \u00d7 10\u22128) and phosphomimic Y23E  in ANXA2-KO BMECs rescued the diminished reOmpB and cell binding force, respectively, whereas the phosphodefective mutant Y23F (p = 0.90) failed to do so  in the plasma or other major hematological parameters [ANXA2 phosphodefective mutant Y23A, not ANXA2 phosphomimic mutant Y23E, yields a predominantly negative effect for the translocation of ANXA2 to the membrane surface. The Y23A mutant can still bind to S100A10 but only in the cytosol, not in the plasma membrane [ANXA2 functions as a binding receptor for rickettsial OmpB, in the present study we further reveal that EPAC1 regulates rickettsial adhesion to EC surfaces by targeting the ANXA2 Y23. Depletion of ANXA2 in ECs reduced the enhanced binding force between reOmpB and ECs in the presence of I942. The WT ANXA2 construct rescued EPAC1-specific activator I942-induced enhancement of reOmpB binding forces to a ANXA2-KO BMEC surface. Furthermore, Y23E mutant rescued the diminished reOmpB and cell binding force in ANXA2-KO BMEC, whereas the Y23F mutant failed. However, there was no increasing in the binding force in the phosphomimic Y23E mutant-transfected ANXA2-KO BMEC in response to I942, further suggesting that EPAC1 modulates Y23 phosphorylation of the binding receptor ANXA2. Currently the accurate pathway how EPAC1 modulates the phosphorylation of specific residues(s) in ANXA2 remains to be identified. Furthermore, the potential crosstalk between cAMP-EPAC1 system and other identified factors involved in rickettsial adhesion and/or invasion should be investigated in future studies. and S25 ,56. Phosid rafts ,57 and tartments ,58. Afteechanism . We reporameters . Specifimembrane . Given tThe colloidal probe-based AFM assay was developed to study protein\u2013protein interactions and to quantify the interacting forces in the range from picoNewtons to nanoNewtons . During EPAC1-KO BMEC is still higher than the forces between BSA and EPAC1-KO BMEC. These data may suggest that intracellular EPAC1 is not the sole regulator on endothelial surface receptor for OmpB-mediated rickettsial binding. Such unknown host surface protein(s) remain to be identified. Furthermore, although BSA has been widely employed as a mock control using AFM or FluidFM to measure protein-protein interaction in studying bacterial adhesion [The binding forces between reOmpB and adhesion , identifEPAC1 modulates rickettsial adhesion, involving regulation of tyrosine 23 phosphorylation of the binding receptor ANXA2. This finding provides experimental and theoretical support for therapeutic strategies for rickettsiosis targeting the host cAMP-EPAC system. In conclusion, we have revealed a novel mechanism by which EPAC1-KO mice were derived as described previously [ANXA2-KO mice on the C57BL/6J background were a generous gift from Dr. Katherine Hajjar  [R. australis. Therefore, this organism was chosen as the TRG rickettsial agent of choice [All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB). Wild-type (WT) mice (C57BL/6J) were obtained from The Jackson Laboratory . C57BL/6 eviously . ANXA2-KNY, USA) . All micf choice .R. australis (strain Cutlack) was prepared as described [escribed . UninfecEPAC1-KO, and ANXA2-KO mice using established protocol [Mouse brain microvascular endothelial cells (BMECs) were isolated from wild-type, protocol ,51,62.ANXA2, ANXA2 mutant Y23E, and ANXA2 mutant Y23F were purchased from GENEWIZ .A rabbit polyclonal antibody against SFG/TRG rickettsiae was obtained from the laboratory of Dr. David Walker . AlexaFluor 594-conjugated goat anti-rabbit IgG and ProLong Gold Antifade Reagent with DAPI were purchased from Invitrogen . Recombinant rickettsial OmpB (reOmpB) (amino acids 1363\u20131655) was purchased from MyBioSource . Unless otherwise indicated, all reagents were purchased from Thermo Fisher Scientific. Expression constructs for GFP-tagged mouse WT ANXA2, ANXA2 Y23E, and ANXA2 Y23F constructs (1 \u00b5g/500 \u00b5L for 1 \u00d7 105 cells), respectively, using Magnetofection PolyMag and PolyMag Neo . The efficacy of transfection was evaluated with GFP fluorescent microscopy [Transfections were performed using a published protocol . Cells wcroscopy .TACGGGTCAGTCAAACCCTACACCAACTTCGATGCTGAGAGGGATGCTCTGAACATTGAGACAGCAGTCAAGACCAAAGGAGTGGATGAGGTCACCATTGTCAACATCCTGACAAACCGCAGCAATGTGCAGAGGCAGGACATTGCCTTCGCCTATCAGAGAAGGACCAAAAAGGAGCTCCCGTCAGCGCTGAAGTCAGCCTTATCTGGCCACCTGGAGACGGTGATTTTGGGCCTATTGAAGACACCTGCCCAGTATGATGCTTCGGAACTAAAAGCTTCCATGAAGGGCCTGGGGACTGACGAGGACTCCCTCATTGAGATCATCTGCTCACGAACCAACCAGGAGCTGCAAGAGATCAACAGAGTGTACAAGGAAATGTACAAGACTGATCTGGAGAAGGACATCATCTCTGACACATCTGGAGACTTCCGAAAGCTGATGGTCGCCCTTGCAAAGGGCAGACGAGCAGAGGATGGCTCAGTTATTGACTACGAGCTGATTGACCAGGATGCCCGGGAGCTCTATGATGCCGGGGTGAAGAGGAAAGGAACCGACGTCCCCAAGTGGATCAGCATCATGACTGAGCGCAGTGTGTGCCACCTCCAGAAAGTGTTCGAAAGGTACAAGAGCTACAGCCCTTATGACATGCTGGAGAGCATCAAGAAAGAGGTCAAAGGGGACCTGGAGAACGCCTTCCTGAACCTGGTCCAGTGCATCCAGAACAAGCCCCTGTACTTCGCTGACCGGCTGTACGACTCCATGAAGGGCAAGGGGACTCGAGACAAGGTCCTGATTAGAATCATGGTCTCTCGCAGTGAAGTGGACATGCTGAAAATCAGATCTGAATTCAAGAGGAAATATGGCAAGTCCCTGTACTACTACATCCAGCAAGACACCAAGGGTGACTACCAGAAGGCACTGCTGTACCTGTGTGGTGGGGATGACTGA-3\u2019.5\u2019-ATGTCTACTGTCCACGAAATCCTGTGCAAGCTCAGCCTGGAGGGTGATCATTCTACACCCCCAAGTGCCANXA2 mutant Y23E, the sequence of bases encoding Y23 is replaced with GAA. For ANXA2 mutant Y23F, the sequence of bases encoding Y23 is replaced with UUC.For R. australis  was injected through the tail vein, rickettsial virulence (by plaque assay) was measured in a 1 \u00b5L blood sample collected from the orbital venous sinus (OVS) at different times until 1 h post-infection (p.i.). Rickettsial antigens and mouse EPAC1 were detected with a rabbit polyclonal antibody against SFG/TRG rickettsiae (1:1000) and mouse monoclonal antibody against EPAC1 (1:500) , respectively, overnight at 4 \u00b0C, followed by incubation with AlexaFluor 594 goat anti-rabbit (1:1000) and AlexaFluor 488 goat anti-mouse (1:2000) antibodies for 15 min. Normal mouse or rabbit IgG was used as an antibody negative control during IF staining  were coated with reOmpB using published protocols ,64. Brieg-poly(ethylene glycol) (PLL-g-PEG) to block nonspecific adhesions prior to calibration using the method of Sander et al. [The FluidFM system coupling Nanosurf Core AFM  and Fluidic Pressure Controller  was used for this assay. A micropipette  (Cytosurge AG) was coated with 0.1 mg/ml poly(L-lysine)-r et al.  and pre-r et al. . The forr et al. . Ten celt-test or One-Way ANOVA . p values are as follows: ** p < 0.01 and * p < 0.05. Statistical significance is considered as p < 0.05.Values are reported as mean \u00b1 SEM. The data were analyzed using the Student\u2019s"}
+{"text": "The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C.\u00a0elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C.\u00a0elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C.\u00a0elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.Proximity labeling provides a powerful  Hin\u00a0vivo . Proximiin\u00a0vivo , 2, 7. T limited , 10 and  limited , 12 and  tissues  or pretr tissues .Caenorhabditis elegans has proven a useful workhorse to investigate metazoan biology, offering powerful genetics, in\u00a0vivo cell biology, an anatomy described at electron micrograph resolution, and a defined number of cells whose gene expression can be profiled at single cell resolution  tRNA synthetase (MuPheRS)  , 27. MuPC.\u00a0elegans results in robust biotinylation signals in the intestine, with the strongest signals generated by TurboID , we generated transgenic animals expressing a TurboID-mNeongreen-3xFLAG (TbID-mNG) fusion protein in various tissues (B). We compared protein biotinylation levels in age-synchronized young adults expressing the neuronal rab-3p::TbID-mNG transgene with wild-type controls. In the absence of exogenously added biotin, transgenic animals showed only a slight increase in biotinylated proteins compared with controls. Adding exogenous biotin increased protein biotinylation specifically in animals expressing the rab-3p::TbID-mNG transgene (C). To optimize biotin availability to worm tissues, we treated animals with exogenous biotin for different time intervals, using biotinylation in neurons as a readout. A 2-h incubation was sufficient to achieve robust protein labeling (D). To identify an optimum biotin concentration for protein labeling, we treated animals with varying concentrations of biotin for 2\u00a0h. We observed a substantial increase in biotinylation in worms treated with 1\u00a0mM biotin but higher biotin concentrations did not appear to further increase biotinylation (E). We also used the E.\u00a0coli biotin auxotrophic strain MG1655 as a food source for worms instead of standard OP50 , indicating that TurboID is functional in all major C.\u00a0elegans tissues.Like pan-neuronal expression, intestinal, hypodermal, and muscle-specific expression of TurboID-mNG conferred robust biotinylation activity A, indicaC.\u00a0elegans (A\u2013C). A significant advantage of PL compared with IP is that extracts can be collected under strong denaturing conditions , since the biotin tag is covalently attached. This allowed us to achieve >95 % solubilization of proteins . After extensive washing and elution, we fractionated the affinity-purified biotinylated proteins by SDS-PAGE, visualized the proteins by Coomassie staining, and analyzed gel slices by LC-MS/MS analysis (B). Using a threshold of at least two unique peptides, we cumulatively identified >4000 proteins expressed in one or more tissues . In addition, we observed a strong correlation between the two replicates at the level of spectral counts (B). Consistent with previous findings, we identified most proteins in the intestine, followed by the hypodermis and muscle cells (B), (B), and 1274 proteins were detected in only one tissue (C). Proteins identified by MS/MS when TurboID was targeted to neurons included broadly expressed neuronal proteins, such as CLA-1, UNC-31, and proteins expressed in subsets of neurons, such as the glutamate decarboxylase UNC-25, which is known to be expressed in 26 neurons, and OSM-10, which is expressed in four pairs of neurons (D). Muscle-specific samples were enriched in CPNA-1, CPNA-2, PQN-22, and F21C10.7 (D); intestine-specific samples were enriched in ACOX-2, CGR-1, C49A9.9, and C49A9.3 (D), and hypodermis-specific samples included F17A9.5, PAH-1, R05H10.1, and CPN-2 (D). In addition, we identified tissue-specific enrichment for many proteins whose expression was previously uncharacterized . For some of these proteins we validated the tissue-selective expression highlighted by our MS/MS data by making transgenic reporters. The reporters confirmed the expression profile predicted by MS/MS, validating the method's specificity .We next optimized a protocol to extract and affinity purify biotinylated proteins from \u00a0elegans , A\u2013C. A ins A\u2013C, . We achiins A\u2013C, . Westernins A\u2013C, A. After analysis B. Using B and C, . Using tB and C, , we lookB and C, A. In addl counts B. Consisle cells B, 12). . C.\u00a0eleglls (B), B, and 12e tissue C. Protei neurons D. MuscleF21C10.7 D; intest C49A9.3 D, and hynd CPN-2 D. In addcterized , Fig.\u00a02EC.\u00a0elegans nervous system includes 118 classes of neurons, with most classes consisting of a single pair of neurons that form left/right homologs (gcy-8 promoter (A). Western blot analysis of extracts from these animals revealed a biotinylation signal significantly higher than that in extracts from wild-type controls (B). Correlation plots of mass spectrometry data obtained for affinity-purified extracts made from animals expressing the gcy-8p::TbID-mNG transgene showed reproducible results between replicates (A). As expected, these samples were enriched for proteins specifically or selectively expressed in AFD neurons when compared with similarly processed extracts from nontransgenic controls, or from animals expressing a rab-3p::TbID-mNG transgene (C and S4B). Enriched proteins included the transmembrane guanylate cyclases GCY-8 and GCY-18 and the homeobox transcription factor TTX-1 (D and S4B). Our MS data also identified other proteins enriched in the AFD-specific TurboID samples compared with the pan-neuronal TurboID controls (E and S4C). To examine if these proteins were selectively expressed in AFD neurons, we generated transgenic reporter lines (F). nex-4 and F37A4.6 were expressed specifically in AFD, albeit F37A4.6 at low levels; T06G6.3 was expressed in a small subset of neurons that included AFD (F). Our list of AFD-enriched proteins, identified by mass spectrometry, was consistent with AFD-specific gene expression identified by RNA Seq . ELKS-1 is expressed throughout the nervous system and localizes to the presynaptic active zone, in proximity to other presynaptic proteins such as \u03b1-liprin and RIM . Western blot analysis of extracts from ELKS-1::TbID animals did not show an increased biotinylation signal compared with wild-type controls (B). However, mass spectrometry analysis of streptavidin-purified proteins from this knock-in strain revealed enrichment of known synaptic proteins including UNC-10/RIM, SYD-1/SYDE1, SYD-2/\u03b1-liprin, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, and the RIM-binding protein RIMB-1 (D). We measured enrichment by comparing mass spectrometry data obtained for control extracts processed in parallel from wild-type and from transgenic animals expressing free TurboID-mNG throughout the nervous system, rab-3p::TbID-mNG (C and S4E) or both nervous system and other tissues (G). Between-replicate correlation plots revealed reproducible results between experimental repeats (D). We also identified several previously uncharacterized proteins as enriched in ELKS-1::TbID-mNG samples . We expressed mNeongreen translational fusion transgenes for several of these proteins exclusively in the AFD neuron pair and showed that they colocalized with ELKS-1::mScarlet at presynaptic active zones where AFD is known to synapse with AIY interneurons . Together, our findings show that TurboID-mediated proximity labeling is an effective method to reveal protein interactors in C.\u00a0elegans at endogenous levels with specificity and sensitivity.We next asked if TurboID can highlight the interactome of a specific  and RIM . To havesynapses A. Westercontrols B. Howeven RIMB-1 D. We meaTbID-mNG C and S4E tissues G. Betwee repeats D. We alsrneurons  . Human ANKS1B, also known as AIDA-1, is a risk locus for autism and neurodevelopmental defects (C.\u00a0elegans ortholog of human KIAA0930. C16E9.2 was recently given the name sentryn (STRN-1). STRN-1 together with the SAD kinase and liprin-\u03b1 promote dense-core vesicle (DCV) pausing at presynaptic regions  also suggest that these proteins are expressed in neurons. Such hypotheses raised by the MS data need to be directly tested, for example, using colocalization studies with fluorescently tagged reporters.Some proteins identified in the ELKS-1 proximity labeling experiments are usually considered nonneuronal. We speculate some of these proteins may also be expressed in neurons. For example, we find components of the muscle dense body, including the myotilin ortholog KETN-1/MYOT, the ALP-Enigma protein ALP-1, the sorbin homolog SORB-1, and ZYXin ZYX-1  enrichedC.\u00a0elegans, adding biotin to plates seeded with bacteria (E.\u00a0coli OP50 or NA22 strains) is sufficient to promote TurboID labeling, but long incubation times are required to achieve detectable protein biotinylation . We find that a 2-h incubation with exogenous biotin is sufficient for strong TurboID signals in worm neurons (D). Since in the lab C.\u00a0elegans is typically grown on E.\u00a0coli as a food source, feeding biotin-auxotrophic E.\u00a0coli to worms provides some control over the start of biotinylation by TurboID. For time-sensitive experiments, it may be possible to soak worms in a buffer containing biotin for shorter periods, e.g., 30 or 60\u00a0min and still achieve robust protein biotinylation, although further analysis using MS is required to confirm this.An important variable for TurboID protocols is the delivery of biotin or biotin derivatives. In cultured mammalian cells expressing TurboID or miniTurbo, adding biotin for as little as 10\u00a0min yields robust biotinylation . In C.\u00a0e neurons D. Since C.\u00a0elegans proteome comprises integral membrane proteins; however, only 10\u201311% of the proteins for which we detect peptides correspond to integral membrane proteins. Membrane protein identification is a long-standing problem in mass spectrometry: integral membrane proteins typically yield fewer tryptic peptides, due to a paucity of positively charged residues. In TurboID experiments underrepresentation is compounded, since biotinylation occurs on solvent exposed lysine residues. It will be interesting in future work to explore the recovery of membrane proteins when TurboID is targeted to plasma membrane of C.\u00a0elegans cells.About 27% of the A) and MS analysis (data not shown). These carboxylases; PCCA-1/PCCA , PYC-1/PC (pyruvate carboxylase), MCCC-1/MCCC (methylcronotoyl coenzyme A carboxylase), POD-2/ACACA (acetyl coenzyme A carboxylase) were the most abundantly detected proteins in our MS experiments in every condition tested, including controls. Depending on how broadly and strongly we expressed TurboID in C.\u00a0elegans tissues, these carboxylases account for 8%\u201385% of peptides identified in MS analysis. We aim to knock-in tags into each of these four genes , using CRISPR/Cas9 to allow their depletion by affinity purification, thereby increasing the sensitivity of proximity labeling, particularly when the TurboID-tagged protein is expressed in a small number of cells or at low levels.Four endogenously biotinylated carboxylases dominate the biotinylated proteome in TurboID expressing lines, as seen by both Western blot A and MS C.\u00a0elegans community to map the proteomes of specific cells and subcellular structures and to characterize the interactomes of proteins of interest.Despite extensive washing and use of denaturing conditions, streptavidin purified samples retain substantial nonspecific background. Notably, the mass spectrometer identifies many proteins even when we apply our affinity purification protocol to extracts made from control animals that do not express TurboID. These nonspecific contaminant proteins may be sticky and/or expressed at high enough levels to come through to our pipeline. Alternatively, there may be endogenous biotinylation of many proteins beyond the highly abundant carboxylases PCCA-1, PYC-1, POD-2, and MCCC-1. A future priority is to create a comprehensive list of such contaminants. In summary, our findings show that TurboID works well in a wide array of contexts in the worm. TurboID will be a reliable and useful tool for the E.\u00a0coli MG1655. C.\u00a0elegans husbandry otherwise followed standard laboratory culture conditions (Worms were grown at room temperature (22 \u00b0C) on nematode growth medium (NGM) plates seeded with the biotin auxotroph nditions .C.\u00a0elegans strains used in this study include:N2, the wild-type Bristol straindbIs37[rab-3p::mNeongreen::3XFLAG]AX7647 dbIs24[rab-3p::CeTurboID::mNeongreen::3XFLAG]AX7526 dbIs25[ges-1p::CeTurboID::mNeongreen::3XFLAG]AX7542 dbIs28[myo-3p::CeTurboID::mNeongreen::3XFLAG]AX7578 dbIs33[dpy-7p::CeTurboID::mNeongreen::3XFLAG]AX7623 AX7606 dbIs32[gcy-8p::CeTurboID::mNeongreen::3XFLAG]dbEx1234AX7917 dbEx1241AX7922 AX7928 dbEx1247AX8059 dbEx1266[R10E8.8p::mNeongreen::3XFLAG]AX7935 dbEx1251; dbEx1253AX7937 dbEx1255; dbEx1253dbEx1256[T06G6.3p::mNeongreen::3XFLAG]; dbEx1253AX7939 dbEx1296AX8110 dbEx1297AX8111 dbEx1300AX8117 elks-1(syb1710). The syb1710 allele is a knock-in that expresses ELKS-1 tagged C-terminally with CeTurboID::mNeongreen::3XFLAG.PHX1710 C.\u00a0elegans  pEntry vector, which was a gift from Dr Dominique Glauser (University of Fribourg).DNA encoding N linker::CeTurboID::c-Myc and mNeongreen::3XFLAG were stitched together by fusion PCR using primer sequences that encoded a short Gly-Ser linker. The resulting PCR product (N linker::CeTurboID::c-Myc::mNeongreen::3XFLAG) was cloned into position 2 of a Multisite Gateway pDONR vector (ThermoFisher).https://www.addgene.org/.The codon-optimized TurboID plasmid is available at Addgene C.\u00a0elegans genomic DNA by PCR and cloned into position 1 of a Multisite Gateway Donor vector (Thermo Fisher). The resulting pEntryslot1 vectors containing these promoters were each mixed with the dg398 pEntryslot2_mNeongreen::3XFLAG::stop vector, a pEntryslot3 vector containing the let-858 or tbb-2 3\u2032UTR and pDEST (ThermoFisher) in an LR reaction. The resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25\u00a0ng/uL.Promoters for B0379.1 (\u223c0.9\u00a0kbp), T01G5.1 (\u223c2.1\u00a0kbp), Y53G8AL.1 (\u223c2.5\u00a0kbp), nex-4 (\u223c1.6\u00a0kbp), F37A4.6 (\u223c2.5\u00a0kbp), T06G6.3 (\u223c2.2\u00a0kbp), and R10E8.8 (\u223c1.6\u00a0kbp) were amplified from C03H5.6 (\u223c2\u00a0kbp) and C11E4.6 (\u223c6\u00a0kbp) were amplified from C.\u00a0elegans genomic DNA by PCR and cloned into a position 2 Gateway donor vector. cDNA for H06I04.1 isoform a was synthesized by IDT  and cloned into a position 2 Gateway donor\u00a0vector. The resulting pEntryslot2 vectors were mixed with dg397 pEntryslot3_mNeongreen::3XFLAG::stop::unc-54 3\u2032UTR entry vector and a pDEST vector in an LR reaction. elks-1 cDNA (\u223c2.5\u00a0kbp) was PCR-amplified from a C.\u00a0elegans cDNA library and cloned into a position 2 Gateway donor vector, mixed with gcy-8 promoter (position 1) and mScarlet (position 3) and a pDEST vector in an LR reaction. Resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25\u00a0ng/\u03bcL.ORFs for Around 100 L4 stage transgenic animals expressing an extrachromosomal array were picked and transferred to unseeded NGM plates. The plates were placed in a UV cross-linker  with lids removed and exposed to UV (energy setting 300). The irradiated worms were then transferred to seeded plates (10 worms/plate). In total, 200 of their F1 progeny expressing the transgene were picked and individually transferred to a seeded plate. Plates containing many transgenic F2 progeny, indicating potential integration of the transgene, were selected, and eight F2 animals from the candidate plates singled and examined for 100% transmission in the next generation. Integrants were outcrossed to N2 worms at least four times.F-myo-3p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGTGTAGGCAATCAGTCAAACCGAATAAAAR-myo-3p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTCTAGATGGATCTAGTGGTCGTGGGTTF-dpy-7p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGAATCTCATTCCACGATTTCTCGCAACACATR-dpy-7p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTATCTGGAACAAAATGTAAGAATATTCTTAF-gcy-8p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGGGTTCAACAAGGGTATTGTATTGCAATCAGTGR-gcy-8p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTTGATGTGGAAAAGGTAGAATCGAAAATCCF-T01G5.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACCTGAACACAACATTTTCTGR-T01G5.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGACATGAAATTGTATCTGAAAGCF-Y53G8AL.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGTAGTTATGGAAAAGCAACGTCGGAGR-Y53G8AL.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTCTAAAATAGCATTGGTTCTGAAACTTTGF-B0379.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGAAACAATATTATTTTTGTTTCACAGR-B0379.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGTTGTTGTTGTCTCGATGGAAAAGF-nex-4p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCCTGAGAATTACTGAAGTTTAAGCR-nex-4p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTCGAGTTACTTCAATGCTCACGF-F37A4.6p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGATTCAGAAAATTCAGAAAGGCATTCR-F37A4.6p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCATTATAGGAAGACTGAGATTCCAAGCF-T06G6.3p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCATTTTTTGCTCTAAAGGTGGAATAGR-T06G6.3p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTTTGAAAAAAGTTCAGAGTAGTAGAGF-C03H5.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGTTAGAATGTATACATCCAACATR-C03H5.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTATGTACGATTCATCAAACCACCTACF-C11E4.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCCTCAAAGACTTTGTCATATCR-C11E4.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAATTTCTTTGGTTCTCAGTAGTTTGCTGF-H06I04.1-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCAAAGAAACTGGAAAAATGGCGGR- H06I04.1-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAAAATGGTCCAGATCTTGCTTCATTGGTCF-R10E8.8p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACATAAAATACGTTTTAGTAGCTGTCAGCACR-R10E8.8p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTTTTTGTCTGAAAATCGAACATTAAAAATAACAGGF-elks-1-cDNA-Entry2-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGAAAAATGGCACCTGGTCCCGCACCATACAGCR-elks-1-cDNA-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAGGCCCAAATTCCGTCAGCATCGTCGTGrab-3 promoter (\u223c1.2 kb) and ges-1 promoter (\u223c3.4 kb) were used from de Bono lab plasmid collection.N/C terminus Gly-Ser rich linkersCeTurboIDIntronsC-mycmNeongreen3XFLAGGGAGGTGGTGGATCAGGCTCGGGAGGTCGAGGCTCAGGATCCGGTTCCGGCTCCGGCTCTGGTTCCGGTTCGGGTTCCGGTTCTGGAAAGGATAACACCGTTCCACTTAAGCTTATCGCCCTTCTTGCCAACGGAGAATTCCACTCTGGAGAGCAACTTGGAGAGACTCTTGGAATGTCCCGTGCTGCCATCAACAAGCATATCCAAACCCTTCGTGATTGGGGAGTTGATGTTTTCACTGTTCCAGGAAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGATACTCCCTTCCAGAGCCAATCCCACTTCTTAACGCCAAGCAAATCCTTGGACAACTTGATGGAGGATCCGTCGCTGTCCTTCCAGTTGTTGATTCCACCAACCAATACCTTCTTGACCGTATCGGAGAGCTTAAGTCTGGAGACGCCTGCATCGCTGAGTACCAACAAGCTGGACGCGGATCTCGCGGACGCAAGTGGTTCTCCCCATTCGGAGCCAACCTTTACCTTTCTATGTTCTGGCGTCTTAAGCGTGGACCAGCTGCTATCGGACTTGGACCAGTTATCGGAATCGTTATGGCTGAGGCCCTTCGTAAGCTTGGATATAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTTCGTGTTAAGTGGCCAAACGATCTTTACCTTCAAGACCGTAAGCTTGCTGGAATCCTTGTCGAGCTTGCTGGAATCACCGGAGACGCCGCTCAAATCGTTATCGGAGCTGGAATCAACGTTGCCATGCGTCGTGTTGAGGAGTCTGTTGTTAACCAAGGATGGATCACTCTTCAAGAGGCTGGAATCAACCTTGATCGTAACACCCTTGCTGCCACCCTTATCCGTGAGCTTCGTGCTGCCCTTGAGCTTTTCGAGCAAGAGGGACTTGCCCCATACCTTCCACGCTGGGAGAAGCTTGACAACTTCATCAACCGCCCAGTTAAGCTTATCATCGGAGATAAGGAATCTTCGGAATCTCTCGCGGAATCGACAAGCAAGGAGCTCTTCTTCTTGAGCAAGATGGAGTCATTAAGCCATGGATGGGAGGAGAGATTTCCCTTCGTTCCGCTGAGAAGGCCGGAGGAGAACAGAAGCTTATAAGTGAGGAGGACCTGGGATCCGCTGGATCCGCTGCTGGATCCGGTGAGTTCATGGTGTCGAAGGGAGAAGAGGATAACATGGCTTCACTCCCAGCTACACACGAACTCCACATCTTCGGATCGATCAACGGAGTGGATTTCGATATGGTCGGACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGAACTGGAAACCCAAACGATGGATACGAGGAACTCAACCTCAAGTCGACAAAGGGAGATGCAATTCTCGCCATGGATTCTCGTGCCACACATCGGATACGGATTCCACCAATACCTCCCATACCCAGgtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagATGGAATGTCACCATTCCAAGCTGCCATGGTGGATGGATCGGGATACCAAGTTCACCGAACAATGCAATTCGAGGATGGAGCCTCGCTCAGTGAACTACCGATACACATACGAGGGATCGCACATCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGAGAGGCTCAAGTTAAGGGAACAGGATTCCCAGCTGATGGACCAGTGATGACAAACTCACTCACAGCTGCTGATTGGTGCCGATCGAAAAAGACATACCCAAATGATAAGACAATATCTCGACATTCAAGTGGTCGTACACTACTGGAAACGGAAAGCGATACCGATCGACAGCCCGAACAACATACACATTCGCTAAGCCAATGGCCGCCAACTACCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAATCAACCAATGTACGTGTTCCGAAAGACAGAACTCAAGCACTCAAAGACAGAGCTGAACTTCAAAGAGTGGCAAAAGGCCTTCACAGATGTGATGGGAATGGATGAACTCTACAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGC.\u00a0elegans expressing fluorescent proteins were acquired using a Leica  SP8 inverted laser scanning confocal microscope with 10\u00d7 0.3 NA dry, 63\u00d7 1.2 NA water or 63\u00d7 1.2 NA oil-immersion objectives, using the LAS X software platform (Leica). The Z-project function in Image J  was used to obtain the figures used in the panels. Animals were mounted on 2% agarose pads and immobilized with 100\u00a0\u03bcM of sodium azide.Confocal microscopy images of transgenic C.\u00a0elegans grown on E.\u00a0coli MG1655 were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22\u00a0\u00b0C) in M9 buffer supplemented with 1\u00a0mM biotin, and E.\u00a0coli MG1655 for 2\u00a0h. Two hours later, the worms were washed three times in M9 buffer and flash frozen after the M9 buffer was completely aspirated and 4\u00d7 Bolt LDS sample buffer supplemented with fresh DTT. The samples were then thawed, boiled for 10\u00a0min at 90 \u00b0C, vortexed for 10\u00a0min, centrifuged for 30\u00a0min at 15,000\u00a0rpm at 4 \u00b0C, and the supernatant collected. Proteins were transferred to a PVDF membrane (Thermofisher Scientific) following electrophoresis using Bolt 4\u201312% Bis-Tris Plus gels (Thermofisher Scientific). Membranes were blocked for 1\u00a0h at room temperature with TBS-T buffer containing 5% BSA and incubated for 2\u00a0h at room temperature with HRP-conjugated or fluorescently labeled streptavidin or with HRP-conjugated antibodies. Membranes were then washed three times with TBS-T. The following antibodies or protein-HRP conjugates were used for this study: IRDye 800CW Streptavidin (1:7000 in TBS-T) (LI-COR Biosciences), Anti-FLAG M2-Peroxidase (1:5000 in TBS-T) (A8592 Sigma), anti-alpha tubulin-HRP  (DM1A Abcam ab40742), Streptavidin-HRP (1:5000 in TBS-T) . Membranes were imaged using ChemiDoc the Imaging System .Synchronized populations of C.\u00a0elegans were bleached and the eggs transferred to NGM plates seeded with E.\u00a0coli MG1655 to obtain synchronized populations of worms. The animals were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22 \u00b0C) in M9 buffer supplemented with 1\u00a0mM biotin, and E.\u00a0coli MG1655 for 2\u00a0h unless stated otherwise. Two hours later, the worms were washed three times in M9 buffer and allowed to settle on ice after the last wash. After completely aspirating the M9 buffer, two volumes of RIPA buffer supplemented with 1\u00a0mM PMSF and cOmplete EDTA-free protease inhibitor cocktail (Roche Applied Science) were added to one volume of packed worms. The animals were again allowed to settle on ice and then added dropwise to liquid N2 to obtain frozen worm \u201cpopcorn.\u201d A Spex 6875D cryogenic mill was used to grind frozen C.\u00a0elegans to a fine powder, which was then stored at \u201380\u00a0\u00b0C. Worm powder was thawed by distributing it evenly along the length of a 50\u00a0ml falcon tube and rolling it on a tube roller at 4 \u00b0C. After the sample was completely thawed, it was centrifuged  to collect the sample at the bottom of the tube. SDS and DTT were added to the sample to final concentrations of 1% and 10\u00a0mM respectively, from stock solutions of 20% SDS and 1M DTT. The tubes were gently inverted a few times and immediately incubated at 90 \u00b0C for 5\u00a0min. After heat treatment, the samples were sonicated continuously for 1\u00a0min twice, with brief cooling between the two sonication steps. Sonication used a probe sonicator microtip (MSE Soniprep 150 plus) and an amplitude setting of 16/max. The samples were cooled to room temperature following sonication and adjusted to 2 M urea using a stock solution . The samples were then centrifuged at 100,000g for 45\u00a0min at 22 \u00b0C, and the clear supernatant between the pellet and surface lipid layer transferred to a new tube.Gravid adult g for 5\u00a0min (or until the buffer was completely eluted from resin). Around 4\u00a0ml of clarified sample was then loaded onto the equilibrated spin column and desalted by centrifugation at 1000g for 5\u00a0min (or until the sample was completely eluted from resin) to remove free biotin. The desalting step was repeated once more using freshly equilibrated columns. Protein concentration in the samples was measured using Pierce 660\u00a0nm protein assay reagent supplemented with Ionic Detergent Compatibility Reagent (IDCR) (Thermo Fisher Scientific).Zeba spin desalting columns (7K MWCO) (Thermofisher) were equilibrated three times with 5\u00a0ml RIPA buffer containing 1% SDS and 2 M urea, freshly supplemented with protease inhibitors  by centrifugation at 1000C.\u00a0elegans total protein lysate) were equilibrated in RIPA buffer by briefly incubating them three times in the buffer and using magnetic separation to retain beads while discarding buffer (note: we were able to reduce the amount of protein lysate to 5\u201310\u00a0mg in subsequent experiments without compromising the mass spectrometry data). Desalted, clarified samples were combined with Dynabeads in a 50\u00a0ml falcon tube and incubated overnight in a tube roller at room temperature. Beads were magnetically separated using a neodymium magnet taped to the tube and incubated on a rocking platform. Unbound lysate was removed and the Dynabeads transferred to 2\u00a0ml LoBind protein tubes (Eppendorf). Beads were washed five times with 2% SDS wash buffer  and five times with 1M KCl wash buffer; beads were transferred to new tubes after each wash.Dynabeads MyOne streptavidin C1  to saturation in sample buffer (NuPAGE LDS sample buffer 4\u00d7) containing reducing agent (NuPAGE sample reducing agent 10\u00d7). Elution sample buffer was centrifuged at 13,000\u00a0rpm for 5\u00a0min to remove undissolved biotin and the elution sample buffer saturated with dissolved biotin was transferred to a new tube. Hundred microliter of elution buffer was applied to Dynabeads, vortexed gently, heated for 5\u00a0min at 90 \u00b0C, before the vortexing and heating steps were repeated. Dynabeads were separated magnetically and elution buffer containing biotinylated proteins was transferred to a new 1.5\u00a0ml LoBind protein tube (Eppendorf). Seventy microliter of sample was electrophoresed on a NuPAGE 4\u201312% Bis-Tris protein gel (Invitrogen), which was then stained with InstantBlue (Expedeon) for visualization. The gel was sliced into 20 fractions and sent for mass spectrometry analysis.via a nano-flow electrospray ionization source with a hybrid quadrupole orbitrap mass spectrometer . Data collection was performed in data-dependent acquisition (DDA) mode with an r\u00a0=\u00a070,000 (@ m/z 200) full MS scan from m/z 380\u20131600 with a target AGC value of 1e6 ions followed by 15\u00a0MS/MS scans at r\u00a0=\u00a017,500 (@ m/z 200) at a target AGC value of 1e5 ions. MS/MS scans were collected using a threshold energy of 27 for higher energy collisional dissociation (HCD) and a 30\u00a0s dynamic exclusion was employed to increase depth of coverage. Acquired raw files were then searched in MaxQuant (C.\u00a0elegans reference proteome from UniProt KB (including SwissProt and TrEMBL entries) downloaded on September 27, 2019. In total, 28,474 accessions were actually searched. We used trypsin, which cleaves peptides at the C-terminal side of lysine and arginine residues, to generate peptide fragments for mass spectrometry. Most parameters were kept at their default value. Carbamidomethyl (C) (+57.0214637236) was set as fixed modification. Variable modifications included were \u201cOxidation (M)\u201d (+15.9949146221), \u201cAcetyl (Protein N-term)\u201d (+42.0105646863), \u201cDeamidation (NQ)\u201d (+0.9840155848), \u201cGln->pyro-Glu\u201d (-17.0265491015), \u201cPhospho (STY)\u201d (+79.9663304084), as well as two custom modifications  to account for TurboID-induced biotinylated peptides. Match Between Runs  and Second Peptide Search were activated. All FDRs were set to 1%. Identified data was then reprocessed in R using the evidence.txt results table, but a decision was made to exclude indirect, MBR-based identifications to focus on higher confidence hits. The decoy used to establish the FDR was the reverted search database. MaxQuant applies a PSM-level FDR globally as well as per class of modified peptides (\"Site decoy fraction), and also at protein level. All three FDR levels were set to 1%.Polyacrylamide gel slices (1\u20132\u00a0mm) containing the purified proteins were prepared for mass spectrometric analysis using the Janus liquid handling system . Briefly, the excised protein gel pieces were placed in a well of a 96-well microtitre plate, destained with 50% v/v acetonitrile and 50\u00a0mM ammonium bicarbonate, reduced with 10\u00a0mM DTT, and alkylated with 55\u00a0mM iodoacetamide. After alkylation, proteins were digested with 6\u00a0ng/\u03bcl Trypsin  overnight at 37 \u00b0C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile. The digest was analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300\u00a0nl/min. A C18 Acclaim PepMap100 5\u00a0\u03bcm, 100\u00a0\u03bcm\u00a0\u00d7 20\u00a0mm nanoViper (ThermoScientific Dionex), trapped the peptides prior to separation on an EASY-Spray PepMap RSLC 2\u00a0\u03bcm, 100\u00a0\u00c5, 75\u00a0\u03bcm\u00a0\u00d7 250\u00a0mm nanoViper column (ThermoScientific Dionex). Peptides were eluted using a 60-min gradient of acetonitrile (2% to 80%). The analytical column outlet was directly interfaced MaxQuant  (versionC.\u00a0elegans, for which there is no requirement for review and approval from an institutional animal care and use committee. Transgenic experiments were carried out following IST guidelines for such work.The work used the free-living nematode via the PRIDE partner repository at http://www.ebi.ac.uk/pride with the dataset identifier PXD027068. Submission details: Project Name: Interactome analysis of C.\u00a0elegans synapses by TurboID-based proximity labeling. Project accession: PXD027068. We have uploaded the mass-labeled MS/MS data on MS Viewer, available at https://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. The key to access the repository is bbfl7oetve. Only \u223c100,000 of the \u223c2.5 million spectra obtained in the course of our work are in the database, since replicates are omitted, and the best spectrum retained for each peptide. All remaining data are contained within the article.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium This article contains The authors declare that they have no conflicts of interest with the contents of this article."}
+{"text": "In the article titled \u201cLong Noncoding RNA TUG1/miR-29c Axis Affects Cell Proliferation, Invasion, and Migration in Human Pancreatic Cancer\u201d [Correct forward sequence should read as follows:5\u2032-gatccGCTTGGCTTCTATTCTGAATCCTTTCAAGAGAAGGATTCAGAATAGAAGCCAAGCTTTTTTG-3\u2032.Correct reverse sequence should read as follows:5\u2032-CAAAAAAGCTTGGCTTCTATTCTGAATCCTTCTCTTGAAAGGATTCAGAATAGAAGCCAAGCG-3\u2032."}
+{"text": "KO-heart) die by 9 weeks of age due to extensive cardiomyocyte apoptosis and severe HF, which suggests that fortilin sustains cardiomyocyte viability. The lack of fortilin is also associated with drastic upregulation of p53 target genes in the hearts. The heart-specific deletion of p53 in fortilinKO-heart mice extends their life spans from 9 to 18 weeks by mitigating cardiomyocyte apoptosis. Our data suggest that fortilin is a novel cardiac p53 inhibitor and that its inadequate expression in failing hearts and subsequent overactivation of the p53 apoptosis pathway in cardiomyocytes exacerbates HF.Heart failure (HF) has reached epidemic proportions in developed countries, affecting over 20 million people worldwide. Despite modern medical and device therapies, 60\u201370% of HF patients still die within 5 years of diagnosis as it relentlessly progresses through pervasive apoptotic loss of cardiomyocytes. Although fortilin, a 172-amino-acid anti-p53 molecule, is one of the most expressed proteins in the heart, its precise role there has remained unknown. Also unclear is how cardiomyocytes are protected against apoptosis. Here, we report that failing human hearts express less fortilin than do non-failing hearts. We also found that mice lacking fortilin in the heart (fortilin Heart failure (HF)\u2014a complex clinical syndrome secondary to structural and functional impairments of the heart muscle\u2014has reached epidemic proportions in developed countries, currently affecting over 20 million people worldwide. Despite modern medical and device therapies, 30\u201340% of HF patients die within 1 year and 60\u201370% die within 5 years of diagnosis [Fortilin  is a highly conserved, 172-amino-acid, 20\u2009kDa protein that blocks apoptosis \u201310, and To evaluate the role of fortilin in the heart and in human HF, we subjected tissue lysates of human hearts from subjects with non-failing hearts (NFHs) and HF patients with non-ischemic cardiomyopathy (NICM) and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository  to an auflox/flox mice using standard homologous recombination techniques [fortilin KO mice  by crossing fortilinflox/flox mice with mice over-expressing Cre-recombinase under the control of the cardiac-specific Myh6 promoter [+/+, Jackson Laboratory)  mice as the control. As expected, fortilin was not detectable in the hearts of fortilinKO-heart mice, whereas it was normally expressed in all other tissues at both message  Fig. . In cardces Fig.  is excisces Fig. , while fces Fig. . We usedage Fig.  and protage Fig.  levels.KO-heart mice started to die as early as 6 weeks of age and all were dead by 9 weeks of age  in fortilinKO-heart mice, but not in fortilinWT-heart mice  than in fortilinKOp53WT and fortilinKOp53KO mice (the latter two had comparable expression), both at the message  and protein  levels. In addition, total p53 expression levels were significantly higher in fortilinKOp53WT than in fortilinKOp53KO mice, both at the message  and protein levels . p53 expression levels also were significantly higher in fortilinKOp53WT than in fortilinWTp53WT mice, both at the message  and protein levels . On the other hand, phosphorylated p53, an active form of p53, was most abundant in the hearts of fortilinKOp53WT mice, followed by those of fortilinWTp53WT and then by those of fortilinKOp53KO , suggesting that the lack of fortilin increases not only total p53 protein but also phosphorylated and activated p53 protein.To test the hypothesis that the lack of fortilin causes the heart to fail through overactivation of the p53 apoptotic pathway, we knocked out \ufeffp53 by crossing fortilinlox mice  and geneice Fig.  and S2b.protein levels of total p53 to be significantly higher in the heart of fortilinKOp53WT mice than in those of fortilinWTp53WT mice as we observed above. However, this process does not explain why the message levels of p53 were also significantly higher in the hearts of fortilinKOp53WT mice than in those of fortilinWTp53WT mice .Because fortilin binds p53 and facilitates its proteasome-mediated degradation at the protein level , it is ep53-pomoter-GLuc/SEAP cells). We then transiently silenced fortilin by treating the cells with lentiviral vector containing shRNA against fortilin (sh-fortilin). These cells were irradiated with a moderate X-ray dose (8\u2009Gy) and subjected to the GLuc-SEAP assay  and the secreted alkaline phosphatase (SEAP) gene under the control of the constitutional CMV promoter . Echocardiography showed that the hearts of fortilinKOp53KO mice had better overall heart function . These data suggest that fortilin sustains heart function by negatively regulating p53 and that the lack of fortilin leads to inappropriate overactivation of the p53 pathway, apoptosis and loss of cardiomyocytes, dilated cardiomyopathy, HF, and death.Immunohistochemical staining of myocardium showed that the lack of p53 drastically reduced the expression levels of both BAX and cleaved lamin, which are apoptosis markers Fig. . ConsistKO-heart mice increased their survival by about 10 weeks .Although the deletion of p53 in the heart of fortilineks Fig. , it did ice Fig. . We founway Fig. , a vs. cKO-heart mice, we treated fortilinKOp53KO mice with either KIRA6, a selective IRE1\u03b1 inhibitor [KOp53KO mice to live modestly longer compared to vehicle treatment . At week 10 of treatment (about 18 weeks of age), both body weights and HW/BW ratios of KIRA6-treated fortilinKOp53KO mice were modestly but significantly greater than those of vehicle-treated fortilinKOp53KO mice  level and by activation of the p53 promoter by knockdown of fortilin in the cardiomyocyte cell line . Because there are more endothelial cells and fibroblasts combined than cardiomyocytes in the murine hearts [KOp53KO heart. To evaluate the status of activated and functional p53 in the heart, we stained the tissue with anti-phospho-p53 antibody. We found that phosphorylated p53 were significantly less in fortilinKOp53KO mice than that in fortilinWTp53WT mice, suggesting that despite the similar total p53 signals between fortilinKOp53KO and fortilinWTp53WT hearts by both immunohistochemistry  and Western blots , functional p53 is significantly less in fortilinKOp53KO hearts than in fortilinWTp53WT hearts.There were p53 signals detectable in the Western blot of the total heart lysates from fortiline hearts \u201332, it iBecause cardiomyocytes are not capable of proliferating in normal conditions, their continuous apoptotic loss leads to irreversible progression of HF. Protecting cardiomyocytes against apoptosis represents a new approach in HF gene therapy, distinct from the ones focusing on calcium metabolism and \u03b2-adrenergic receptor signal transduction . The cursh-fortilin) and random control sequence (lentivirussh-control) were purchased from Sigma . The lentivirussh-fortilin was experimentally shown to silence rat fortilin . Lentiviral particles that contained (a) the murine p53 promoter sequence fused to the GLuc cDNA, (b) the SEAP gene under the constitutional SV40 promoter, and (c) the puromycin resistance gene were obtained from GeneCopoeia  . The actual promoter sequence is as follows: GGATCTGTGGCTAGCTGGGGTTGGTCATCACCACCGCATGGCGGAGGCACCGGTTCAAAGTCTGTATTTTTCTCCGCTGGGGAACCTTGGGGTACCGGAGCTGGGGCCAGGTCAGGAGGGAGGCTATCCGGAGCTAAGAGTCGCTCCTCCGACGTCTTCATTCTGTAGAGTAAGCCCCCGGAAGGCAGAGGTCGGGCAAGTCTCGCTGAGCCGGCTACCAGCTGCCGAGGCTAGAGTGCATTACCGTTCCCAGGGATGCTCAGAGACCGGAGTCCGCTTTCCTCTTCCGGAAAATGTAAGCCGAACCTAAAGCAATCACCAGGGAACGAGTGTCCAAAGCCAAGCGCCTAGGGTCGCTAGGCGCCGCCAGGGCTTCTTGCTCTCGCGGGAGTCGGGCCACCTTCCGATAGGCTCTCCGCATCCTCCTCCGATTCCGAGCGGGAAGGCGGGAAGGAACGACTTTGCCTACACCTCAAGCGCTGGAGAATTCCTAGAGGTTTCTGGGAGTTGTAGTCTGAACTCTGGGCCTTGGCGAAAACTACACGAGCGCCCCCTACCGTCCCCTGGGGGTAATTCTTAAAGCGCCTATCCTCCCTGGCCTGCAGAGGGCGCATAATTTCTACAGTTTTTGCCCCTCTTGACTATCTTGTTTTGAATCCCKinase-Inhibiting RNase Attenuator 6 (KIRA6) , an inhiGTAACCTCAGGTTTCCTTTCTCCCCATCTCTCCCCCCTTCTTGTTCCTCTCTTTCCCTTTCTCCCCCGCCCTCCCTTCATTCATTCGACATTTATTATCAAGTTCTTACTGCCTAACCCAGGACTATACAAGGCATTGGGAAAAAAATAGCAATGTTTTCTAGTTCTTAATCTCCATAAAGTTTTCGTTGCTGTGCAATTAAAGGCTGTGAAAACAGTCTTTACAGAGAGTGATAAGGACTGTACAGGAAATTAAACACGGTGGTGCGATACCAAGTATCTCGGAGAACACGTTAGATTGAGATACTATGAAAAGCCTTTCTAAAGTGACATTTTAGCTAATGAGGGGAAAAAGAACTTAGGGGCCCGTGTTGGTTCATCCCTGTACTTGGAAGGCCTAAAGCAGGAAGACGGCCGCGAATTCCAGGCCAGCCTTGGCTACAAAGACTCTGTCTTAAAAATCCAAAAAGATGGCTATGACTATCTAGCTGGATAGGAAAGAGCACAGAGCTCAGAACAGTGGCGGTCCACTTACGATAAAAACTTAATTCTTTCCACTCTTTATACTTGACACAGAGGCAGGAGTCCTCCGAATCGGTTTCCACCCATTTTGCCCTCACAGCTCTATATCTTAGACGACTTTTCACAAAGCGTTCCTGCTGAGGGCAACATCTCAGGGAGAATCCTGACTCTGCAAGTCCCCGCCTCCATTTCTTGCCCTCAACCCACGGAAGGACTTGCCCTTACTTGTTATGGCGACTATCCAGCTTTGTGCCAGGAGTCTCGCGGGGGTTGCTGGGATTGGGACTTTCCCCTCCCACGTGCTCACCCTGGCTAAAGTTCTGTAGCTTCAGTTCATTGGGACCATCCTGGCTGTAGGTAGCGACTACAGTTAGGGGGCAC.\u00ae CRL-1446\u2122) cell line (H9C2 hereafter) was directly obtained from American Type Culture Collection . Cells were maintained in Dulbecco\u2019s modified Eagle\u2019s medium  with 10% fetal bovine serum (FBS)  at 37\u2009\u00b0C in an atmosphere containing 5% CO2. H9C2 cells stably harboring the lentiviral construct that contained both the p53 promoter sequence driving the expression of GLuc and the SEAP gene under the control of the constitutional SV40 promoter were generated by transducing the cells with Lentivirusp53-promoter-GLuc/SEAP and selecting them with puromycin (H9C2p53-promoter-GLuc/SEAP). They were maintained in puromycin-containing (0.3\u2009\u00b5g/mL) media.MycoFluor\u2122  was used to detect mycoplasma contamination when appropriate. The H9C2(2-1) (ATCCp53-promoter-GLuc/SEAP cells were transiently transduced by either (lentivirussh-fortilin) or (lentivirussh-control) at the multiplicity of infection (MOI) of 1. The next day, cells were irradiated using the RS-2000 X-ray irradiator  at 8\u2009Gy. Seventy-two hours after the irradiation, conditioned media were collected and subjected to both SEAP and luciferase assays using the Secrete-Pair Dual Luminescence Assay Kit according to the manufacturer\u2019s instruction (GeneCopoeia). The degree of activation of the p53 promoter was assessed by dividing the luciferase activity units by the SEAP activity units and expressing it in arbitrary units (A.U.).On the first day, H9C2Mice were given standard mouse chow  and water ad libitum, maintained on a 12-h light\u2013dark cycle and seen by a scientist daily to observe their behavior and health. Any animal that displayed substantive signs of distress anytime during the protocol, including but not limited to poor grooming, hunched back posture, or a loss in weight exceeding 10% of the weights at the initiation of the experiments, were identified and treated accordingly, including removal from the experiment and euthanasia. For mouse experiments where grouping was based on pharmacological treatments, mice of the same genotype were randomly assigned to treatment and control groups.flox/flox mice, in which the fortilin gene was flanked by the LoxP sequence to allow tissue-specific deletion, were generated using the standard homologous recombination technique described previously: we micro-injected a mutant C57BL6 embryonic stem cell (ESC) line that contained two LoxP sequences flanking all six fortilin exons into C57BL6 blastocysts [flox/flox mice were fully in the C57BL/6J genetic background from the beginning. Fortilinflox/flox mice were then crossed with a transgenic flipase strain to remove the neomycin resistance gene cassette. To generate fortilinKO-heart mice, fortilinfloxflox mice were crossed with C57BL/6J mice overexpressing the Cre transgene under the control of cardiac-specific \u03b1-myosin heavy chain (Myh6) promoter [+/+, Jackson Laboratory, Ann Arbor, ME, Stock # 011038, already backcrossed to C57BL/6J for eight generations by the donating laboratory). Genotyping of fortilinKO-heart and fortilinWT-heart mice was performed on tail-derived genomic DNA using standard PCR-based methods. The presence of the \u03b1MHC-Cre transgene was detected using the following primer sets: 5\u2032-ATGACAGAC AGATCCCTCCTATCTCC-3\u2032 (M1) and 5\u2032-CTCATCACTCGTTGCATCATCGAC-3\u2032 (M2). \u03b1MHC-Cre+/\u2212 mice yielded a 300\u2009bp amplified fragment and \u03b1MHC-Cre\u2212/\u2212 mice yielded no amplicons. To determine the hemi- (\u03b1MHC-Cre+/\u2212) or homo- (\u03b1MHC-Cre+/+) zygosity of the MHC-Cre transgene, RT-qPCR was performed using the primers and probe sets described previously [\u2013\u0394\u0394CT method [Forward primer (oIMR1084): 5\u2019-GCGGTCTGGCAGTAAAAACTATC-3\u2019Reverse primer (oIMR1085): 5\u2019-GTGAAACAGCATTGCTGTCACTT-3\u2019Probe (13593): 5\u2019-FAM-AAACATGCTTCATCGTCGGTCCGG-IBFQ-3\u2019FAM, 6-carboyfluorescein; IBFQ, Iowa Black\u2122 double-quenched probe (Integrated Data Technologies (IDT), Coralville, IA)\u03b1MHC-Cre-transgene:Forward primer (oIMR1544): 5\u2019-CACGTGGGCTCCAGCATT-3\u2019Reverse primer (oIMR3580): 5\u2019-TCACCAGTCATTTCTGCCTTTG-3\u2019Probe (TmoIMR0105): 5\u2019-JOE-CCAATGGTCGGGCACTGCTCAA-IBFQ-3\u2019Apob (apolipoprotein B) [The target gene is otein B) .Internal control to calculate \u0394CT for the \u03b1MHC-Cre-transgene:Fortilinstocysts . The respromoter  (\u03b1MHC-Creviously  (see belT method  to deterflox/flox mice yielded a 405\u2009bp fragment and wild-type fortilin mice (fortilinWT/WT mice) yielded a 280\u2009bp fragment.The presence of the LoxP-fortilin-LoxP knock-in construct was verified using the following primer sets: 5\u2032-TGGACCCTGACTTTCATCACCTC-3\u2032 (F1) and 5\u2032- GTCATCTAACCTTACCCCAGTAAGC-3\u2032 (F2): fortilinKO-heart mice were crossed with p53flox/flox mice available from the Jackson Laboratory . The status of the \u03b1MHC-Cre-transgene was evaluated by both PCR and RT-qPCR as described above, and the status of fortilin and p53 loci was determined using the following PCR-based genotyping strategies:Forward primer: 5\u2019-TGGACCCTGACTTTCATCACCTC-3\u2019Reverse primer: 5\u2019-GTCATCTAACCTTACCCCAGTAAGC-3\u2019Expected size of the mutant (floxed fortilin gene) allele: 405\u2009bpExpected size of the wildp-type allele: 280\u2009bpFortilin locus:Forward primer: 5\u2019-GGTTAAACCCAGCTTGACCA-3\u2019Reverse primer: 5\u2019-GGAGGCAGAGACAGTTGGAG-3\u2019Expected size of the mutant (floxed fortilin gene) allele: 390\u2009bpExpected size of the wild-type allele: 270\u2009bpP53 locus:To generate a strain of C57BL/6J mice that lack both fortilin and p53 in the heart, fortilinKOp53KO mice were daily and intraperitoneally injected with either 5\u2009mg/kg of KIRA6 in solution  or the same solution without KIRA6 as vehicle (a) for the subsequent 10 weeks for the sacrifice group (N\u2009=\u20095 each) or (b) until death for the survival analysis group (N\u2009=\u20095 each). Mice were examined daily for their behavior and health. Mice in the sacrifice group were weighed and underwent transthoracic echocardiography before they were sacrificed, and their hearts were harvested and processed as described elsewhere in \u201cMethods\u201d section.For the KIRA6 rescue experiment, 8-week-old male fortilin2 and then by 0.5\u20133.0% isoflurane and placed on a heated platform, as described previously [KO-heart mice, the baseline echo was obtained when they were 7 weeks of age.Mouse transthoracic echocardiography was performed using a Vevo 2100 High-Resolution Imaging System  on mice anesthetized initially by 4% isoflurane in 1\u2009L/min of Oeviously . We imagAfter being weighed, mice were sacrificed by isoflurane inhalation until effective, followed by exsanguination. Both the heart and lungs were routinely harvested. The liver, kidney, and spleen were harvested, when appropriate, for protein and RNA analyses. The harvested hearts and lungs were rinsed with phosphate-buffered saline (PBS), drained on absorbent paper, and weighed. The ratio of heart weight to body weight (HW/BW ratio) as well as that of lung weight to body weight (LW/BW ratio) was then calculated. For the sagittal cross-sectional analysis of the heart, the entire heart was fixed in 4% paraformaldehyde and subjected to paraffin embedding, sectioning, and hematoxylin and eosin staining according to the standard protocol. The right ventricle and atria were removed from the left ventricle (LV). The LV was then divided into three sections of equal size . The mid-ventricular part of the LV was fixed in 4% paraformaldehyde before being embedded in paraffin for immunohistochemistry. The apical portion of the LV was flash-frozen for subsequent protein extraction, and the basal portion of the LV was placed directly into Tri-Reagent  and frozen for subsequent RNA extraction.\u2013\u0394\u0394CT method [\u00ae RT-PCR kit  in the ABI Step One Plus Real-Time PCR system and the following primer and probe sets (Integrated DNA Technologies):Fortilin:Forward primer: 5\u2032-TCCGACATCTACAAGATCCGG-3\u2032,Reverse primer: 5\u2032-ATCTTGCCCTCCACCTCCA-3\u2032,Probe: 5\u2032-FAM-AGATCGCGG/ZEN/ACGGGCTGTGC-IBFQ-3\u2032ZEN\u2122\u2009=\u2009an internal quencher to enhance the quenching activity of the 3\u2019 quencher IBFQ (IDT)Mouse Col1:Forward primer: 5\u2032-GAAACCCGAGGTATGCTTGA-3\u2032,Reverse primer: 5\u2032-GTTGGGACAGTCCAGTTCTT-3\u2032,Probe: 5\u2032-FAM-TGTGCGATGACGTGCAATGCAATG-IBFQ-3\u2032Mouse Myh7Forward primer: 5\u2032-CCATCTCTGACAACGCCTATC-3\u2032,Reverse primer: 5\u2032-GGATGACCCTCTTAGTGTTGAC-3\u2032,Probe: 5\u2032-FAM-TCAGTCCATCCTCATCACCGGAGA-IBFQ-3\u2032Mouse ANFForward primer: 5\u2032-TCCGATAGATCTGCCCTCTT-3\u2032,Reverse primer: 5\u2032-CTCCAATCCTGTCAATCCTACC-3\u2032,Probe: 5\u2032-FAM-AAAGCAAAC/ZEN/TGAGGGCTCTGCTCG-IBFQ-3\u2032Mouse Acta1Forward primer: 5\u2032-CTCCCTGGAGAAGAGCTATGA-3\u2032,Reverse primer: 5\u2032-CGATAAAGGAAGGCTGGAAGAG-3\u2032,Probe: 5\u2032-FAM-CATCGGCAATGAGCGTTTCCGTTG-IBFQ-3\u2032Mouse Serca2Forward primer: 5\u2032-CATCAGTATGACGGGCTTGTAG-3\u2032,Reverse primer: 5\u2032-CTCGGTAGCTTCTCCAACTTTC-3\u2032,Probe: 5\u2032-FAM-AGCCACCATCTGTGCTCTGTGTAA-IBFQ-3\u2032Mouse p53Forward primer: 5\u2032-CAGCTTTGAGGTTCGTGTTTG-3\u2032,Reverse primer: 5\u2032-AGTTCAGGGCAAAGGACTTC-3\u2032,Probe: 5\u2032-FAM-TCTTCTTCT/ZEN/GTACGGCGGTCTCTCC-IBFQ-3\u2032Mouse BaxForward primer: 5\u2032-TTGCTGATGGCAACTTCAACTGGG-3\u2032,Reverse primer: 5\u2032-TGTCCAGCCCATGATGGTTCTGAT-3\u2032,Probe: 5\u2032- FAM-TTTGCTAGC/ZEN/AAACTGGTGCTCAAGGC-IBFQ-3\u2032Mouse PumaForward primer: 5\u2032-ATGGCGGACGACCTCAAC-3\u2032,Reverse primer: 5\u2032-AGTCCCATGAAGAGATTGTACATGAC-3\u2032,Probe: 5\u2032-FAM-AGCAGCATC/ZEN/GACACCGACCCTCAC-IBFQ-3\u2032Mouse NoxaForward primer: 5\u2032-TGCACCGGACATAACTGTGGTTCT-3\u2032,Reverse primer: 5\u2032-TGAGCACACTCGTCCTTCAAGTCT-3\u2032,Probe: 5\u2032-FAM-AAAGAGCAG/ZEN/GATGAGGAGCCCAAGC-IBFQ-3\u2032Mouse 18S rRNAForward primer: 5\u2032- GCCGCTAGAGGTGAAATTCT-3\u2032,Reverse primer: 5\u2032-TCGGAACTACGACGGTATCT-3\u2032,Probe: 5\u2032-JOEN-ACCAGAGCG/ZEN/AAA GCATTTGCCAAG-IBFQ-3\u2032JOEN\u2009=\u20096-carboxy-4\u2032,5\u2032-dichloro-2\u2032,7\u2032-dimethoxyfluoresceinMouse RT-qPCR was performed as described previously . We usedT method  to calcuAnti-fortilin Anti-p53 Anti-GAPDH Anti-Bax Anti-PUMA Anti-NOXA Anti-Tropomyosin SDS\u2013PAGE and Western blot analyses were performed as described previously , 37\u201340 oAll antibodies were used with appropriate IRDye680LT- or IRDye800CW-conjugated secondary antibodies . The signal intensities of protein bands were quantified using the Odyssey Infrared Imaging System (LI-COR) and normalized to the signal intensity of the loading control protein (GAPDH or tropomyosin) and expressed in A.U. To quantify p53 expression in response to X-ray irradiation, 0.5% (v/v) TCE was added to a polyacrylamide gel before polymerization. After standard SDS\u2013PAGE, the gel was UV-irradiated on the Bio-Rad ChemiDoc MP Imaging System  for 2\u2009min. The image was electronically captured, and the cumulative band densities were calculated to assess loading conditions as previously described . The sigTo evaluate the expression levels of fortilin in human hearts, we obtained de-identified tissue lysates of human hearts from patients with NFHs, non-ischemic cardiomyopathy (NICM), and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository . An automated capillary-based quantitative Western blot system called JESS\u2122  was used to (a) detect fortilin  and (b) visualize total proteins loaded as described previously , 20. ComKOp53KO mice that were treated with either vehicle or KIRA6 were subjected to WES\u2122 (Protein Simple), an automated capillary-based quantitative Western blot system, to detect p-IRE1\u03b1  and glyceraldehyde 3-phosphate dehydrogenase ; the latter served as the loading control. JESS\u2122, but not WES\u2122, can visualize the total proteins loaded for normalization. Compass Software (v3.1) was used to calculate a fortilin expression index by dividing the area under the curve of a fortilin peak by that of a GAPDH peak loaded in the same capillary . The fortilin expression index was expressed in A.U.To quantify phosphorylated IRE1\u03b1, the mouse heart lysates from fortilinCleaved lamin A . The cleavage of lamin is a well-characterized event in apoptosis .BAX p-IRE1\u03b1 p53 phosphorylated p53 Mouse hearts were fixed in 4% paraformaldehyde and embedded in paraffin before they were sectioned at 5\u2009\u00b5m thickness. Immunohistochemistry of mouse hearts was performed as described previously  using thTM DNA Fragmentation Detection Kit  following the manufacturer\u2019s instructions. TUNEL-positive cells within about 0.145\u2009mm2 of the ROI were counted, and TUNEL indices were calculated as the number of TUNEL-positive cells per unit area (in mm2). Results are expressed in A.U.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of heart tissue was performed on formalin-fixed, paraffin-embedded samples as previously described , 46 usinTo assess the presence of cardiomyocyte hypertrophy, the heart tissue that had been formalin-fixed and paraffin-embedded was sectioned at 5\u2009\u00b5m thickness and stained with laminin . After stained sections were digitally captured using the EasyScan Digital Slide Scanner, cardiomyocyte areas were measured using ImageJ and expressed in A.U. as described previously . At leasThis study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving animals were approved by the Institutional Animal Care and Use Committees of our institution. Human tissue samples used for this study were procured from the Duke Human Heart Repository (DHHR), which is a Duke University Health System Institutional Review Board (DUHS IRB) approved tissue repository. Samples were procured by the DHHR using written informed consent or a waiver of consent for discarded tissues. No HIPAA information was provided with any of the samples used in this study. Human myocardium was acquired from the left ventricular free wall of explanted hearts following cardiac transplantation. Non-failing (NF) left ventricular tissue was acquired from donors whose hearts were not utilized for transplant, thus becoming available for research.N) is indicated in either figures or the main text. The degree of the spread of data was expressed by the standard deviation (\u00b1SD). The difference between the control and study groups was analyzed using unpaired two-tailed Student\u2019s t-test for two groups or one-way analysis of variance (ANOVA) followed by Fisher\u2019s pairwise multiple comparisons for multiple groups. P\u2009<\u20090.05 was considered to be statistically significant. For survival analyses, Kaplan\u2013Meier survival curves were generated, and the Log-Rank (Mantel\u2013Cox) test was used to compare the curves. The numbers of mice used in in vivo experiments were determined by (i) power analysis, assuming an \u03b1 error rate of 0.05, a \u03b2 error rate of 0.20, and an expected difference of 25%, in Minitab 17  or (ii) our previous dataset and experience from similar experiments performed as part of past research. A similar variance was observed between the groups that were statistically compared. No data were excluded unless outliers were identified and verified by the outlier tests (Minitab 17). Although the scientists were not blinded to allocation during experiments and readouts evaluation, all readouts from the experiments were predetermined, highly objective, and obtained according to the validated protocols.All measurements were taken from distinct samples  and the size of biological samples (SUPPLEMENTAL MATERIAL"}
+{"text": "Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and   \u2022lncRAP2 is a cytosolic lncRNA conserved in mouse and human needed for adipogenesis\u2022lncRAP2 complexes with mRNA stability and translation regulators, including Igf2bp2\u2022lncRAP2-Igf2bp2 stabilizes lipid metabolism mRNAs to potentiate energy expenditure\u2022lncRAP2-Igf2bp2 genetic and expression variation is linked to BMI, type 2 diabetes Biological sciences; Molecular physiology; Molecular biology; Omics Previous studies revealed that many lncRNAs are specifically enriched in white and/or brown adipocytes and play vital roles in adipocyte biology , includiin situ hybridization (smFISH) visually determines RNA abundance and location, revealing whether a lncRNA diffuses to trans sites beyond its chromosomal locus or remains tethered in cis in the nucleus (in\u00a0vivo (rather than in solution after cell lysis)  . And fool lysis) .Here, we use these and other tools to interrogate the mode of action of lncRAP2, a white adipocyte-selective RNA that is essential for adipogenesis . We showin situ hybridization (smFISH) reveals that lncRAP2 diffuses from the nucleus, spreading throughout the cytoplasm at 14\u00a0\u00b1 3 transcripts per white and 9\u00a0\u00b1 3 transcripts per brown adipocyte   in 3T3-Lvia posttranscriptional control of metabolically important proteins, impacting sensitivity to diet-induced obesity and type 2 diabetes risk  (Igf2bp2 with those of depleting Ppar\u03b3 or linc-ADAL (lincRNA for adipogenesis and lipogenesis) in mature white adipocytes  D and S3Fipocytes   , including 138 whose mRNAs were found to be direct Igf2bp2 targets by RIP-seq. For both direct Igf2bp2 targets and non-targets, the changes in protein levels occurring upon lncRAP2 depletion closely correspond to those occurring upon  t test) F. These depleted A, such adepleted G. By conIgf2bp2 depletion in mature adipocytes for 1,205 genes quantifiable by both RNA-seq and mass spectrometry. The correlation between the two types of responses was strong for direct Igf2bp2 targets with differentially regulated protein vs. non-targets, both after lncRAP2 depletion  and after Igf2bp2 depletion   I and S4B t test) . We founepletion J. In sum2 \u2265 0.8) with variants within lncRAP2, but not so with any other locus within 500kb, including the IDE gene  associated with increased body mass index, higher fasting insulin levels, and insulin resistance in women with polycystic ovary syndrome in a Chinese population  maps to IDE gene A and S5AIDE gene A, insulaIDE gene B.Figure\u00a0\u22128). Additional genetic variants mapping to the lncRAP2 transcription start and end sites  and gene body (rs967878) also represent risk alleles for type 2 diabetes (p\u00a0< 109 to 10\u22128), thus evidencing association of multiple lncRAP2 alleles with diabetes  (Igf2bp2 levels decrease as body mass index increases (Igf2bp2 trends lower in adipocytes of leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) obese mice  (The pathogenesis of type 2 diabetes can involve obesity-linked adipose dysfunction . To expl t test) E. Human ncreases D and S5Encreases F. We alsese mice F, which ese mice . We spec, ttest) G. Thus, Obesity has become pandemic , increasOur data show that multiple genetic variants associated with propensity to type 2 diabetes are confined to an lncRAP2 linkage disequilibrium and chromatin interaction domain. Analyzing hundreds of phenotypes interrogated by genome-wide association studies reveals that these variants, mapping to the lncRAP2 gene body and transcription start and end sites, are specifically associated with body fat mass, insulin secretion, and type 2 diabetes, implicating lncRAP2 alleles in the development of obesity and diabetes. Supporting this notion, lncRAP2 is suppressed in the white fat of mice and humans consuming a high-fat diet, and in humans lncRAP2 levels decrease progressively with increasing obesity, whereas lncRAP2 levels are restored upon weight loss.lncRAP2 is enriched in white relative to brown adipocytes and is critical for adipogenesis . AlthougIgf2bp2 levels in pancreatic islets .Further information and requests for resources and reagents should be directed to the lead contact, Marko Knoll (This study did not generate new unique reagents.C57BL/6J mice were bred in house or purchased from Jackson Laboratories (stock # 000664). All mice were housed under a 12\u00a0h light/dark cycle at constant temperature (20\u00b0C). All procedures were performed according to protocols approved by the Committee on Animal Care at the Massachusetts Institute of Technology.2 asphyxiation and interscapular brown adipose tissue (BAT) and subcutaneous  white adipose tissue (scWAT) was harvested into room temperature plain DMEM . The fat pads were transferred into a well of a 6 well plate and minced with scissors for 5\u00a0min. Minced tissues were then transferred into a 50\u00a0mL conical tube with 3\u00a0mL Hank's balanced salt solution  supplemented with 0.2% collagenase A  and 2% BSA  using a 1\u00a0mL pipet tip with the tip cut off to allow aspiration of larger pieces. The tissues were incubated agitating (350rpm) and repeated vortexing every 5\u00a0min for 10\u00a0s at 37\u00b0C for 30\u00a0min or 20\u00a0min for scWAT. Following collagenase digestion, 10\u00a0mL room temperature plain DMEM was added and cells were filtered through a 70\u00a0\u03bcm mesh filter . Mature adipocytes and the stromal vascular fraction (SVF) were separated by centrifugation at 700\u00a0g for 5\u00a0min. The supernatant was removed and the SVF resuspended in 10\u00a0mL room temperature plain DMEM followed by additional filtering through a 30\u00a0\u03bcm mesh filter (Miltenyi Biotec # 130-041-407) and subsequent centrifugation at 700\u00a0g for 5\u00a0min. The SVF from subcutaneous white fat pads (scWAT) of 8 mice were then resuspended in 10\u00a0mL DMEM supplemented with 10% heat inactivated new born calf serum  and plated on two 10\u00a0cm dishes . The SVF from interscapular brown fat pads of 6\u20138 mice were then resuspended in 6\u00a0mL DMEM supplemented with 10% HI NCBS and plated on 3 wells of a six well plate . After 4 and 24 h, the medium was replaced by fresh, pre-warmed DMEM/10% HI NBCS at 37\u00b0C and with 5% CO2. Cells were grown to confluence and then passaged no more than two times before seeding the pre-adipocytes for differentiation.6\u20138 male 2\u20134\u00a0week old mice were sacrificed by COPre-adipocytes derived from BAT were cultured to confluence and then subsequently overgrown for 4\u20136 additional days until growth arrested. The cells were then induced to differentiate by culturing them for two days in induction medium consisting of DMEM supplemented with 10% fetal bovine serum  and 850\u00a0nM insulin (Sigma-Aldrich #I1882), 0.5\u00a0\u03bcM dexamethasone (Sigma-Aldrich #D4902), 250\u00a0\u03bcM 3-isobutyl-1-methylxanthine , 1\u00a0\u03bcM rosiglitazone , 1\u00a0nM 3,3,5-triiodo-L-thyronine  and 125\u00a0nM indomethacin (Sigma-Aldrich #I7378). Subsequently, the induction medium was replaced with DMEM supplemented with 10% FBS and 160\u00a0nM insulin and 1\u00a0nM T3 for another two days. The cells were then cultured in DMEM 10% FBS and 1\u00a0nM T3 until day 8 of differentiation, and the medium was replaced every other day. Pre-adipocytes from scWAT were cultured similar but the induction medium and following medium did not contain T3.Mature adipocyte cell layers were washed twice in plain pre-warmed DMEM and stimulated with 1\u00a0\u03bcM isoproterenol (Sigma-Aldrich I6504). After 6\u00a0h of stimulation, the cells were washed once with cold PBS and RNA was harvested using TRizol or QIAzol lysis reagent as described below. For immunoblotting, the cultures were harvested after stimulation with isoproterenol after washing with cold PBS on ice and adding 40\u00a0\u03bcL RIPA buffer per well of a 6 well plate or 100\u00a0\u03bcL RIPA buffer to a 10\u00a0cm dish.Was performed as described . BrieflyFollowing addition of fresh medium, cells were stimulated with isoproterenol. Cell culture medium was collected after 24h of stimulation and stored at\u221220\u00b0C. Glycerol release was measured using the Adipolysis Assay Kit  following the instructions of the manufacturer.Total RNA was isolated from tissues or cells using TRizol or QIazol reagent (LifeTechnologies/Ambion) and a miRNAeasy kit (Qiagen). 300\u00a0ng were reverse transcribed using Superscript II reverse transcriptase (LifeTechnologies/Invitrogen) using random hexamers (LifeTechnologies/Invitrogen). The cDNA was diluted 1:10 and 2.5\u00a0\u03bcL for a 96 well plate or 1\u00a0\u03bcL for a 384 well plate were used for quantitative Real-time PCR. qPCR was carried out on an ABI7900HT Fast real-time PCR system (Applied Biosystems) and analyzed using the delta delta Ct method normalized to 18S if not stated otherwise. Results are shown as pooled data from 3\u20134 independent experiments displaying the average and SEM.Poly A+ RNA sequencing (TrueSeqStrandedPolyA) was performed on RNA samples using a Nextseq genome sequencer (Illumina). RNA-seq paired-end reads were aligned to the mouse genome  using STAR v2.6.1  providedLysates were centrifuged for 10\u00a0min at 13,000\u00a0g to remove debris, and NuPAGE sample buffer and reducing buffer (LifeTechnologies) were added after measuring and adjusting the samples for protein concentration . 2\u201320\u00a0\u03bcg protein per sample were separated for 2\u20134\u00a0h at 60\u2013100\u00a0V using 8% 26 well NuPAGE Bis-Tris Midi gels in MOPS or MES buffer (LifeTechnologies) and in Criterion cells (Bio-Rad) using respective adapters (LifeTechnologies). Protein was wet transferred to polyvinylidene fluoride (PVDF) membranes  in Criterion blotter cells (Bio-Rad) using 2 x NuPAGE Transfer buffer (LifeTechnologies) with 10% methanol for 25\u00a0min at 1 A. After blocking the membrane in filtered  TBS-T  with 3% BSA  for 1 h, blots were incubated in primary antibody diluted in TBS-T 3% BSA sealed in hybridization bags and gently shaking at 4\u00b0C overnight, then washed three times for 10\u00a0min in TBS-T, incubated in secondary antibody  diluted in TBS-T 3% BSA gently shaking for one hour at room temperature, and then washed again three times for 10\u00a0min in TBS-T. Antibody binding was visualized using ECL Plus Western Lightning reagent (PerkinElmer NEL 102) and blots were exposed to film . Films were scanned without adjustments using an Epson scanner. All immunoblotting data shown were reproduced with almost identical results in at least one and typically two to three additional independent experiments.Cells were washed in PBS and fixed in 3.7% formaldehyde solution for 1 h, followed by staining with Oil Red O for 1 h. Oil Red O was prepared by diluting a stock solution (0.5\u00a0g of Oil Red O (Sigma) in 100\u00a0mL of isopropanol) with water (6:4) followed by filtration. After staining, plates were washed twice in water and photographed.The 5\u2032 and-3\u2032 ends of lncRAP2 was determined using the FirstChoice RLM-Race Kit from Ambion following the manufacturers instructions. Primers were designed accordingly and can be found in To separate the nuclear from the cytoplasmic fraction differentiated 3T3-L1 adipocytes were harvested and 1 million cells were used to isolated the fractions using the PARIS kit (Life technologies) according to the manufacturers instructions. Separated fractions were then analyzed using Real-time PCR and gene specific primers.Single molecule RNA FISH and fluorescence microscopy were described previously . Brieflyhttp://hannonlab.cshl.edu/fastx_toolkit/index.html), discarding non-clipped reads or reads <25nt after linker clipping (\u201c-l 25 -c\u201d parameters), andRibosome profiling sequencing reads were clipped to remove 3\u2032 linkers using fastx_clipper from the FASTX-Toolkit  using STAR v2.6.1 with default parameters and \u201c--seedSearchStartLmax 20 --sjdbOverhang 39 --outSJfilterOverhangMin 30 8 8 8 --sjdbScore 2\u201d. To measure gene-level and region-level  expression from uniquely-aligned reads, we quantified read counts using HTseq-count. Only genes for which a read count could be obtained in each sample and replicate were retained, and counts were then normalized as counts per million mapped reads (cpm) using only reads from these genes.Gene-level translation efficiency was calculated as the ratio of normalized Ribo-seq read density (cpm) to the normalized RNA-seq read density (cpm).We computed the ribosome release score using the RRS program  default. For mRN7 re-suspended in 2mL 1X PBS, then lysed in nuclear isolation buffer  for 20\u00a0min. Nuclei were pelleted by centrifugation at 2,500\u00a0g for 15\u00a0min. Supernatant was discarded and nuclei were re-supended in 1mL RIP buffer containing the HALT protease and phosphatase inhibitor (Thermo scientific) and split into two fractions and mechanically sheared using a Dounce homogenizer with 20 strokes. Nuclear membrane and debris were pelleted by centrifugation at 13,000\u00a0rpm for 10\u00a0min at 4\u00b0C. The supernatant was pre-cleared by adding 30\u03bcL slurry of protein A/G beads  and incubation for 2\u00a0h at 4\u00b0C on a rotator. Beads were removed by centrifugation at 2500\u00a0g for 1\u00a0min and 10% of the supernatant was removed to a new tube (10% input) and the rest was incubated with antibodies to Suz12 , IgG , Ezh2 , hnRNPU , CoREST  or no antibody for 3h at 4\u00b0C on a rotator. Then 60\u03bcL slurry of protein A/G beads were added for 2h at 4\u00b0C on a rotator. Beads were pelleted by centrifugation at 2500\u00a0rpm for 30s and washed 3 times in 500\u03bcL RIP for 10\u00a0min each followed by one wash with 1X PBS. For the isolation of RNA, the beads were re-suspended in TRIzol after the last wash step and isolated according to the manufacturers instructions. The RNA pellet was re-suspended in 10\u03bcL dH2O and was directly used for reverse transcription using random hexamers and SuperScript II (Invitrogen). Analysis was done by real-time PCR and sequencing.RNA coimmunprecipitation was done as described . Mature RNA FISH probes were biotinylated using Biotin-XX, SSE (Thermo Fisher). As controls, we also used probes against H19  and BlooRaw reads (40bp unpaired and non-strand-specific) were mapped to mm9 using bowtie2 . Peak caChIRP-MS was performed as described  with theMass spectrometry fragmentation spectra were correlated against a custom peptide database, formed by downloading all RefSeq species-specific (mm9) entries, and against a database of common contaminants  using Mascot (Matrix Science) 2.5.1 and the Sequest algorithm (Thermo). The resulting Mascot search results were uploaded into Scaffold (Proteome Software), and a minimum of two peptides and peptide threshold of 95% and protein threshold of 99% were used for identification of peptides and positive protein identifications. Proteins enriched in ChIRP-MS experiments were identified based on several criteria. First, only proteins identified by 2 or more unique peptides in the differentiated sample were considered. Then proteins whose total peptide count was at least 10-fold greater in the exon probes versus intron probes were considered enriched. Finally, proteins introduced during sample preparation and purification  were excluded from further analysis. To distinguish lncRAP2-specific interactors from proteins that generally bind RNA during its processing, we compared the proteins enriched by lncRAP2 with those enriched by Dleu2 , Neat1 aAfter proteins were separated on an Acryl-amid gel (see Western blot), the gel was fixed in 40% EtOH with 10% HAc for 1\u00a0h then washed 2\u00a0\u00d7 20\u00a0min in 30% EtOH and once for 20\u00a0min in water. The gel was sensitized for 1\u00a0min in 0.02% Na2S2O3 following 3\u00a0\u00d7 20\u00a0s wash in water and 20\u00a0min incubation in cold (4\u00b0C) 0.1% AgNO3 solution. The gel was washed 3\u00a0\u00d7 20\u00a0s in water and developed in a new chamber in 3% Na2CO3 with 0.05% formaldehyde. As soon as bands became visible the reaction was stopped by a short wash in water and then incubation in 5% HAc. All lanes were cut out and processed .Gene lists were analyzed for enrichment of genes grouped by biological process ontology or by curated annotations from the Molecular signatures database with GSEA  using deGenomic sequences from regions of interest (e.g. UTRs) were searched for matches to a database of TF recognition sites  for TFs Processed ChIA-PET, Hi-C, and capture Hi-C datasets were downloaded and visualized in triangle heatmap mode using the UCSC genome browser or in arc mode using the Washington University Epigenome browser, with default normalization and resolution settings and fixed display values.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genome-wide read density maps were generated by MACS2 using the \u201c--bdg\" option, normalized by RSeQC (http://CRAN.R-project.org/package=gplots).All sequencing reads were quality-checked with FastQC  was assessed by a Fisher's exact test using the fisher.test R implementation with default parameters. For unpaired data, a Shapiro-Wilk normality test was first performed using the shapiro.test R implementation with default parameters; for normally distributed data we then used a two-sided ttest (t.test R implementation with default parameters) to assess confidence on the measured difference of their mean values. For unpaired data that don't follow a normal distribution, we used a non-parametric Wilcoxon rank-sum test to determine if they belong to the same distribution. Variance was represented as mean\u00a0\u00b1 SEM of n\u00a0= 3 replicates unless otherwise specified.>hg19_chr10: 94,176,182-94,180,397ATCCGTTACCCAACGGCTGTCGAAAGAGGAGCACGACGTGAACTTCCACCGACAATCGTTTAAATACTGCAGGCGAAGGACGGGTTCTTATTGTTCCTTCAGGCAAACCGGCAGCTGAGTCCAGCTTGTGAACTTGAACTTGAACTTGCTGAAGAAGCTCCCGGCGGCCCTCTGCTGGCCGCCGCCTTTGCCAGAGGGAGAGGCTGGAGTCACCCTGTTGGAACCCATTTCTGGGGCTGGCACCTGTTGGGCTGCCCGGCCGCGCGTACCTGGTCCCATCGGGGGCTCTGCCCACTCCGCTGATGACGCGGGTAGAAGGGAGGCCGCAGGGACACTCTGGGGGGACTGTGCCGGGCGGGCACCCCCCAGCTGCTCACTGTGGGGTGCGGCACCGAGGCCTGGTTGGGCTGCAAGGAGACCGACTGGGATTCCCGGGCTGGTGGCCGGGGAGACGGGGTAGAGGTGAGAAGCAAGAGCTCAGGAGGCCTCAGGCCCCAGCGCTGTGGGGCTGCCGTTGTCGTTCTGGGTGGAGGTCTGGCCAAACCGGCTTTTGCCCCGAGTGAGGAATTCCTGCTCATTTTGGTGTTAGTGGAGAGGTCGCTGTTCACAGCGGGGGTGGGGTTCGTCCCCTCCAGGCTAGTAGGGAGCTGGCTGGTGCATTGCTGTGTTGCTGCCCTTCTGCCCTGTCTCCTGATCCTGTTCAGACTCTGGGTGGCTCCTTTGAGTCTTCTCAGGCCGTGAAAGATGGGAAAGGCAGGTTGTTGAAACCACTGCTGCTCCGGGCTGGCAGAGACCCTGAGCTGTACCTCCTGGGATGAAAGGAGAACCCAGGAGCAGGCAGGTGACCACTGCTTCCTCATACTCCTCAGCAAGTCCTGGATCTCTTCCTGTCTGTAACCCACGGATACCATGAACTTAGTTTGCAGGGGGTCCTCCCAGTGGGAGAGTGGCTGCACACAGCGCTTAAGTTCCTCACCTTCATGACTCGCATTCATCCATGGACCCCTCACAGTGTGCTCTGAAGGGCCTCTCTTCCTGCAATTGACAATGAGAAATTTCTTCAGCAAGTTTTCACTCTCCGGGGACATGGGGGAGGGAATGGAATACATCCTGCTCAGTACCTGCTCCTATAGCTCCTTGAAGTTCTGTCCATCGAAAAATAAGGATCCATTTACCAGTATATAGAGGATAACTCGCAGACGCCATACATCCACCAGCTCATATTTGTGGCCCTAGAAGAATTCCAGGGCAGCCTAAGGGGACTGCCACAAAAGGTATCCAGCTTGTTGCGAAAGGCGAATTTATTCCTGAACCCAATCTGTGATGTTCATGTTAACATGGACAATACATTTCTGGTGACAATACACAATATCTGTGGACAATGCACTTCTGGTAACAGCTTGCATAGCAGACACTTTTGGTGGAATTTGCTTCAGGCCTCTTTCCTGCTGCTGTGAGCAGGTATTCACAAACTTCTCGCTAGCGTGCTTTGTGATGAGGCAGAGATTCCTCACTATCGATCACAGCAAATACTTCACTATGTTGGGGTGATCTGAGCCTTCATGATTCCTACTTTGTGGATGGTGTCTGGAGGCTGGAGGAGATCTGCTGAGTCTCATCAATGAACTTCACGGCGGCTGCTTTCCCAGTCAGGATGTCCCAGGCCAACCCCACCTTAGCCAGGTTGTCCTTGACGATGGTCTCAAGGATCCTCGCTGCCAATAAGGATCCCTCCTCAGCAGAGATGGCTGAGAGGCCCTGCGGCGTGTGGGACTTACTACTGGGCTTGGAGTCAAGGTGTCCCAGGTCGGCTTAATTTTGGGAAAAGCTGGTGAGAAGCTTGGTCCAAATCACCTGAAAAGAAAATCACTTGCATAGTTTACCTGAAGAACGGATTTCTATAATGGAATCAAAACACAATACTGGACAAAAATCAAATAAGCAAAAGCAACAAACTGAAAAGGAAATGCTGAGTAAAAAAGAACTTAAAAATTAGAAAAATAAGAGAAAAAAGAACAAGAAGAACCAGTAACCTCAGCCAAGCCAGGGACTTACATACGTGCTTGGAATTCCAAAGCAGCAGTTCCTTATGAGGAAGAACTAAGCCTAGTAACAAGGCTGAGGATAATCTATGTGGCTTTCTCATGCTTTGGTCTCAAGAACTCTTTACTCTTAAAGAAAATATATTGAGGACCACAAAGAGGATTTTTTATTGATATGGGTTACAGTTATGAATATTTACCTTATTAGAAATTAAAACCTCTAGGATGCTTCAATGGCCTTTTCTAGTTTGAAAAGATAACAGGCTGGGTGTGGTGGCTCACGCCTGTAATTCCAGCACTTTGGGAGGCCGAGGTGGGCAAATCACCTGAGCTCGGGAGTTCGAGAAAAGGTATAAAAATGTTTGGCTTTTAAAGAGCCCACAATATCTACACTTAAAATATTTCATTTTTTTCTTTAAACTCTAAATGATTGGTTTCAAAATGATGCCACAACTTAGCTGGCATTATGATAGTGTATAAGTATGTTCTGTTGTGTACGACACAATGAGCTTCATTATTACCATCTGCGACACTGAGGGGAAGATAGCTTTCATCATATTTTCTCATTTAAAGTTTTGCCCAGTTTCATTTGCATAGATTCCCTTTTTCCATGAGCTGCTATGTCAGTCTCAGCATCTTTCAATGTAGAGTTTGCAGCTATGAGTTGAGAAAGCACATTTTCTACTCTTTTTAAGTGAATAATCCACTGTGCCTGGTGTACCTCTCCTTCAGCATAGGATAGGGACATCCAGGTACTGGACCCGTCACTGGCACCTCAGTGGGGAGAACCCAGATGCCCCTACATGATGTTTAAAGATGCTTTATATACATAAAAGTGCACAAATCATCAGCCCACAGCTTGGTGACTGTTCACATATTGAACTCATCTATTTATCTAGTATCCAGGTCAAGAAACAGCCATTACAGCCCCCCAAGATCCCACACCCCTTTTCCAGTCACTTTCTCTGCAGTGATAACCACTCTTCTGTATTTTGACAGCATAGATTCATTTTGCTTATTTTTGAACTTTACATACATGGATTCATACAGTATTGGATCCTTTGTGTCTGCTTCCTTTGCTTAATTTGTTTTTGTTTGTTTGTTTGTTTGTTTGTTTTTCTTGAGACAGAGTCTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCACGATCTCAGATCACTGCAACCTCCACCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTATAGGAGCTTGCCACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTTACCTCATGTTCCGCCTGCCTCAGCCTCCCAAAGTGCTGGAATTACAGGCATGAACAACCACGCCTGGTCCTTTGCTCAATATTTTTGTGAGATCCATCCATATTGTTTATTATTCTAAATGCTCATTGTGTGACTGTAACACAATTTGTTAATTTGTTTATTCATTTTACTGTTACTGGGCAGTTGAGTAGTTCTCAGTTTTCAGATGCTATAGTGCTGCCATAAACATTCTTGTTCAGGTTTTTGGGGGACATATATATGGCTTTCTGTTGGATATATATAAATATATTAAGGGTGTGGCTGAACAACCATTTGACAGTTTATGCTAACAAGGTGACTCGTGGTAGGCCCCTTAGGCCAGGTGATATCAGCCTGACCTCCAGAGAGTGGGGTGGGGGCTGGAGACTGAGTTCAACCACATGGACAATAAGTCTATCATGTAATGAAGCCCCAGTAAAAACTCTGGATGCTGAAGCTCAGGTGAGTGTCCCTGATTGGCAGTACTCTATATGTGTTGTCTCACACATCCAAATCAGCAGGGTAATGCATTCTGAGGACCCCAGAGGCTTCACATTTGGAACCCTCTCAGACTCTGCTCTATCAATCTCTTTCTTTGGCTAATTTTGATCTCTATCCTTTCCCTGAAATAAACTGTAACTGTGAGTATAACAG."}
+{"text": "Messenger RNA (mRNA) comprises three major parts: a 5'-UTR (UnTranslated Region), a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g.) neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs) found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region) in the 3'-UTR.We compared the cis-acting signal of Tau with mRNAs in the axonal regions of neurons using the ClustalW program for alignment of sequences and the Mfold program for analysis of secondary structures.We found that at least 3 out of 12 mRNA analyzed  have a sequence similar to the cis-acting signal of Tau in the 3'-UTR. This could indicate that these messengers are localized specifically in the axon. The Mfold program showed that these mRNAs have a similar \"bubble\" structure in the putative sequence signal.Hence, we suggest that a U-rich sequence in the 3'-UTR region of the mRNA could act as a signal for its localization in the axon in neuronal cells. Sequences homologous to the DTE sequence of BC1 mRNA could direct the messenger to the dendrites. Messengers with homologues of both types of sequence, e.g. \u03b2-actin, might be located in both dendrites and axon. A messenger RNA (mRNA) comprises three major parts, a 5'-UTR (UnTranslated Region), a coding region and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Many studies have shown that certain sequences in the 3'-UTR are involved in localizing the mRNAs in different cells such as neurons, epithelial cells, oocytes and early embryos ,2. Such The mRNA of tau has been studied in detail . Tau is Recently, many mRNAs have been shown to be located in neuronal axons: \u03b2-actin, tropomyosin 3 (Tpm3), cofilin, vimentin, immunoglobulin heavy chain biding protein (Bip), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulated protein (grp75) and synuclein . The objThe 3'-UTR of tau mRNA contains 3884 bases; the U-rich region (in bold) is responsible for the localization of this mRNA in the axon hillock and growth cone.UUUUUUUUUUUUUACUUUAGCGGUUGCCUCCUAGGCCUGACUCCUUCCCAUGUUGAACUGGAGGCAGCCAAGUUAGGUGUCAAUGUCCUGGCAUCAGUAUGAACAGUC AGUAGUCCCAGGGCAGGGCCACACUUCUCCCAUCUUCUGCUUCCACCCCAGCUUGUGAUUGCUAGCCUCCCAGAGCUCAGCCGCCAUUAAGUCCCCAUGCACGUAAUCAGCCCUUCCAUA CCCCAAUUUGGGGAACAUACCCCUUGAUUGAAAUGUUUUCCCUCCAGUCCUAUGGAAGCGGUGCUGCCUGCCUGCUGGAGCAGCCAGCCAUCUCAGAGACGCAGCCCUUUCUCUCCUGUC CGCACCCUGCUGCGCUGUAGUCGGAUUCGUCUGUUUGUCUGGGUUCACCAGAGUGACUAUGAUAGUGAAAAGAAAAAGAAAAAGAAAAAAGAAAAAAGAAAAAAAAAAAAGGACGCAUGU UAUCUUGAAAUAUUUGUCAAAAGGUUGUAGCCCACCGCAGGGAUUGGAGGGCCUGAUAUUCCUUGUCUUCUUCGUGACUUAGGUCCAGGCCGGUCGAGUGCUACCCUGCUGGACAUCCCA UGUUUUGAAGGGUUUCUUCUUCAUCUGGGACCCCUGCAGACACUGGAUUGUGACAUUGGAGGUCUAUACAUUGGCCAAGGCUGAAGCACAGGACCCGUUAGAGGCAGCAGGCUCCGACUG UCAGGGAGAGCUUGUGGCUGGCCUGUUUCUCUGAGUGAAGAUGGUCCUCUCUAAUCACAACUUCAAGUCCCACAGCAGCCCUGGCAGACAUCUAAGAACUCCUGCAUCACAAGAGAAAAG GACACUAGUACCAGCAGGGAGAGCUGUGGCCCUAGAAAUUCCAUGACUCUCCACUACUAUCCGUGGGUCCUUUCCAAGCCUUGCCUCGUCACCAAGGGCUUGGGAUGGACUGCCCCACUG AUGAAAGGGACAUCUUUGGAGACCCCCUUGGUUUCCAAGGCGUCAGCCCCCUGACCUUGCAUGACCUCCUACAGCUGAAGGAUGAGGCCUUUAAAGAUUAGGAACCUCAGGCCCAGGUCG GCCACUUUGGGCUUGGGUACAGUUAGGGACGAUGCGGUAGAAGGAGGUGGCCAACCUUUCCAUAUAAGAGUUCUGUGUGCCCAGAGCUACCCUAUUGUGAGCUCCCCACUGCUGAUGGAC UUUAGCUGUCCUUAGAAGUGAAGAGUCCAACGGAGGAAAAGGAAGUGUGGUUUGAUGGUCUGUGGUCCCUUCAUCAUGGUUACCUGUUGUGGUUUUCUCUGUAUACCCCCAUUUACCCAU CCUGCAGUUCCUGUCCUUGAAUAGGGGUGGGGGUACUCUGCCAUAUCUCUUGUAGGCAGUCAGCCCCCAAGUCAUAGUUUGGAGUGAUCUGGUCAGUGCUAAUAGGCAGUUUACAAGGAA UUCUGGCUUGUUACUUCAGUGAGGACAAUCCCCCAAGGCCCUGGCACCUGUCCUGUCUUUCCAUGGCUCUCCACUGCAGAGCCAAUGUCUUUGGGUGGGCUAGAUAGGGUGUACAAUUUG CCUGGAACCUCCAAGCUCUUAAUCCACUUUAUCAAUAGUUCCAUUUAAAUUGACUUCAAUAUAAGAGUGUAUCCAUUUGAGAUUGCUUGUGUUGUGGGGUAAAGGGGGGAGGAGGAACAU GUUAAGAUAAUUGACAUGGGCAAGGGGAAGUCUUGAAGUGUAGCAGUUAAACCAUCUUGUAGCCCCAUUCAUGAUGUUGACCACUUGCUAGAGAGAAGAGGUGCCAUAAGGCUAGAACCU AGAGGCUUGGCUGUCCACCAACAGGCAGGCUUUUGCAAGGCAGAGGCAGCCAGCUAGGUCCCUGACUUCCCAGCCAGGUGCAGCUCUAAGAACUGCUCUUGCCUGCUGCCUUCUUGUGGU GUCCAGAGCCCACAGCCAAUGCCUCCUCAAAACCCUGGCUUCCUUCCUUCUAAUCCACUGGCACAUCAGCAUCACCUCCGGAUUGACUUCAGAUCCACAGCCUACACUACUAGCAGUGGG UAAGACCACUUCCUUUGUCCUUGUCUGUUCUCCAGAAAAGUGGGCAUGGAGGCGGUGUUAAUAACUAUAGGUCUGUGGCUUUAUGAGCCUUCAAACUUCUCUCUAGCUUCUGAAAGGGUU ACUUUUGGGCAGUAUUGCAGUCUCACCCUCCGAUGGCUGUAGCCUGUGCAGUUGCUGUACUGGGCAUGAUCUCCAGUGCUUGCAAGUCCCAUGAUUUCUUUGGUGUUUUGAGGGUGGGGG GAGGGACAUGAAUCAUCUUAGCUUAGCUUCCUGUCUGUGAAUGUCCAUAUAGUGUACUGUGUUUUAACAAACGAUUUACACUGACUGUUGCUGUACAAGUGAAUUUGGAAAUAAAGUUAU UACUCUGAUUAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCAGGCCCCUGGGGCCGUCACUGAUCAUGGAGAGAAGAGAGAGUGAGAGUGUGGAAAAAAAAAAAAAAAGAAUGACCUGGCCCCUCACCCUCUGCCCUCCCCGCUGCUCCUCAUAGACAG GCUGACCAGCUUGUCACCUAACCUGCUUUUGUGGCUCGGGUUUGGCUCGGGACUUCAAAAUCAGUGAUGGGAAAAAGUAAAUUUCAUCUUUCCAAAUUGAUUUGUGGGCUAGUAAUAAAA UAUUUUUAAGGAAGGAAAAAAAAAACACGUAAAACCAUGGCCAAACAAAACCCAACAUUUCCUUGGCAAUUGUUAUUGACCCCGCCCCCCCCUCUGAGUUUUAGAGGGUGAAGGAGGCUU UGGAUAGAGGCUGCUUCUGGGGAUUGGCUGAGGGACUAGGGCAACUAAUUGCCCACAGCCCCAUCUUAGGGGCAUCAGGGACAGCGGCAGACAUGAAAGACUUGGGACUUGGUGUGUUUG UGGAGCCGUAAGGCGUAUGUUAACUUUGUGUGGGUUUGAGGGAGGACUGUGAUAGUGAAGGCUGAGAGAUGGGUGGGCUGGGAGUCAGAGGAGAGAGGUGAGGAAGACAGGUUGGGAGAG GGGGCAUUGCGUCCUUGCCAAGGAGCUUGGGAAGCACAGGUAGCCCUGGCUGCAGCAGUCUUAGCUAGCACAGAUGCCUGCCUGAGAAAGCACAGUGGGGUACAGUGGGUGUGUGUGCCC CUUCUGAAGGGCAGCCCAUGGGAGAAGGGGUAUUGGGCAGAAGGAAGGUAGGCCCCAGAAGGUGGCACCUUGUAGAUUGGUUCUCUGAAGGCUGACCUUGCCAUCCCAGGGCACUGCUCC CACCCUCCAGGAGGAGGUCUGAGCUGAGGAGCUUCCUUUUCGAUCUCACAGGAAAACCUGUGUUACUGAGUUCUGAAGUUUGGAACUACAGCCAUGAUUUUGGCCACCAUACAGACCUGG GACUUUAGGGCUAACCAGUUCUUUGUAAGGACUUGUGCCUCUUGCGGGAACAUCUGCCUGUUCUCAAGCCUGGUCCUCUGGCACUUCUGCAGUGGUGAGGGAUGGGGGUGGUAUUCUGGG AUGUGGGUCCCAGGCCUCCCAUCCCUCGCACAGCCACUGUAUCCCCUCUACCUGUCCUAUCAUGCCCACGUCUGCCACGAGAGCCAGUCACUGCCGUCCGUACAUCACGUCUCACCGUCC UGAGUGCCCAGCCUCCCAAGCCCAAUCCCUGGACCCCUGGGUAGUUAUGGCCAAUCUGCUCUACACUAGGGGUUGGAGUCCAGGGAAGGCAAAGAUUUGGGCCUUGGUCUCUAGUCCUAC GUUGCCAGAAUCCAACCAGUGUGCCUCCCACAAGGAACCUUACAACCUUGUUUGGUUUGCUCCAUCAGGCGUUUGGCGCCAUCGUGGAUGGAGUCCGUGUGUGCCUGGAGAUUACCCUGG ACACCUCUGCThis cis-acting signal of tau was compared base by base with the other afore mentioned mRNAs using simple alignment. We also made comparisons with another sequence that is specific for the localization of BC1 mRNA in dendrites, the Dendritic Target Element (DTE) .In the \u03b2-actin mRNA of chicken a cis-acting signal \"zipcode\" has been described; a zipcode binding protein binds to this sequence and this is a prerequisite for the localization of the mRNA. The sequence is a tandem repeat of an ACACCCACACCC motif. The mRNA of \u03b2-actin has been located in the axon of the neuron and in dendritic spines ,14. \u03b2-acCofilin is a cytoskeleton modulating protein; it is also known as actin depolymerizing factor (ADF). The potential role of cofilin is to modulate the changes of actin organization that accompany neurite initiation, axonogenesis and growth cone guidance . The posVimentin has been located by RT-PCR in the axons of dorsal root ganglia (DRG) neurons. A possible sequence signal in vimentin mRNA shares some U with tau but also contains more purines, which might indicate that the protein is not exclusive to the axonal region .Bip is a protein that binds to the immunoglobin heavy chains in pre-\u03b2 cells. Its mRNA shares some U with the tau sequence; nevertheless, its sequence suggests that this mRNa, like vimentin, is probably not exclusive to the axon .The heat shock proteins and grp75 messengers have similarities with the tau sequence, but once again they are probably not exclusive to the axon. They could interact with other proteins in different parts of the cell.Synuclein is a soluble unfolded protein that can aggregate into insoluble fibrils under several pathological conditions including Parkinson's and Alzheimer's diseases . The posThese analyses carried out by alignment allowed us to show that the cis-acting signals of the mRNAs examined have some homology with that of the tau messenger.The highest homology scores are:The secondary structures of these four mRNAs, which showed the closest homologies to the tau sequence, were analyzed using the program Mfold Figs. , 2, 3, 4The first messenger to be analyzed in the 3'-UTR with respect to its localization/stabilization was tau ,10. The Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonging to the heat shock protein (HSP) family. It is highly expressed in conditions of glucose deprivation of glucose. Its messenger was located in the axon and it has a U-rich region. It might not be confined exclusively to the axon because this protein responds to a metabolic stress .Alfa-synuclein is involved in neurodegenerative diseases and its presence has been observed in the pre-synaptic and nuclear compartments, though the location in the nucleus has not been well documented. The synuclein messenger possesses a U-rich region; nevertheless a C interrupts the potential signal sequence. When it is wrongly folded, this protein may aggregate in the cell forming fibrils, typical of Alzheimer's and Parkinson's diseases. The aggregation of synuclein is similar to tau in Alzheimer patients, which could indicate similar intracellular behavior by both proteins .The 3'-UTR of the cofilin messenger has a U-rich region very similar to the signal sequence of tau, which on the face of it suggests that it might be located exclusively in the axon. Nevertheless, recent studies demonstrate that it participates in the shrinkage of dendritic spines associated with the long-term depression of hippocampal synapses, suggesting that it is also found in dendrites. Moreover, it is involved in neuronal development, axogenesis, guidance of the growth cone and dendrite formation. Although the cofilin messenger is present in axons, the possible participation of the protein in events related to the unplugging of synapses because of its association with actin further suggests that it is not confined to the axon but also occurs in the dendrites .The 3'-UTR of the \u03b2-actin messenger is very short and shows low homology when aligned with the tau cis-acting signal. However, when it was aligned with the dendritic target element, it showed better homology. The \u03b2-actin messenger was shown to possess a zipcode that leads it towards the dendrites instead of the axon .Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative conditions, including Parkinson's and Huntington's diseases, which are characterized by accumulation of protein aggregates . These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones, suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by \u03b2-amyloid and tau protein aggregates. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. A direct association between the chaperones and tau protein has been demonstrated. Many results suggest that the up-regulation of molecular chaperones may suppress the formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . When weOn the basis of the results we suggested a model for mRNA localization in the axon Fig. .The mRNAs containing the U-rich region could be complexed with a protein responsible for transport toward the axon, just as HuD complexes with and stabilizes the tau messenger . HuD itsThe mechanisms determining whether a messenger such as \u03b2-actin is transported preferentially to the axon or the dendrites are poorly understood. The existence of two potentially conflicting location signals in the 3'-UTR  raises questions about how the final destination of such mRNAs is determined within the neuron.In the 3'-UTRs of some mRNAs in neurons there are cis-acting signals that direct mRNAs such as tau and GAP-43 to the axon. In general, these signals are rich in uridine and do not contain guanidine. Comparison of the Dendritic Target Element (DTE) with the 3'-UTRs of several axon-located messengers showed some homology in a specific region of the 3'-UTR. Most of the 3'-UTRs studied possess homologies with the signals involved in the localization of mRNAs in axons and dendrites. This might explain why as much \u03b2-actin is present in dendrites as in axons, though the distribution mechanisms in such cases are not understood. In addition, we found a DTE homology in the 3'-UTR of HSP70 and 90. The significance of this is not clear; some messengers are transported towards the axon or towards the dendrites as required.A sequence homologous to DTE in tau occurs near the end of the 3'-UTR, next to the polyadenylation site, which indicates that only the axon signal sequence  is functional, because mRNA degradation starts at the poly(A) site. The 3'-UTR of MAP2  possesseVery U-rich sequences in the 3'-UTR might be signals that direct some mRNAs exclusively to the axon. If we understand which signals/sequences the neuronal cell uses for the correct location of its mRNAs, it might become possible to determine which factors lead to mislocalization of messengers and of proteins, as has recently been suggested in relation to certain neurodegenerative diseases such as Alzheimer's.Rattus norvegicus genome and were located using the following GeneBank accession numbers. \u03b2-actin; NM_031144, tropomyosin 3 (Tpm3); NM_057208, cofilin; NM_017147, vimentin; NM_031140, immunoglobulin heavy chain biding protein (Bip); M14050, heat shock protein 60 (HSP60); X53585, heat shock protein 70 (HSP70); L16764, heat shock protein 90 (HSP90); S45392, glucose regulated protein (grp75); s78556, synuclein; NM_031688; NM_057114 and NM_053576 and tau; X79321. The 3'-UTRs of the mRNAs were analyzed using the program ClustalW [All the mRNAs analyzed in this study belong to the ClustalW , and theClustalW .The author(s) declare that they have no competing interests."}
+{"text": "Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics. Complex cellular processes that control cell state and decision-making are orchestrated through highly interconnected regulatory networks. Quantitative genetic interaction mapping enables the systematic discovery of how gene-gene interactions give rise to complex cellular processes . Uncovermultiplex strategy for assessing genetic interactions using CRISPR-Cas9 (MoSAIC).The CRISPR-Cas9 system enables efficient genome engineering of mammalian cells through a programmable guide-RNA (gRNA) that targets Cas9 to a desired locus for editing \u20138. Thus 2 in high-Glucose Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) containing 10% fetal bovine serum and 1% Penicillin/Streptomycin (Life Technologies). HEK 293T cells containing eGFP were a gift from Stephen Goff (Columbia University). 293FT cells were obtained from Life Technologies and were maintained in the same medium formulation and supplemented with 0.1 mM non-essential amino acids, 2 mM L-glutamine and 500 ug/ml Geneticin.HEK 293T cells were obtained from the American Tissue Collection Center (ATCC) and grown at 37\u00b0C, 5% COhttp://www.broadinstitute.org/rnai/public/resources/protocols).Lentivirus was produced in 293FT cells and stable Cas9-eGFP cells were transduced as previously described  inducible Cas9 cells were generated as follows. 293T cell clones stably expressing eGFP-Cas9 under dox inducible promoter were generated by transduction of PLX301-eGFP-Cas9/Bsd  using LT1 transfection reagent (Mirus) followed by selection with 10mg/ml Blasticidin (Bsd). 293T cells were infected with lentiviral particles at MOI of 0.3 followed by clonal selection. We selected a clone with highest differential Cas9 expression following 48 hour induction using immunostaining of FLAG-tagged Cas9, followed by flow cytometry.The eGFP-Cas-9 clone was infected with lentivirus containing gRNA constructs targeting eGFP and STAT1 or eGFP-only. Twenty-four hours post-infection, the media was changed and supplemented with 10 ug/ml blasticidin (Life Technologies) and cells were selected for three days, prior to doxycycline induction of Cas9. Cells were harvested on days 14, 21, and 28 post-induction. Gene knockout efficiencies were measured by either flow cytometry or SURVEYOR assay. Flow cytometry was performed using a LSRII or LSR Fortessa to quantify fraction of eGFP positive cells.MV.1, MV.3, MV.5, MV.6, MV.7 originated from lentivector v_w0, originally called plxsgRNA (Addgene 50662). A point mutation was made in the PGK promoter to eliminate the BsmB1 restriction site for all down-stream cloning (v_w0).MV.2 originated from pLenticrispr (49535). Vs.d1 was amplified with primers containing eGFP gRNA 1 / STAT1 gRNA2 and cloned into the pLenticrispr vector to generate an all-in-one vector containing two gRNAs.To clone MV.1 backbone, pLenticrispr was used as a template with vs_p39(f) and vs_p40 (r) to amplify an insert containing the reverse direction chimeric RNA, filler region with BsmB1 restriction sites and a forward direction chimeric RNA sequence. The chimeric- filler-chimeric was cloned into v_w0. To clone in gRNAs, vs.d5 (dsDNA) containing reverse direction H1 promoter, LoxP site and forward direction U6 promoter, was amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites. The PCR product containing both gRNAs and both promoters was cloned into the MV.1 backbone to generate MV.1.1 and MV.1.2.To clone MV.3 backbone, H1 promoter expressing short tracr RNA was cloned into v_w0 from px261 (Addgene 42337). To clone in gRNAs, vs.d11 (containing U6 promoter) was amplified with primers vs_p79 and vs_p80/ vs_p81 / vs_p82 and PCR products were cloned into MV.3 backbone.To clone MV.5 backbone, the U6 promoter, filler region and chimeric RNA was cloned into v_w0 from lenticrispr_v1 (Addgene #49535) using vs_p59/vs_p40 primers. To clone in gRNAs,v_w2 containing chimeric RNA-LoxP-site and H1 promoter were amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites.To clone MV.6 backbone, U6 promoter, the filler region with BsmB1 restriction sites and the chimeric RNA v2, was cloned into v_w0 using vs_d10. To clone in gRNAs,v_w2 containing chimeric RNA-loxP-site and H1 promoter were amplified with primers containing eGFP gRNA 1 and STAT1 gRNA 2 as well as BsmB1 restriction sites.In MV.7, used the backbone established in MV.6. To clone in gRNAs, oligo pairs (vs_p75 / vs_p76 and vs_p77/vs_p78) containing -BsmB1 Overhang-gRNA1-chimeric RNA-gRNA 2 were synthesized, annealed and ligated into backbone.Unless noted, all DNA constructs and primers were obtained from IDT (Geneblocks) and used for PCR and assembly steps as described above.vs_d1: chimeric RNA\u2014LoxP site\u2014U6 promoter:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATAACTTCGTATAGCATACATTATACGAAGTTATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCvs_d5: reverse H1 promoter\u2014LoxP site\u2014U6 promoter forward:TAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCGAATTATAACTTCGTATAGCATACATTATACGAAGTTATGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCv_w2: chimeric RNA\u2014LoxP site\u2014forward H1 promoter:GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTATAACTTCGTATAGCATACATTATACGAAGTTATAATTCGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACAGATCTAvs_d10: Xho1 site\u2014forward U6 promoter\u2014BsmB1 filler region (36nt)\u2014Chimeric RNA version 2\u2014Nhe1:ATTCGAACTCGAGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACGGTTTTCTTGCTCTTTTTTGTACGTCTCTGTTTTAGAGCCGGAAACGGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGCTAGCGCTAACvs_d11: forward U6 promoter:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCvs_p26 :AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGvs_p64 :TATTTTAACTTGCCGTTTCCGGCvs_p59 (forward):ACGGACTCGAGGAGGGCCTATTTCCCATGATTCvs_p40 (reverse):GATCACGGAGCTAGCCTGCCATTTGTCTCAAGATCTAGAATTCvs_p75:CACCGGAGCTGGACGGCGACGTAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTGAAGTTCGAGGGCGACACCCvs_p76:AAACGGGTGTCGCCCTCGAACTTCAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTTACGTCGCCGTCCAGCTCCvs_p77:CACCGGAGCTGGACGGCGACGTAAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTCCCCGGGGAAGTTCGAGGGCGACACCCvs_p78:AAACGGGTGTCGCCCTCGAACTTCCCCGGGGAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTTTACGTCGCCGTCCAGCTCCvs_p79 (forward):AGGGATCCTGAGGGCCTATTTCCCATGAvs_p80 (reverse):GCGCTAGCTAAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGGTGTTTCGTCCTTTCCACAAvs_p81 (reverse):TTAGCGCTAGCTAAAAGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGGTGTTTCGTCCTTTCCACAAGvs_p82 (reverse):AGCGCTAGCTAAAAGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGGTGTCGCCCTCGAACTTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACTTTACGTCGCCGTCCAGCTCGTTTTGGGACCATTCAAAACAGCATAGCTCTAAAACGGTGTTTCGTCCTTTCCACAeGFP gRNA 1:GAGCTGGACGGCGACGTAAAeGFP gRNA 2:GAAGTTCGAGGGCGACACCCSTAT1 gRNA 1:GATCATCCAGCTGTGACAGGSTAT1 gRNA 2: CCTGTCACAGCTGGATGATCeGFP sequence:GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCWe used the crystal structure of the sgRNA-targetDNA-cas9 complex to model the UA31CG-AU32GC-sgRNA . The corIn order to comprehensively map genetic interactions in a gene network, all possible single and double knockouts (KO) need to be simultaneously interrogated. MoSAIC achieves this in a single step through PCR of a common DNA template with gRNA primer pools . The firTo optimize the system for simultaneous targeting of Cas9 to multiple loci, we designed and tested two MoSAIC-compatible strategies:1) dual promoter, dual gRNA transcripts, and 2) single promoter, single RNA transcript . We explored several designs that use RNA Pol III promoters U6 and H1 in different positions and orientations , having We began by benchmarking a previously described approach utilizing two unidirectional U6 promoters to express dual gRNAs  designs. We founWe then explored whether the promoter choice and orientation impacted KO efficiency in a position-dependent fashion  sequences is sufficient for multiplexed Cas9-mediated KO in the absence of the SpRNase III RNA cleavage enzyme [Previously, studies demonstrated that targeting Cas9 to multiple loci could be achieved by co-expressing the RNA cleavage enzyme ge sites . Anothere enzyme . In ordee enzyme  Fig 4A)Csy4 alonWe further explored MV7 designs that incorporated transcripts containing two tandem gRNA-chimeric RNA sequences and thus did not require a tracrRNA . This deS. pyogenes CRISPR-Cas9 crystal structure (PDB-4OO8) [MoSAIC is designed such that gRNA pairs serve as barcodes that can then be PCR amplified and identified using next-generation sequencing. We achieved this by altering the second chimeric RNA sequence such that placement of a reverse sequencing primer results in PCR amplification of both gRNAs with an amplicon size that is NGS compatible . Primer DB-4OO8)  to prediHere we report on the development of a generalizable strategy for multiplex targeting of Cas9 for mammalian genome engineering. We describe the implementation of this approach to combinatorially target two genes simultaneously and monitor the mutational efficiency of several gRNA designs. We find that gRNA pairs expressed from dual U6-H1 promoters lead to optimal Cas9-mediated genome editing, which can be combined with single transcript multiple gRNA designs (MV7) to increase editing efficiency. In addition, we find that a unidirectional single-transcript gRNA system resulted in low KO efficiency. While the precise reason for this observation has not been fully elucidated, we speculate that MV3.3 and 3.4 designs that lead to contiguous unprocessed gRNA products may cause a lower Cas9 targeting efficiency by virtue of Cas9 binding to either of the two gRNA positions, but not simultaneously. In addition, we observed that unidirectional dual gRNA designs resulted in increased KO efficiency for the second gRNA position. While further experiments are needed, we speculate that higher KO efficiency of the second gRNA may be the result of transcription read-through of first position gRNA, leading to increased levels of second gRNA transcripts and Cas9 targeting.To enhance multiplexing of our system, we identified specific chimeric RNA variants at positions 11\u201312 and 17\u201318 corresponding to the tetraloop repeat-anti-repeat duplex that are amenable to alteration. We showed that these chimeric RNA variants still maintained Cas9 targeting while also allowing compatible barcode amplification in a pooled format to enable multiplex assessment of cell populations using NGS. In this manner, the gRNA pairs serve as unique molecular barcodes linking their abundance in the cell population with their screened phenotype. This strategy is also compatible with the use of different Cas9 variants, including CRISPRi  and CRISMoSAIC overcomes several key technical hurdles associated with high-throughput generation and measurement of dual loci perturbations in mammalian cells. Additionally, to facilitate subsequent iterative introduction of gRNA constructs and enable higher-order combinatorial genetic perturbations, the integrated lentiviral vector design includes loxP \u201clanding-pad\u201d sequences. The MoSAIC system expands the toolbox for genetic modification of mammalian genome and extends our knowledge of Cas9 targeting design parameters. Recently, Wong et al described the CombiGEM approach to generate combinatorial gRNA libraries for Cas9-based genomic interrogations by barcoded sequencing . The Com"}
+{"text": "Herein we present the data necessary for generation of alternative means to produce equimolar mixtures of peptides   Specifications TableValue of the data\u2022Excellent reproducibility of peptide concentration data produced by the analysis of tryptic digestions is the necessary for method comparisons. The costs associated with a particular approach in terms of reagents and time may be a pivotal factor.\u2022The Hi3 method is a very valuable approach in cases of relatively simple proteomes. The speed of method development for core laboratories can be critical. The Hi3 method is relatively straightforward, once reproducible results are obtained.\u2022The QConCAT design is very elegant but there are critical issues, such as the protein construct solubility and stability, which require careful attention to obtain optimal results. The database attached is necessary for analysis of the mass spectral results.1The data are compiled in the bar graphs for the quantitative proteome analyses of trivalent influenza vaccines and standards using three methods: Hi3, QconCAT and synthetic peptides.http://www.thegpm.org/crap/) and, since the viruses are grown in chicken eggs, the entire chicken  proteome  as well as selected full length influenza proteins (mostly from GISAID http://platform.gisaid.org/epi3/frontend#4f5b25, and the World Health Organization http://www.who.int/influenza/vaccines/virus/en/.Databases contained either the customized QconCAT protein sequence or the full sequences of the proteins represented therein, along with trypsin, several human keratins (cRAP from 2Data was obtained by the comparison of mass spectral signal from the three most intense fully tryptic peptides identified from the samples versus the internal standard protein. Experiments were designed to compare the mass spectral signal strengths from equimolar tryptic peptides identified by the Hi3 method by five different QconCAT designs as well as from synthetic peptides. The samples analyzed were commercial trivalent influenza vaccines as well as monovalent influenza reference standards. The mass spectra were obtained by reversed phase separation using a C18 UPLC column, Waters nanoAcquity UPLC, directly coupled to a Waters Synapt HDMS mass spectrometer. The mass spectrometer was programmed to carry out data-independent MSMS and incorporated a lock spray of glu-fibrinopeptide. The data was processed using Protein Lynx Global Server 3.0 for the identification of the three most intense peptides from a given protein identified in a custom database which has been attached. The intensities from all charge states of a peptide are included in the software calculations; however, the intensities resulting from in-source fragmentation and modified peptides must be added manually.2.12.1.1CATATGGATGACGATGATAAACTGGTGAACGAACTGACCGAATTCGCGAAACTGGGCGAATACGGCTTCCAGAATGCACTGATCGTTCGTCATCTGGTTGACGAACCACAAAACCTGATTAAAGACGCATTTCTGGGCTCCTTTCTGTACGAATACTCTCGCGTTGTTGGTCTGTCTACCCTGCCTGAGATTTACGAAAAACTGCCACTGGTCGGTGGTCATGAAGGCGCAGGCGTCGTCGTTGGTATGGGCGAAAACGTGAAATCCATCAGCATTGTAGGTTCCTACGTTGGCAACCGTGCCAATGAACTGCTGATCAACGTCAAATCCACCCAGAACGCGATCGACGAGATTACGAATAAAATGAACTACTACTGGACCCTGGTTGAGCCGGGCGATAAAGAACAGCTGTCCAGCGTGTCCTCTTTCGAACGTATGAACACGCAGTTTACCGCTGTAGGCAAAAGCACCCAGGCTGCCATCGACCAGATCAACGGTAAAATCGATCTGTGGTCCTATAATGCCGAGCTGCTGGTTGCCCTGGAAAATCAGCACACTATTGACCTGACTGATAGCGAAATGAACAAGGAGTTCTCTGAAGTGGAAGGTCGTTGGGACCTGTTCGTGGAACGTCTGTCTGGCGCGATGGACGAACTGCACAACGAAATCCTGGAACTGGATGAGAAACTGTCTACTCACAACGTTATCAACGCGGAAAACGCTCCGGGTGGCCCGTACAAAATTGTGGTGGACTACATGGTGCAGAAGAACCTGAACTCCCTGAGCGAGCTGGAAGTGAAAACTTTCTTCCTGACTCAGGGTGCCCTGCTGAACGACAAATACAACGGCATCATTACGGACACCATCAAATATGGTAACGGTGTTTGGATCGGTCGCGGTGACGTGTTCGTTATCCGCACGCTGCTGATGAACGAGCTGGGTGTTCCGTTCCACCTGGGCACCAAGCTGGTGGATTCTGTAGTCTCTTGGAGCAAAGTAATCGAGGGCTGGAGCAACCCGAAGATTCTGTTTATTGAAGAAGGCAAAGGTGTAACCCTGCTGCTGCCGGAACCGGAATGGACCTACCCGCGCCTGAACGTTGAAACTGATACTGCGGAAATTCGTTATGGTGAAGCGTATACCGACACCTATCACAGCTACGCTAACAAAGAGTGGACCTATATCGGTGTTGATGGTCCGGATAACAACGCTCTGCTGAAAGGCGGTCTGGAACCTATCAACTTCCAAACCGCAGCGGACCAGGCGCGTATCTCTCAAGCAGTACACGCTGCGCACGCAGAAATCAACGAAGCTGGCCGTCTGACCGAGTGGACTTCTTCCAATGTTATGGAAGAACGTAACGTTCTGCAGCCGTCTTCCGTAGATTCTCAGACCGCAATGGTTCTGGTAAACGCTATCGTTTTCAAGTAAGGATCC.2.1.2CATATGGACGATGACGACAAGCATGTCAAACTGGTGAACGAACTGACTGAATTCGCAAAAACTTGCGTGCGTTTCGAAAAACTGGGCGAATACGGTTTCCAGAACGCACTGATCGTGCGTTACACCCGTAAACTGAAACACCTGGTGGATGAACCTCAAAACCTGATCAAACAGAACTGCCGTCGTCCAATCAAAGTAGTGGGTCTGTCTACCCTGCCGGAGATTTACGAAAAAATGGAAAAGCGTCCGGTGAAGCTGCCTCTGGTCGGCGGTCACGAAGGCGCTGGTGTTGTTGTTGGTATGGGCGAGAACGTGAAAGGTTGGAAGGTTGTTAAATCTATTAGCATCGTTGGCTCTTACGTAGGTAACCGCGCCGACACTCGTGATCTGAAGAGCACCCAGAATGCCATCGATGAAATCACCAACAAAGTCAACTCTCGCGAAGGTCGTATGAACTACTATTGGACCCTGGTTGAACCGGGCGACAAGATTACCTTTCGTGAACTGCGCGAACAGCTGTCTTCTGTGTCCAGCTTCGAACGTTTTGAAATCCGTGAATGCCGCACGTTCTTTCTGACGCAGGGTGCACTGCTGAACGACAAACATTCCAACCGCGTACTGAAATACAACGGTATCATCACTGATACCATTAAATCCTGGCGCAGCTTTAAATACGGTAACGGCGTCTGGATCGGTCGTACGAAATCTGACCTGAAATCCACCCAGGCGGCCATTGATCAGATCAACGGCAAACTGAACCGTGATACCAAAATCGATCTGTGGTCCTACAACGCGGAACTGCTGGTGGCCCTGGAAAACCAACACACCATCGACCTGACCGACTCTGAAATGAACAAACTGTTCGAGCGTATTGAAAAAGAATTTTCCGAGGTGGAGGGTCGCATCCAAGATCGTCGCCCGTACCGCACTCTGCTGATGAATGAACTGGGTGTTCCATTCCACCTGGGTACCAAACAGGTTTGCCGCAACGGCCGTCTGGTTGACAGCGTAGTAAGCTGGTCTAAAGAAATCCTGCGTACCTTCAAAGTTATCGAAGGTTGGTCTAACCCGAAATCTAAACTGCTGCAGCGCCTGTCTGGTGCGATGGACGAACTGCACAACGAGATCCTGGAACTGGATGAGAAAGTTGACGACCGTCACATCCGCCTGTCCACCCACAATGTAATTAACGCAGAAAACGCTCCGGGCGGTCCGTACAAAATTGGTACTCGCTCCGGTCGTATTGTGGTTGACTATATGGTACAGAAGTCTGGCAAAGCAACTAAAGGCGTTACGCTGCTGCTGCCGGAACCGGAATGGACTTATCCGCGTCTGTCCTGTCGTTTCGTCAAGCTGAACGTTGAAACCGACACGGCGGAAATTCGTCTGATGTGTAAAGTGAAATATGGCGAAGCGTATACCGATACTTACCATTCTTACGCTAACAAGATCCTGCGCCTGTATCGTGGCGGCCTGGAACCGATTAATTTCCAGACTGCGGCGGATCAGGCTCGTGAGCTGATCCGTAGCCTGAAAATCTCCCAGGCAGTACACGCTGCTCACGCTGAAATCAACGAAGCGGGCCGTGAGGTAGTTCGTTTCGAGAAACTGACCGAATGGACCAGCAGCAATGTTATGGAGGAGCGTAAAATCAAATAAGGATCC.2.1.3CATATGGATGACGACGACAAAGCAAGCGGTAAACTGGTGAACGAGCTGACTGAGTTCGCTAAAGCGAGCGGTAAGCTGGGTGAATACGGTTTCCAGAACGCACTGATCGTACGTGCTTCTGGTAAACACCTGGTCGACGAGCCTCAAAACCTGATTAAAGCTAGCGGCAAAGACGCCTTCCTGGGTAGCTTCCTGTACGAGTACAGCCGTGCTTCTGGCAAAGTAGTAGGTCTGAGCACTCTGCCAGAAATCTACGAGAAAGCGTCTGGTAAGCTGCCTCTGGTCGGTGGTCATGAAGGTGCTGGTGTAGTAGTAGGTATGGGTGAGAACGTGAAAGCATCCGGTAAAAGCATCAGCATCGTCGGTTCCTACGTCGGTAACCGTGCAAGCGGTAAAGCTAACGAGCTGCTGATCAACGTCAAGGCAAGCGGCAAATCTACCCAGAACGCAATCGACGAGATCACTAACAAAGCATCTGGTAAAATGAACTACTACTGGACCCTGGTCGAGCCGGGCGACAAAGCCAGCGGTAAGGAGCAGCTGTCTTCTGTGTCTTCCTTCGAGCGTGCATCTGGTAAGATGAACACCCAGTTCACGGCAGTGGGTAAAGCATCCGGCAAGACTTTCTTCCTGACTCAGGGTGCACTGCTGAACGATAAAGCTTCCGGTAAATACAACGGCATCATCACGGACACCATCAAAGCGTCTGGCAAGTACGGCAACGGTGTGTGGATCGGTCGTGCTTCCGGTAAGGGTGATGTTTTCGTGATCCGTGCATCTGGCAAATCCACTCAGGCAGCAATCGACCAAATCAACGGTAAGGCTTCCGGCAAGATCGACCTGTGGTCTTACAACGCTGAACTGCTGGTTGCTCTGGAAAACCAGCATACTATCGACCTGACCGACTCTGAAATGAACAAAGCCTCCGGTAAAGAATTCTCCGAAGTGGAAGGCCGCGCGTCTGGTAAATGGGACCTGTTTGTGGAACGCGCCTCTGGTAAGACTCTGCTGATGAACGAACTGGGTGTGCCATTTCACCTGGGTACGAAAGCATCTGGCAAGCTGGTGGATTCTGTGGTATCCTGGTCTAAAGCCTCTGGTAAAGTTATCGAAGGCTGGTCCAACCCGAAAGCCAGCGGCAAGATCCTGTTTATTGAAGAAGGTAAAGCGTCCGGTAAACTGTCCGGCGCGATGGACGAACTGCACAACGAAATTCTGGAACTGGATGAAAAAGCTTCTGGCAAACTGTCCACCCACAACGTTATTAACGCGGAAAACGCCCCGGGCGGCCCGTACAAAGCTAGCGGTAAAATCGTTGTTGATTATATGGTTCAGAAAGCTTCCGGCAAAAACCTGAACAGCCTGTCCGAACTGGAAGTTAAAGCCTCCGGCAAAGGCGTTACCCTGCTGCTGCCGGAACCGGAATGGACCTACCCGCGTGCCAGCGGCAAACTGAATGTTGAAACCGATACCGCTGAAATTCGCGCCAGCGGTAAATATGGCGAAGCTTATACCGATACCTATCACTCCTATGCGAACAAAGCGAGCGGCAAAGAATGGACCTATATTGGCGTAGATGGCCCGGATAACAACGCGCTGCTGAAAGCCTCTGGCAAAGGCGGCCTGGAACCGATTAATTTTCAGACCGCGGCTGATCAGGCTCGTGCGAGCGGTAAAATTTCTCAGGCGGTTCATGCGGCGCACGCGGAAATTAATGAAGCGGGCCGTGCGTCTGGCAAACTGACCGAATGGACGTCTTCTAATGTTATGGAAGAACGCGCATCCGGCAAAAATGTTCTGCAACCGTCTAGCGTTGATTCCCAGACCGCGATGGTTCTGGTTAATGCGATTGTTTTCAAAGCGTCCGGCAAATAAGGATCC.2.1.4CATATGGCTGGTCGTGCGTCTGGTAAACTGGGTGAGTACGGTTTTCAGAACGCGCTGATCGTACGTGCGTCTGGTAAAGTAGTCGGTCTGTCTACCCTCCCGGAGATCTACGAAAAAGCGTCCGGTAAAGAGGTCCTGGTTCTGTGGGGTATTCACCACCCGTCTACTTCTGCAGATCAGCAGTCTCTGTACCAGAACGCAGACGCTTACGTATTCGTTGGCTCTTCTCGTGCGTCTGGCAAAATCGACCTGTGGTCTTACAACGCCGAACTGCTGGTTGCCCTGGAAAACCAGCACACTATCGACCTGACCGACTCCGAAATGAACAAAGCGTCCGGTAAACTGTCTGGTGCGATGGACGAACTGCACAACGAAATCCTGGAACTGGACGAGAAAGCCAGCGGTAAAACCTTCTTCCTGACTCAGGGTGCGCTGCTGAACGACAAAGCTTCTGGCAAAACCCTGCTGATGAACGAACTGGGTGTTCCGTTTCACCTCGGTACCAAAGCGTCTGGTAAAGGTGTTACCCTGCTGCTGCCGGAACCGGAATGGACTTATCCACGTGCCTCTGGTAAAGGTGGTCTGGAACCGATCAACTTTCAGACGGCCGCAGATCAGGCACGTGCTTCTGGTAAAATCTCTCAGGCTGTTCACGCCGCGCACGCAGAAATCAACGAAGCAGGTCGTGCTTCTGGCAAACTGAACGTTGAAACCGACACCGCGGAAATCCGTGCCTCTGGTAAACTGGTTGACAGCGTTGTTTCTTGGTCCAAAGCGTCCGGTAAATACAACGGTATCATCACCGACACCATCAAAGCCTCTGGTAAATTCACCTCCTCTGCCAACGGTGTTACGACCCACTACGTATCTCAGATCGGTGGTTTCCCGGATCAGACCGAAGACGGTGGTCTGCCGCAGTCTGGTCGTGCTTCTGGTAAATCTACCCAGGCGGCGATTGACCAGATCAACGGTAAAGCGTCCGGCAAATCTACGCAGAACGCGATCGACGAGATCACCAACAAAGCCTCTGGTAAACTGCCGCTGGTAGGTGGTCACGAAGGTGCAGGTGTTGTAGTGGGTATGGGTGAGAACGTGAAAGCGAGCGGTAAACTGGTTAACGAACTGACGGAGTTCGCCAAAGCCTCTGGTAAAAACCTGAACAGCCTCAGCGAACTGGAAGTGAAAGCCTCTGGCAAATACGGTAACGGTGTTTGGATCGGTCGTGCTAGCGGTAAATCTGGTTACAGCGGTATCTTCTCTGTTGAGGGTAAAGCCTCCGGTAAATACGGTGAAGCCTACACGGATACCTACCACTCTTACGCCAAAGCGAGCGGTAAACTGACCGAATGGACGAGCTCTAACGTTATGGAGGAACGTGCCTCTGGTAAAGACGCTTTCCTGGGCTCCTTCCTGTACGAATACTCTCGTGCGTCTGGTAAATCTATCTCTATCGTCGGCTCCTACGTAGGTAACCGTGCGTCTGGTAAAATGAACTACTACTGGACCCTGGTTGAACCGGGTGACAAAGCTTCCGGTAAATCTCAGCAGGCGGTTATCCCGAACATCGGTTTTCGTCCACGTGCTTCTGGCAAACTGAACTGGCTGACCCACCTGAACTTCAAAGCCTCCGGTAAAATGAACACCCAGTTCACGGCGGTTGGTAAAGCGTCTGGTAAAGCCAACGAACTGCTGATCAACGTGAAAGCCTCCGGTAAACACCTCGTAGACGAACCGCAGAACCTGATCAAAGCGTCTGGCAAAAACGTACTCCAGCCGTCTTCTGTTGACTCTCAGACCGCTATGGTTCTGGTCAACGCGATCGTATTCAAAGCCAGCGGTAAAGGTAACTCTGCCCCGCTGATCATTCGTGCCTCTGGTAAAGGTTGGGCTTTTGACGACGGTAACGACGTTTGGATGGGTCGTGCGTCTGGTAAAGGCGACGTATTCGTTATCCGTGCGTCTGGTAAAGCGGACACCATCTCTTCTCAGATCGAACTGGCCGTTCTGCTGTCTAACGAGGGTATCATCAACTCCGAAGACGAGCACCTGCTGGCTCTGGAACGTGCCTCTGGTAAACTGGCAGCGGCCCTGGAACACCACCACCACCACCACTAATAGGGATCC.2.22.2.12.2.2FASTA Protein Database used: 2015_2016_FluQuant.fasta"}
+{"text": "The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an  Transcriptional bursting is a potential source of cell-to-cell variability but the molecular mechanisms are unclear. Here the authors use single molecule imaging to analyse the kinetics of bursting on DNA and observe that bursting is an intrinsic property of RNA polymerases on DNA. Our results show transcriptional bursting occurs with the minimum components of bacterial transcription, suggesting an intrinsic molecular mechanism of transcriptional bursting. Based on the kinetic analysis, we propose that transcriptional bursting is intrinsically caused by interplay between RNA polymerases (RNAPs) on DNA. Our analysis can quantitatively explain the gene-nonspecific kinetics previously observed in noisy gene expression \u22121 at our experimental conditions , to correct for the biased transcription rate for each nucleotide speciesin vitro transcription system -labelled RNAP holoenzyme and a DNA template containing a lacUV5 promoter and single- or six-target sites for the oligo probe (NTP] (in vitro single-molecule measurement (13kA) is \u223c0.2\u2009s\u22121 (in vitro single-molecule measurements originated from an arrest state. This was supported by atomic force microscopy (AFM) images of transcription elongation as well  or RNAP enters an arrest state at a rate of kA/(kF+kA). In this model, one simulation time is consistent with 1/kF=100\u2009ms. Arrest state is rescued by pushing of a trailing RNAP (collision) at a rate of kR. When RNAP moves from the 71th and 72th box, the 57th box of the nascent mRNA is \u2018exposed' and the mRNA is \u2018visualized' and counted , we performed a simulation based on a cellular automaton model 1617. In 16.045\u2009s\u22121 . The treectively . On the ectively . Furtheron model  as kR iskA) varies by a few orders of magnitude depending on measurement conditions not only in this study (kA=0.00005\u223c0.2\u2009s\u22121), but also in the previous study , is consistent with the suggested kinetic scheme under the gene-nonspecific constraint, in which only koff (10\u22125\u223c102\u2009s\u22121) is varied to change the gene expression level10kI). The kI is a critical parameter to control not only the mean of mRNA distribution but also the Fano factor as our experimental data suggest.To explain the discrepancy of the Hill coefficients between experimental data 1.9) and our model (1.0), however, we may need other sources of cooperativity such as indirect interplay via DNA stress generated by RNAP. As previously reported, RNAP generates local torsional stress on DNA, which is sufficiently long-lived to untwist the upstream region even on linear DNA is koff , was lar and our in vivo. In such models, where the regulatory proteins turn on the gene, the koff corresponds to their unbinding rate, which is generally constant as long as the binding sequence is unchanged, whereas in fact koff is largely changed in a gene-nonspecific mannerkoff in the previous models must be gene specific, which is contrary to the global feature of transcriptional bursting observed in vivo. On the other hand, to explain the gene-specific feature of transcriptional bursting as observed in yeastAt the same time, we note that models where the binding and unbinding of regulatory factors such as gyrase, and possibly enhancers in eukaryotic cells, cannot by themselves fully explain the gene-nonspecific kinetics of transcriptional bursting observed in vitro data, especially the trend between Fano factor and mean. However, we cannot fully rule out such effects by our in vitro experiments alone. Specifically, the sequence dependence of arrest rate during elongation was not fully examined, because our in vitro experiment .5\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccIn the above sequence, lower case letters indicate the T7A1 promoter region (\u2212163 to +38 from the transcription start site represented by the bold typed a). An underline indicates the oligo probe-binding site. The template sequence from the oligo probe-binding site to the 3\u2032-end (downstream region) is the same as in the original report5\u2032-CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-/5Bio/G/Cy5/CCGGATAAAACTTGTGC-3\u2032 (synthesized by Integrated DNA Technologies).See In the Qdot tracking experiment with fastFISH , we usedaattgtgagcggataacaatttcacacaggaaacagctatggaccgcaagcttCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCCCCTATCCCTTATCTTAACCACTCCAATTACATACACCAGCTTCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACCACCGTTGATATATCCCAATGGCTGCAGCTG GATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGC-3\u2032.Six-target template (641\u2009bp): 5\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAtaatgcagctggcacgacaggtttctatgcttccggctcgtataatgtgtgglacUV5 promoter region (\u2212131 to +53 from the transcription start site represented by the bold typed a). Six underlines indicate the oligo probe-binding sites.In the above sequence, lower case letters indicate the Single-target template (517\u2009bp):aattgtgagcggataacaatttcacacaggaaacagctatgCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACCACCGTTGATATATCCCAATGGCTGCATTTCAAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTTCAAACTGCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGC-3\u20325\u2032-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAtaatgcagctggcacgacaggtttcccgactggaaagcgggcagtga gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtata atgtgtgglacUV5 promoter region (\u2212131 to +41 from the transcription start site represented by the bold typed a). Single underline indicates the oligo probe-binding sites. We prepared the templates for biotinylation at the both ends by PCR withIn the above sequence, lower case letters indicate the 5\u2032-/5Bio/CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-/5Bio/GCCGGATAAAACTTGTGC-3\u2032.See lacZ (1-1179 when 1 indicates the first nucleotide of the gene)  . The ups5\u2032-CCACAACGGTTTCCCTCTAG-3\u2032 and5\u2032-ATAATGCGAACAGCGCAC-3\u2032.PCR-amplified DNA fragments were purified by Wizard PCR Preps DNA Purification System (Promega). The sequence of the DNA template region in plasmids was verified by sequencing. Except as otherwise noted, the primers were synthesized by Hokkaido System Science Co., Ltd, Japan.Escherichia coli RNAP holoenzyme purchased from New England Biolabs. For the Qdot-tracking experiment, we designed the plasmid for co-overexpression of tagged E. coli RNAP subunitsrpoA-rpoB-rpoC-rpoZ between the NdeI and HindIII sites in pT7-7 by In-Fusion Cloning Kit (Clontech). The genes were provided by NBRP-E. coli at NIG . HaloTag  and His-tag fragments were attached to the 3\u2032-end of rpoC. The Plasmid was transformed into BL21 \u03bbDE3. A single colony was inoculated into 1\u2009litre of Luria-Bertani liquid medium (LB) containing 100\u2009\u03bcg\u2009ml\u22121 ampicillin at 37\u2009\u00b0C until OD600 reached 0.3\u20130.5. The protein production was induced by the addition of isopropyl-\u03b2-D-thiogalactoside to 0.2\u2009mM and cells were grown overnight at 23\u2009\u00b0C. Cells were collected by centrifugation , rinsed with PBS and stored at \u221280\u2009\u00b0C. To proceed with protein purification, pellets were resuspended in 80\u2009ml lysis buffer  and protease inhibitor (Roche) was added. Cells were disrupted by sonication, cleared by centrifugation  and filtrated by the 0.22\u2009\u03bcm syringe filter (Millipore). The protein was purified by using 5\u2009ml HiTrap Heparin HP, 5\u2009ml HisTrap HP and Mono Q 5/50 GL, all purchased from GE HealthcarerpoD (\u03c370) gene was inserted in pT7-7 with amino terminus His tag. The Plasmid was transformed into BL21 \u03bbDE3. Cells were collected and disrupted as described above. The protein was purified by using TALON Metal Affinity Resin (Clontech) according to the product manual. The buffer of the eluted \u03c370 was changed to the storage buffer through a 50\u2009kDa MWCO filter  and stored at \u221280\u2009\u00b0C.For the fastFISH and AFM imaging experiment, we used The fluorescent images were recorded by an Olympus IX71 inverted microscope. In the fastFISH experiment, illumination was provided by 532 and 640\u2009nm laser light (Coherent). The lasers were combined into one fibre output by OBIS Galaxy (Coherent). The laser light was expanded to a diameter of 8\u2009mm and focused by a 400\u2009mm focal-length lens into the back focal plane of the objective . The fluorescent photons were collected with a electron-multiplying charge-coupled device (CCD) camera . The effective pixel size was 74\u2009nm. The laser was steered by a piezo mirror (Physik Instrumente) and reflected by a dichroic mirror  just below the objective lens. A dual-view apparatus (Hamamatsu Photonics) equipped with dichroic mirrors (Asahi Spectra) and emission filters  was put in the Cy3 channel. In the Qdot-imaging experiment with fastFISH, illumination was provided by 488 and 532\u2009nm laser light (Coherent). Other optics were the same as described above. To maintain the temperature in the chamber, we warmed the objective by a lens heater .\u22121 PEG mixture solution (NHS-PEG-biotin and NHS-PEG were mixed at the ratio of 1:200 and dissolved in 0.45\u2009M K2SO4 and 0.1\u2009M NaHCO3 pH 9.0) was squeezed between two coverslips and incubated for 30\u2009min at 30\u2009\u00b0C. The NHS-PEG-biotin  and NHS-PEG  were purchased from NOF Corporation, Japan3) for 10\u2009min at room temperatureThe fluorescence imaging experiments were performed inside a sample chamber assembled with a PEG-coated glass. The PEG-coated glass was prepared as follows. Coverslips were cleaned by low-pressure plasma for 5\u2009min with a plasma system . The coverslips were then placed into a freshly prepared 3% solution of N-2-(aminoethyl)-3-aminopropyl-trimethoxysilane  in acetone for 45\u2009min with gentle shaking. The amine-modified coverslips were then rinsed with MilliQ and dried by an air blower. A 20\u2009\u03bcl drops of 200\u2009mg\u2009mlin vitro transcription assays were performed in transcription buffer  at the nucleotide concentration of 100[NTP]  to correct for the biased transcription rate for nucleotide species\u22121 avidin solution was added into a sample chamber and incubated for 3\u2009min. After washing the chamber by transcription buffer, 1\u20132\u2009nM DNA template was added and washed again by transcription buffer. In fastFISH experiment . The fastFISH experiment was performed in 50\u2009nM self-quenched oligo probe, in which a hybridization rate is 0.2\u2009s\u22121 at 25\u2009\u00b0C . All single-molecule observations were performed within 30\u2009min. At least, the data within 30\u2009min have shown no time dependency.All periment  and 4, sat 25\u2009\u00b0C . In Qdotat 25\u2009\u00b0C , a DNA t2 for 5\u2009min. The mica was then rinsed with MilliQ and dried by an air blower. Before acquisition of AFM images, in vitro transcription was performed at the concentration of 10\u201320\u2009nM DNA and 100\u2009nM RNAP in transcription buffer for 30\u2009min at room temperature. The reaction solution was diluted to 1\u20132\u2009nM DNA in 100[NTP] deposition buffer  and added to freshly cleaved mica. After incubation for 2\u2009min, the mica was rinsed with MilliQ and dried by an air blower. AFM imaging was performed with a NanoWizard 3  operating in AC mode. Silicon cantilevers (OMCL-AC160TS) were purchased from Olympus. Images of 512 \u00d7 512 pixels were acquired with a scan size of <3\u2009\u03bcm at a scan rate of two lines per second.AFM images were acquired on mica in air. The mica was pretreated by incubation of 10\u2009mM MgClImages of 512 \u00d7 512 pixels were acquired by commercial software  at 10\u2009Hz acquisition rate. In the fastFISH experiment, the positions of Cy5-labelled DNA and Qdot-labelled RNAP were decided by using a two-dimensional Gaussian distribution fitting3132The plots in m is the mean of mRNA number distribution, mmin and mmax are its minimum and maximum, respectively, kI is transcription initiation rate, khalf is the rate when m equals (mmax\u2212mmin)/2 and n is the Hill coefficient.Here, mmin=0.3\u00b10.1, mmax=3.9\u00b10.1, khalf=0.017\u00b10.002 and n =1.9\u00b10.2. The simulated data .The experimental data .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-8, Supplementary Table 1 and Supplementary ReferencesA stochastic cellular automaton model of transcriptionA stochastic cellular automaton model with mRNA degradation"}
+{"text": "AbstractSerenotheres Ahyong & Ng, 2005 (Pinnotheridae) is currently only represented by one species, Serenotheresbesutensis . A new species is now assigned to this genus, described from a date mussel Leiosolenusobesus Carpenter, 1857  collected in the Solomon Islands. Serenotheresjanussp. n. differs from Serenotheresbesutensis in possessing a conspicuously broader carapace, with the lateral margins of the dorsal lamellum distinctly produced and the posterolateral part deeply concave, the dorsal lamellum being highest at the median cleft, the rostrum is relatively more prominent, the surfaces of the anterolateral margin and hepatic region are less prominently pitted and eroded, the ischiomerus of the third maxilliped is relatively more rectangular, and the P2 merus is proportionately longer.The pea crab genus  Durckheimia De Man, 1889  and Xanthasia White, 1846 , and established two new genera, namely Serenotheres Ahyong & Ng, 2005, for Durckheimiabesutensis Ser\u00e8ne, 1967; and Tridacnatheres Ahyong & Ng, 2005, for Xanthasiawhitei De Man, 1888. They commented that Serenotheres differed from all pinnotherid genera not only by the unusual carapace which has an additional large plate above its normal carapace surface  which overhangs the frontal margin, but also by possessing a two-segmented third maxilliped palp , Smithsonian Institution, Washington D.C.The specimen examined is deposited in the  = third maxillipedMXP3;  = pereiopods 2\u20135P2\u2013P5 (first to fourth ambulatory legs), respectively. Measurements (in millimetres) are of the carapace width and length, respectively. The terminology used essentially follows that in The following abbreviations are used: PageBreakCATACTATTAACAGATCGTAATCTAAATACCTCATTCTTTGACCCAGCCGGTGGTGGAGATCCTGTTCTCTATCAACATTTATTTACCCTTATATTTTATCTTCGGAGCTTGGGCAGGTATAGTAGGAACTTCTTTAAGTTTAATAATTCGAGCTGAACTTAGACAACCAGGCAGACTTATTGGAAATGACCAAATTTATAATGTAATAGTTACAGCCCATGCTTTTGTTATAATTTTCTTTATAGTTATACCAATTATAATCGGAGGCTTCGGAAACTGATTAGTTCCTTTAATACTTGGGGCCCCAGATATAGCATTCCCTCGTATAAACAATATAAGATTTTGACTCTTACCTCCATCTTTATCACTCTTACTTACAAGAAGAATAGTTGAAAGTGGAGTAGGAACAGGATGAACTGTTTATCCTCCTCTAGCTTCAGCTATTGCCCATGCTGGAGCTTCTGTAGATTTAGGAATTTTCTCGCTTCATTTGGCCGGTGTATCGTCAATCTTAGGAGCAGTAAATTTTATTACTACTGTAATTAATATACGATCATATGGAATAATGATAGACCAAATACCACTATTTGTCTGATCAGTATTTATCACCGCAATCCTCCTACTTCTATCCCTACCGGTTCTAGCAGGAGCTATTAC. This record is deposited in Genbank under submission number KX949585.A mtDNA COI barcode was generated from this individual following standard Sanger sequencing protocols as outlined in Taxon classificationAnimaliaDecapodaPinnotheridaehttp://zoobank.org/FD849337-EB57-46F6-965D-8CECBADECD5FUSNM 1421642), in Leiosolenusobesus  , from Njari Island, New Georgia, Solomon Islands, station SOLOM_026; 8.01374\u00b0S, 156.75649\u00b0E, coll. C. Meyer, 9 October, 2014.Holotype \u2640 (8.9 \u00d7 7.9 mm)  large, subrectangular; articles 3\u20135 increasingly smaller, with flagellum very short, not extending beyond orbit  symmetrical from left to right, generally similar in form; margins of carpus and propodus lined with dense, long setae, setation on merus less distinct and more spare; dactylus covered with sparse long setae Figs ; relativMXP3  in having the lateral margins of the dorsal carapace lamellum distinctly produced laterally to form a blunt angular lobe, with the posterolateral margin deeply concave  (cf. Ng and Ahyong 2005) is similar to that of the holotype of Serenotheresjanus sp. n. (8.9 \u00d7 7.9 mm), so the differences observed cannot be explained by size.The type of PageBreakSerenotheresjanus sp. n. indicates a novel lineage among available Pinnotheridae sequences. The closest matches are 86\u201385% in sequence similarity to a handful of other pinnotherid genera including Zaops, Calyptraeotheres, Austinotheres, and Pinnixa   Fig. . The fun"}
+{"text": "Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue\u2019s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites. Post-translational modifications (PTMs) play a critical role in regulating cellular function and response to external stimuli. PTMs, which include phosphorylation, glycosylation, and acetylation, are found in all cell types and lineages and thus likely arose early in evolution. Advances in mass spectrometry-based proteomics have greatly expanded the number of proteins that are known to be modified, but our ability to assign a functional role to specific modifications has lagged. Although acetylation has a well-known role in chromatin remodeling , it is n4\u2013In this paper, we investigated whether lysine acetylation is found on different enzymes and whether this modification is conserved throughout bacterial evolution by performing a comprehensive phyloproteomic analysis. We found that acetylation occurs in conserved lysine residues located in catalytic pockets of enzymes from the glycolytic pathway and the TCA cycle. Moreover, those modifications substantially changed the physical-chemical properties of the enzyme catalytic pocket inhibiting their activity.Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Cyanobacteria, and Fibrobacteres. Each bacterial strain was grown to early stationary phase  database. Acetylated proteins were significantly enriched in glycolysis, pyruvate metabolism, ribosomes, and the TCA cycle, with median enrichment rganisms . AlthougDue to our interest in the mechanism of enzymatic regulation by acetylation, we narrowed our investigation to specific sites of acetylation and in particular those that are conserved across evolution. We focused on the set of enzymes involved in glycolysis and the TCA cycle and asked whether these enzymes contained lysines involved in substrate or cofactor binding and whether those lysines were identified as acetylated in diverse taxa, potentially pointing to a general regulatory mechanism common to all organisms. In glycolysis, there are nine enzymatic reactions for the conversion of glucose-6-phosphate to pyruvate. Seven of the nine enzymes contain catalytically important lysine residues in the active site . In four8\u2013We further investigated TCA cycle enzymes for catalytically essential and acetylated lysines. The transition from glycolysis (pyruvate) to the TCA cycle (acetyl-CoA) (part of the pyruvate metabolism pathway) involves a set of four enzymes that control the formation and therefore the pool of available acetyl-CoA. Three of the four enzymes contain lysine residues that participate in catalysis through substrate/cofactor binding. In both Acs  and phos10.1128/mBio.01894-17.3TABLE\u00a0S3\u00a0TABLE\u00a0S3, XLS file, 0.03 MB.Lysine acetylation on conserved residues in enzyme catalytic regions. For enzymes in the glycolytic and TCA pathways, conserved sites of acetylation are listed. Each site is described by the gene, KEGG ortholog, protein accession number of one reference organism, acetylated sites, and the surrounding protein sequence. Acetylated sites are highlighted in yellow if they are known to bind substrate/cofactors. Download Copyright \u00a9 2017 Nakayasu et al.2017Nakayasu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus\u00a0elongatus enolase (Protein Data Bank [PDB] accession number 5J04) which contains the native substrate (phosphoenolpyruvate [PEP]), the two active site lysines are 2.4 and 2.8\u00a0\u00c5 from oxygen atoms in the substrate . The volume of the empty active site pocket was 162\u00a0\u00c53; with bound PEP, the volume was 70.2\u00a0\u00c53. After removing PEP and building acetyl groups on the K339 and K390 side chain amines, the volume of the pocket was 78.7\u00a0\u00c53. Thus, the acetylated lysines and the native substrate occupy a similar volume and position within the deeply buried active site  were kept; all other nonconserved lysines were permuted  with invariant lysines within their substrate binding site, whose acetylation was conserved and significantly upregulated in the presence of supplemented glucose , the lysine residues were conserved, suggesting that the use of lysine acetylation as a mechanism to control central metabolic activity is an ancient trait. We also note that acetylphosphate has been proposed to be the most primitive acetyl donor (before evolution of coenzyme A) and likely would have been present at the outset of the evolution of fundamental pathways, such as glycolysis and the TCA cycle  and centrifuging under the same conditions. Cells were resuspended with 200\u00a0\u03bcl of 100\u00a0mM NH4HCO3 and lysed by a Bullet Blender (Next Advance) for 4\u00a0min at speed 8 in the presence of approximately 100\u00a0\u03bcl of 0.1-mm zirconia-silica beads at 4\u00b0C. Cell lysates were collected into fresh tubes, while the beads were washed with 200\u00a0\u03bcl of 100\u00a0mM NH4HCO3, and the supernatants were pooled together. The protein concentration was quantified by bicinchoninic acid (BCA) assay , and aliquots of 300\u00a0\u03bcg of proteins were denatured and reduced by adding 8 M urea and 5\u00a0mM dithiothreitol (DTT) and incubating at 60\u00b0C for 30\u00a0min with shaking at 850\u00a0rpm. The reaction mixtures were then diluted 10-fold in 100\u00a0mM NH4HCO3, and 1\u00a0M CaCl2 was added to a final concentration of 1\u00a0mM. Protein digestion was carried out for 3\u00a0h at 37\u00b0C with a 1/50 trypsin-protein ratio. Digested peptides were desalted in 50-mg C18 cartridges  as previously described  database (see \u201cData availability\u201d). Raw mass spectrometry files were converted to the PSI open format mzML . We tracked the functional enrichment for all pathways for all organisms. The data presented are the median enrichment of pathways across all organisms.All peptide spectrum matches were filtered for false-discovery rate using ostKoala . Figure\u00a0https://github.com/samuelpayne/TreeFigureForBiodiversityLibrary, utilizing the ETE 3 toolkit for phylogenomic data . Multiple-sequence alignments were performed using Muscle  via a wemic data . This trmic data  or the amic data . The annhttps://www.wwpdb.org/ website. The structures visualized in http://www.pymol.org). The enolase active site (PDB 5J04 structure) was analyzed with NACCESS 2.1.1 (5J04 structure have relative side chain atom accessibilities ranging from 0.6% to 94%. The distance from K390 N\u03b5 to phosphoenolpyruvate (PEP) O1\u2032 (one of the carboxylate O) is 2.4\u00a0\u00c5, while the distance from K339 N\u03b5 to PEP O2P (one of the phosphate O) is 2.8\u00a0\u00c5. The volume of the empty active site pocket in the PDB 5J04 structure was 162\u00a0\u00c53; with bound PEP, the volume was 70.2\u00a0\u00c53. After removing PEP and building acetyl groups on the K339 and K390 side chain amines, the volume of the pocket was 78.7\u00a0\u00c53.Protein structures from the Protein Data Bank (PDB) were identified via their UniProt accession number or manual search for a protein\u2019s functional annotation using the SS 2.1.1  and CASTSS 2.1.1  to gaugeLactobacillus\u00a0casei maps to K100 of E.\u00a0coli. For Eno, K339 from B.\u00a0subtilis maps to K342 of E.\u00a0coli. For Pgk, K201 of B.\u00a0subtilis maps to K197 of E.\u00a0coli. For LpdA, K56 of B.\u00a0subtilis maps to K54 of E.\u00a0coli. For Tpi, K11 of B.\u00a0subtilis maps to K11 of E.\u00a0coli. For Icd, K226 of B.\u00a0subtilis maps to K235 of E.\u00a0coli. Similar mapping was done for the sites identified by Kuhn et al.  and MP043 (TGA CAA GCT TAG ATA GCG GCG GAT TTA GC). These primers were designed to remove the start codon and add a 5\u2032 NheI site and a 3\u2032 HindIII site. The B.\u00a0subtilis enolase was PCR amplified from B.\u00a0subtilis strain OI3269 using primers MP071 (GTC GGC TAG CCC ATA CAT TGT TGA TGT TTA TGC AC) and MP072 (TGA CCT CGA GGG TAA GGC TTT ATT TGG ATC TCT G). These primers were designed to remove the start codon and add a 5\u2032 NheI site and a 3\u2032 XhoI site. The mutant E.\u00a0coli 7xKtoQ enolase was made by ordering a gBlock from IDT (sequence below) in which lysines at positions 6, 62, 82, 85, 257, 328, and 419 were mutated to glutamines by changing the codons from AAA to CAA. The gBlock contained a 5\u2032 NheI site and a 3\u2032 HindIII site. The respective DNA fragment and the pET28a plasmid were then digested using the respective restriction enzymes, gel purified, and ligated together. The resulting plasmid constructs were then transformed in E.\u00a0coli BL21(DE3).Both the E.\u00a0coli 7xKtoQ enolase sequence (gBlock) follows (restriction sites and mutations are shown by lowercase letters): GTCGgctagcTCCAAAATCGTAcaaATCATCGGTCGTGAAATCATCGACTCCCGTGGTAACCCGACTGTTGAAGCCGAAGTACATCTGGAGGGTGGTTTCGTCGGTATGGCAGCTGCTCCGTCAGGTGCTTCTACTGGTTCCCGTGAAGCTCTGGAACTGCGCGATGGCGACAAATCCCGTTTCCTGGGTcaaGGCGTAACCAAAGCTGTTGCTGCGGTAAACGGCCCGATCGCTCAGGCGCTGATTGGCcaaGATGCTcaaGATCAGGCTGGCATTGACAAGATCATGATCGACCTGGACGGCACCGAAAACAAATCCAAATTCGGCGCGAACGCAATCCTGGCTGTATCTCTGGCTAACGCCAAAGCTGCTGCAGCTGCTAAAGGTATGCCGCTGTACGAGCACATCGCTGAACTGAACGGTACTCCGGGCAAATACTCTATGCCGGTTCCGATGATGAACATCATCAACGGTGGTGAGCACGCTGACAACAACGTTGATATCCAGGAATTCATGATTCAGCCGGTTGGCGCGAAAACTGTGAAAGAAGCCATCCGCATGGGTTCTGAAGTTTTCCATCACCTGGCAAAAGTTCTGAAAGCGAAAGGCATGAACACTGCTGTTGGTGACGAAGGTGGCTATGCGCCGAACCTGGGTTCCAACGCTGAAGCTCTGGCTGTTATCGCTGAAGCTGTTAAAGCTGCTGGTTATGAACTGGGCAAAGACATCACTTTGGCGATGGACTGCGCAGCTTCTGAATTCTACAAAGATGGTcaaTACGTTCTGGCTGGCGAAGGCAACAAAGCGTTCACCTCTGAAGAATTCACTCACTTCCTGGAAGAACTGACCAAACAGTACCCGATCGTTTCTATCGAAGACGGTCTGGACGAATCTGACTGGGACGGTTTCGCATACCAGACCAAAGTTCTGGGCGACAAAATCCAGCTGGTTGGTGACGACCTGTTCGTAACCAACACCAAGATCCTGcaaGAAGGTATCGAAAAAGGTATCGCTAACTCCATCCTGATCAAATTCAACCAGATCGGTTCTCTGACCGAAACTCTGGCTGCAATCAAGATGGCGAAAGATGCTGGCTACACTGCAGTTATCTCTCACCGTTCTGGCGAAACTGAAGACGCTACCATCGCTGACCTGGCTGTTGGTACTGCTGCAGGCCAGATCAAAACTGGTTCTATGAGCCGTTCTGACCGTGTTGCTAAATACAACCAGCTGATTCGTATCGAAGAAGCTCTGGGCGAAcaaGCACCGTACAACGGTCGTAAAGAGATCAAAGGCCAGGCATAAaagcttGTCA.The mutant 2, 0.1\u00a0mM dithiothreitol [DTT], 10% [vol/vol] glycerol) and then stored at \u221280\u00b0C.All enolase constructs were purified on a HisTrap column  on an \u00c4KTA pure chromatography system  using the denaturing protocol provided by the manufacturer. Fractions were collected and run on an SDS-polyacrylamide gel to check for the elution of the desired 6\u00d7His fusion protein. The pure-protein flowthrough was collected and dialyzed against four changes of 1\u00a0liter of TKMD , 10% glycerol, 10\u00a0mM MgCld-2-phosphoglycerate is converted to PEP, resulting in the formation of an intermediate that reacts with a peroxidase substrate, generating a colorimetric (570-nm) product proportional to the enolase activity present. The assay was repeated on three separate days using four dilutions of each enolase construct in acetylated and nonacetylated form to determine enolase activity.Enolase activity was measured using an enolase activity assay kit (Sigma-Aldrich). Briefly, this kit works via a coupled enzyme assay in which https://github.com/ or https://sourceforge.net/. Data format conversion and calibration were performed using msconvert, part of the proteowizard project (source code at http://proteowizard.sourceforge.net/). MSGF+ was used to annotate tandem mass spectral identification (source code at https://github.com/sangtaekim/msgfplus and webserver at https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp). Postprocessing scripts as described above are available at https://github.com/samuelpayne/Biodiversity.Acetylation.Supplement.Coverage and https://github.com/samuelpayne/TreeFigureForBiodiversityLibrary.All data processing tools are publicly available at PXD005851.All primary mass spectrometry files and identification results are freely available at the proteomic data repository PRIDE under accession no."}
+{"text": "The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions.Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different  Plasmodium genus, resulted in at least 438,000 deaths worldwide in 2014 [Malaria, the mosquito-borne parasitic disease caused by members of the One of the most appealing, yet elusive, malaria vaccine candidates is one targeting the asexual blood stage, the clinical stage of the disease. Issues such as antigen polymorphism, lack of MHC I molecules on erythrocytes, and speed of erythrocyte infection all hamper the development of an asexual blood stage vaccine \u20139. Many P. falciparum in antibody-dependent cellular inhibition parasite-growth assay [In one such bioinformatic approach coiled-coil domains present in the asexual blood stage were targeted . Coiled-th assay . CurrentSubunit vaccines based solely on recombinant proteins are generally weakly immunogenic and will most likely not be successful for vaccination in humans. To be an effective vaccine an important consideration is the delivery system. Several recent effective vaccine candidates are based on particulate delivery systems that are able to repetitively express antigens . One proNormally, the sequence of amino acids that form the pentameric and trimeric oligomerization domains are de novo designed sequences that do not have homology to any human proteins to minimize the possibility of inducing an immune response that could be detrimental to the host receiving the vaccine. Here we detail the unusual step in the development of a SAPN vaccine candidate by using a parasite native coiled-coil sequence, the P27 epitope from the Tex1 protein, to form part of the core oligomerization domains. In combination with the intrinsically unstructured epitope P27A, this vaccine candidate is immunogenic in a murine model and induces antibodies that are recognizing the same antigens as sera from seropositive individuals in Burkina Faso, a malaria endemic region.Pept-P27-N (845\u2013871):KKRNVEEELHSLRKNYNIINEEIEEITPept-P27A (223\u2013326):HNNNEKNISYDKNLVKQENDNKDEARGNDNMCGNYDIHNERGEMLDKGKSYSGDEKINTSDN AKSCSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSPept-P27-NC (846\u2013893):KRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMILQIKNKELEPept-P27-C (872\u2013893):KEFEKKQEQVDEMILQIKNKELEPept-P27-N, Pept-P27A, Pept-P27-NC and Pept-P27-C present in the SAPN scaffold were synthesized by solid-phase Fmoc chemistry using Applied Biosystems 431A and 433A synthesizers  as previously described [The single antigens escribed . PeptideSAPN-P27-1:MGHHHHHHASGSWEEWNAKWDEWIRAWVAWRAAWEKWKDDWAYWTLTWKYGELYSKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMIIQIKNKELESAPN-P27-2:MGHHHHHHASLIDYNKAALSKFKERGSWQTWNAKWDVWSNDWNAWRARWQAWVDDWAYWTLTWKYGELYSKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMIIQIKNKELESAPN-P27A-1:MGHHHHHHASGSWQTWNAKWDVWSNDWNAWRADWQAWVDDWAYWTLTWKYGELYSKLAEIERRVEANERALEELAQFVRALSMQAAELERRIEEIARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMSGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSSSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSSAPN-P27A-2:MGHHHHHHASYYIPHQSSLPGSWQTWNAKWDVWSNDWNAWRADWQAWVDDWAYWTLTWKYGELYSKLAEIERRVEANERALEELAQFVRALSMQAAELSEDITKYFRHILYISFYFILVNRARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMSGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSSSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSSAPN-Combo:MGDHHHHHHHHHHAAHAAHAAHAAHAAAARGHNNNEKNISYDKNLVKQENDNKDEARGNDNMCGNYDIHNERGEMLDKGKSYSGDEKINTSDNAKSCSGDEKVITSDNGKSYDYVKNESEEQEEKENMLNNKKRSASGSAKFVAAWTLKAAASGSWEEWNAKWDEWRNDQNDWREDWQAWRDDWAYWTLTWRYGELYSRLARIERRVEELRRLLQLIRHENRMVLQFVRALSMQARRLESKLRNVEEELHSLRKNYNIINEEIEEITKEFEKKQEQVDEMILQIKNKELEE. coli were synthesized by GeneScript USA Inc.  containing the sequence of the constructs SAPN-P27-1, SAPN-P27-2, SAPN-P27A-1, SAPN-P27A-2 and SAPN-Combo. Each gene was cloned into a pPEP-T vector. The resulting constructs were transformed into Tuner (DE3) Competent Cells .Codon optimized genes for 600 of 0.8 cultures were induced with 1\u00a0mM Isopropyl-\u03b2-d-thiogalactopyranoside (IPTG) . Cultures were grown for 4\u00a0h, pelleted at 4000\u00d7\u00a0g and stored at \u221280\u00a0\u00baC until use.Protein expression was carried out in Luria\u2013Bertani (LB) Broth . Briefly, starter cultures were grown for 16\u00a0h at 36\u00a0\u00b0C and inoculated at 1/100 into 1L of LB and grown at 37\u00a0\u00b0C. At an OD2PO4, 20\u00a0mM Tris base, 5\u00a0mM tris(2-carboxyethyl)phosphine, pH 8.0), lysed by sonication, and centrifuged at 30,500\u00d7\u00a0g for 25\u00a0min to clarify the lysate. Purification was carried out on an \u00c4KTApurifier 100  using a 5\u00a0mL HisTrap HP column . Briefly columns were equilibrated with Binding Buffer phosphine, pH 8.0), lysate injected, and then washed with five column volumes of binding buffer. Columns were then washed with 5 column volumes of high phosphate buffer phosphine, pH 8.0), binding buffer, isopropanol wash  to remove LPS [Cell pellets were thawed on ice and resuspended in Imidazole free buffer phosphine and resulting in refolded SAPN in 20\u00a0mM Tris base, 5% glycerol, pH 8.5.Refolding was monitored by dynamic light scattering using a Malvern Zetasizer Nano S  equipped with a 633-nm laser. The hydrodynamic diameter was determined at 25\u00a0\u00b0C. Each sample was run 5 times, and the average result taken.0.025\u00a0mg/mL of each sample was adsorbed onto carbon coated grids  that were subjected to a 10\u00a0s glow discharge, washed with distilled water three times, and negatively stained with 0.5% uranyl acetate . Electron micrographs were taken on an FEI Tecnai T12 Transmission Electron Microscope.Human plasma were collected during the malaria transmission season from adult donors living in Burkina Faso. Plasma from Swiss naive donors who had no malaria history were pooled and used as a negative control. Blood was taken by venipuncture into tubes containing EDTA according to the ethical clearance.BALB/c and C3H mice (5/group) were immunized with 10\u00a0\u03bcg of NP in PBS at the base of the tail  three tiPrevalence (in humans) and titers (in mice) were determined by ELISA; antigens were diluted in PBS and used to coat ELISA plates. Antigen concentration for coating the 96-well flat plates  was 1\u00a0\u03bcg/ml (50\u00a0\u03bcl) for peptides longer than 40 residues, and 5\u00a0\u03bcg/ml for peptides shorter or equal to 40 residues as previously described , 17. SecELISA plates were coated with the appropriate peptide as described above, washed and blocked. Serum samples at appropriate dilutions (at about 50\u201360% of maximum OD as determined by ELISA) were pre-incubated with appropriate competing synthetic peptides at increasing concentrations for 30\u00a0min at room temperature. 50\u00a0\u03bcl of the antibody-peptide mixture was added into the appropriate wells and incubated for 1\u00a0h at room temperature in a humid chamber. ELISA was then completed as described above.Plasmodium species  \u201323. We bEach pentameric coiled-coiled domain will self-assemble with four other pentameric coiled-coiled domains, while each trimeric coiled-coiled domain will self-assemble with two other trimeric coiled-coil domains. To satisfy all of the assembly requirements 15 monomers will self-assemble into a structure known as a least common multiple unit (LCM). In a standard icosahedral structure four LCMs can further self-assemble into a full SAPN structure, meaning that 60 monomers make up each SAPN. However, recent biophysical studies combined with an analysis using viral tiling theory has shown that the SAPNs can possibly also assemble into higher order assemblies containing multiples of 60 chains per particle . Each SAP. falciparum.Another advantage of the SAPN design is that within the SAPN core universal T-helper epitopes can be engineered that ideally lead to more effective immune response. These epitopes range from de novo designed sequences that are engineered to bind to a wide range of MHC II haplotypes to sequences from known infectious agents that have been established to result as strong T-helper epitopes \u201337. TogeSAPN-P27-1, SAPN-P27-2, SAPN-P27A-1, SAPN-P27A-2 and SAPN-Combo is shown in Fig.\u00a0SAPN-P27-2 and SAPN-Combo are displayed in Fig.\u00a0SAPN-P27-1 and SAPN-P27-2) lead to severe aggregation, the constructs with the intrinsically unstructured epitope P27A (SAPN-P27A-1 and SAPN-P27A-2) formed very nice and soluble nanoparticles  from thE. coli and purified under denaturing and reducing conditions using immobilized metal chromatography. Resulting protein was run on SDS-Page gel to verify that the correct protein was purified. The primary product was in the expected size range of 34.3\u00a0kDa.SAPN protein monomers were expressed in Tuner (DE3) SAPN-Combo has a hydrodynamic diameter of 26.4\u00a0nm, which falls into the expected range for refolded SAPNs  in terms of % prevalence and average OD [The prevalence of the human antibody response specific to the so Table\u00a0. This reerage OD , 14.TablSAPN-Combo construct delivered in PBS buffer solution without addition of any adjuvant was tested in BALB/c and C3H mice. End point titers of about 1\u20132\u00a0\u00d7\u00a0106 were obtained when ELISA plates were coated with the homologous construct  while partial inhibition (up to 50%) is observed when SAPN-Combo and single antigens (Pept-P27A and Pept-P27-NC) are used for coating and soluble inhibitors, respectively , thus improving its immunogenicity by coupling to a suitable carrier seems to be a highly promising approach.Pept-P27-C 62% is recognized by 62% of donor\u2019s sera from Burkina Faso  induces a stronger immune response compared to the antibody production against the N-terminal region P27-N , rabbitIn summary, these new SAPNs represent a new vaccine strategy. The epitopes P27 and P27A are expressed primarily on the asexual blood stage of the parasite\u2019s life cycle, while CSP is expressed on the pre-erythrocytic stage. A CSP based vaccine will only prevent infection before clinical symptoms develop. An asexual blood stage vaccine will actually help deal with preventing parasitemia from further developing and reducing clinical symptoms. As both approaches continue to be developed they eventually can be admixed to generate a vaccine candidate that prevents the initial infection, and can eventually prevent any breakthrough infection.Plasmodium species. If confirmed in human clinical trials, it would allow the development of a robust vaccine easy to manufacture, store and deliver to the needed population. These vaccine characteristics have been and remain the \u201choly grail\u201d of vaccine development and formulation.Here we have demonstrated that SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. The SAPNs described in this manuscript represent a key improvement over previous designs. Modification of the nanoparticles allowed P27 to be presented, which was not possible with the first designs of the SAPNs. Since P27 is highly conserved, this might even provide cross-protection among different"}
+{"text": "E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal\u2013epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the  Induced pluripotent stem cells (iPSCs) could be derived from somatic cell by the expression of transcription factors including OCT4, SOX2, KLF4 and c-MYC ,2. The i2+/PKC, PI3K/Akt, and MAPK signaling pathways [Currently, porcine iPSCs have been derived from several groups ,7,8. Howpathways . Lysophopathways . Sphingopathways ,13. Therpathways , but alspathways . Sphere pathways . In this study, porcine iPSCs were generated and cultured in the conditions containing AlbuMAX. The iPSCs show pluripotency and differentiation potential, and abilities of aggregation into both porcine parthenogenetic blastocysts and mouse early embryos. Genes associated with fatty acid metabolism were upregulated in the iPSCs. In addition, AlbuMAX upregulated cAMP levels and promoted mesenchymal\u2013epithelial transition (MET) through cAMP/PKA/CREB signal pathway, leading to enhance the reprogramming efficiency.During porcine embryonic fibroblasts (PEF) reprogramming, we counted the numbers of alkaline phosphatase-(AP) positive cells and analyzed the positive cells of pluripotency surface marker SSEA-1. AP-positive colonies in the medium including AlubMAX  were more than that in control group A. BesideThe LpiPSCs that were morphologically similar to mouse ESCs were domed and three-dimensional A. In ordTo test the capability of aggregation of LpiPSCs into porcine embryos, we injected LpiPSCs into porcine parthenogenetic embryos at the eight-cell stage and examined when embryos developed to the blastocyst stage. The results indicated that LpiPSCs with green fluorescence incorporated into inner cell masses (ICM) of porcine embryos F. Cross-As shown in AP staining during reprogramming, more AP-positive cells were in LpiPSCs than the control. Therefore, AlbuMAX may promote cell proliferation. In order to confirm it, we performed the Bromodeoxyuridine  immunofluorescent staining and flow cytometry analysis of BrdU positive cells in iPSCs. The results revealed that a lot of BrdU positive cells existed in LpiPSCs. The proportions were 35.31% A,B. As aNanog and EpCAM expression levels increased as compared to the control group , transport  and metabolism  were up-regulated significantly in LpiPSCs A, which E-cadherin and EpCAM were upregulated significantly at different stages , such as both sphingosine-1-phosphate and lysophosphatidic acid, which may activate the downstream cAMP/PKA signaling pathway . We hypoE-cadherin promoter. Therefore, we constructed a luciferase reporter and the mutation vector of E-cadherin promoter containing CREB binding sites  and used for the teratoma assay. The animal study proposal  in this study were approved by the Animal Care and Use Committee of China Agricultural University.Retroviral virus vectors (pMXs vector) separately carrying porcine Oct4, Sox2, Klf4 and c-Myc were used to generate iPSCs. The viral production was performed as the method described . RetroviThe reprogramming medium and the porcine iPSCs medium contained Dulbecco's Modified Eagle Medium (DMEM), 15% fetal bovine serum (FBS) , nonessential amino acids, Gluta-MAX, penicillin/streptomycin , \u03b2-mercaptoethanol , human LIF, 2i (CHIR99021 and PD0325901)  and 1% AlbuMAX . The medium in the control group contained 15% fetal bovine serum (FBS), nonessential amino acids, Gluta-MAX, penicillin/streptomycin, \u03b2-mercaptoethanol (Sigma), human LIF, 2i (CHIR99021 and PD0325901) (Selleck) and 1% BSA . The iPSCs were passaged by TrypLE at a ratio of 1:6 every 2\u20133 days.2 incubator.For embryoid body formation, the iPSCs were digested and maintained in the medium mentioned above removal of human LIF, 2i (CHIR99021 and PD0325901) and AlbuMAX on a shaker (40 r/min) in a COTotal RNA was extracted using a Qiagen RNeasy mini RNA kit  and then reverse-transcribed by Oligo-dT primer and M-MLV Reverse Transcriptase . The cDNAs were used for quantitative RT-PCR analysis with LightCycler 480 SYBR Green I Master Kit . The data was analyzed using the comparative CT method. Forward (5\u2032 to 3\u2032)Reverse (5\u2032 to 3\u2032)ReferencespMXs-Oct4GACGGCATCGCAGCTTGGATACACGAGAAGGCGAAGTCGGAAGpMXs-Sox2GACGGCATCGCAGCTTGGATACACGGCTGTTCTTCTGGTTGCpMXs-Klf4GACGGCATCGCAGCTTGGATACACGTCTTTGCTTCATGTGGGpMXs-MycGACGGCATCGCAGCTTGGATACACGAAATAAGGCTGCACCGAGTNanogCATCTGCTGAGACCCTCGACGGGCTTGTGGAAGAATCAGGEF-1\u03b1AATGCGGTGGGATCGACAAACACGCTCACGTTCAGCCTTTEndo-Oct4CAAACTGAGGTGCCTGCCCTTCCAAACTGAGGTGCCTGCCCTTCEndo-Sox2CATCAACGGTACACTGCCTCTCACTCTCCTCCCATTTCCCTCTTTEndo-Klf4CATGAGTTGGGGGAGGGAAGACTCACCAAGCACCATCGTTEndo-MycATCCAAGACCACCACCACTGATCCAAGACCACCACCACTGCdh1TGGGCCGAGTGAGTTTTGAATGACTGTAACCACACCGTCGSall4ATCGACGTTTATCCGAGCCCATCGACGTTTATCCGAGCCCEpCAMTGCTCTTTGAATGCGCTTGGAGAGCCCATCGTTGTTCTGGEsrrbCCGGACAAACTCTACGCCATGCTTGGCCCAACCAATGATGCdc2TTTTCAAAGCTGGCTCTGGGAGGGATGTGGTAGATCCCAGCTTACcncAGAAAGATGCCAGACGGTGGAGGAGGTTTTGGTTTCGGCACcnb1AGATCGCAGCAGGAGCTTTTCCTCGATTCACCACGACGATCcna2CTAACATTGCAGCAGACGGCATCCTTAAGAGGCGCAACCCAcox2AGGACTCAGGACGAGACACATTGAAGGACGGCATGCATCTPrkaa2ATTCTGTCACTGCGGAGAGCAATCCATGGTGTGACTGCCCAcadvlGAAGTCAAATGCCTGCCAGCATGTTGGCGCTCACCATGTAAcadsbACACCAAGTGGCTCATACGGTACCAATCTTCGCGTCTCGGFabp5AGGCACCAGTCCGCTTATTCTTTCGTAGGGCCATTCCCACThe primers were as bellows:The alkaline phosphatase (AP) staining was performed using an Alkaline Phosphatase Detection Kit  according to the manufacturer\u2019s instructions.For immunofluorescence staining, cells were fixed in 4% PFA for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked in the blocking buffer  for 1 h. Primary and secondary antibodies were incubated overnight at 4 \u00b0C and for 1 h at room temperature, respectively. Membrane proteins were not permeabilized in the 0.2% Triton X-100. Nuclei were stained with Hoechst 333342 for 2 min at room temperature. The primary antibodies were as follows: OCT4 , SOX2 , SSEA-1 , Nanog , SOX17 , \u03b1-SMA , \u03b2-III-TUBULIN  and BrdU . The secondary antibodies used here were as follows: goat anti-rabbit Alexa Fluor 594 , goat anti-mouse Alexa Fluor 594 , goat anti-mouse Alexa Fluor 594 , and goat anti-chicken Alexa Fluor 594 .For lipid droplets staining using Nile Red . The cells were fixed with 4% PFA for 20 min at room temperature and washed three times with Dulbecco's Phosphate Buffered Saline (DPBS) and then incubated with 5 ng/ml Nile Red for 5 min at room temperature.For flow cytometry analysis, single cells after digestion of colonies with Tryple were stained with primary antibody and secondary antibody for 1 h, respectively. These cells were then washed three times with DPBS and filtered through a 40-\u03bcm falcon. Fluorescence activated Cell Sorting (FACS) analysis was performed using the Moflo-XDP .7) were subcutaneously injected into the BALB/c nude mice. After two months, teratoma were harvested for histological analysis by hematoxylin and eosin (H&E) staining. For H&E staining, nuclei were stained with the alum hematoxylin and eosinophilic structures were stained with eosin.For teratoma formation, the porcine iPSCs (2 \u00d7 10The maturation and parthenogenetic activation (PA) of porcine oocytes were performed as described . Embryosg for 15 min (4 \u00b0C), supernatant was collected. The protein concentration of samples was measured using the BCA Protein Assay Kit (Beyotime). The samples were boiled at 99 \u00b0C for 5 min and were transferred on ice. The denatured protein samples were used for Sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were in 5% nonfat milk for blocking and probed overnight at 4 \u00b0C with primary antibodies. Secondary antibodies were then probed for 1 h at room temperature. Protein bands were finally detected by SuperSignal West Dura Extended Duration Substrate .Cells were washed in cold DPBS and then lysed in sodium dodecyl sulfate (SDS) sample buffer on ice for 30 min. After centrifugation at 10,000\u00d7 \u00ae reagents were supplemented into all reactions and incubated for 10 min. The luminescence measurement values were used to calculate the cAMP concentration according to the kit manual.The cAMP levels of cells were detected using the cAMP-Glo assay kit according to the protocol (Promega). Cells were treated with an agonist or in the induction buffer for the desired length of time and then lysis buffer was added. The plate was incubated with shaking for 15 min. The cAMP detection solution was prepared and added to all wells, mixed and incubated for 20 min. Kinase-GloE-cadherin promoter were synthesized directly as shown below:Top chainCGTCAAGAGCCATCTGAAGGAGAGGCatcgagACTCTGTCTCAGTTATTTTTCCCTBottom chainagcttGCCTCTCCTTCAGATGGCTCTTGACGAGGGAAAAATAACTGAGACAGAGTcCREB binding cites in the Bases in lower-case were the cohesive ends. Bases in upper-case were the binding sites. In mutation vector construction, mutation cites were designed in the primers and mutation vectors were amplified by rolling circle amplification. CGTAC were transformed to CTGCA in mutation vectors.ForwardCTGCAAGAGCCATCTAGTTATTTTTCCCTReverseAGATGGCTCTTGCAGAGGGAAAAATAACTThe primers were shown as below:Luciferase activity analysis was performed following cell transfection 24 h using the Dual-Glo Luciferase Assay System (Promega)."}
+{"text": "Ash1l upregulates Smad3 expression by directly targeting Smad3 promoter to increase local H3K4 trimethylation. Furthermore, we identify an lncRNA, namely lnc-Smad3, which interacts with the histone deacetylase HDAC1 and silences Smad3 transcription. After TGF-\u03b2 stimulation, activated Smad3 suppresses lnc-Smad3 transcription, thereby recovering the Smad3 promoter accessibility to Ash1l. By revealing the opposite regulatory functions of Ash1l and lnc-Smad3 in Smad3 expression, our data provide insights for the epigenetic control of Treg cell fate to potentially aid in the development of therapeutic intervention for autoimmune diseases.Regulatory T (Treg) cells are important for the maintenance of immune homoeostasis and prevention of autoimmune diseases. Epigenetic modifications have been reported to modulate autoimmunity by altering Treg cell fate. Here we show that the H3K4 methyltransferase Ash1l facilitates TGF-\u03b2-induced Treg cell polarization  Smad3 locus.The transcriptional program activated by Smad2/Smad3 is critical for the induction and function of regulatory T cells. Here the authors show that the expression of Smad3 is modulated by the complementary functions of a methyltransferase Ash1l and an lncRNA lnc-Smad3 on the promoter accessibility of the mouse  Foxp3 locus and promotes its expression, subsequently leading to Treg cell polarization6Regulatory T (Treg) cells are essential for immune homoeostasis by suppressing effector T cell responses during infection, inflammation and autoimmunity134In addition to the well-established functions of transcription factors and cytokines in Treg cell development, other cues, such as epigenetic modifications, are also involved in Treg cell fate810Tnfaip3 promoter region to induce its expression. Ash1l-silenced mice are more susceptible to bacterial infection and autoimmune diseasesLysine methylation is one of the most characterized histone modifications to date. In particular, H3K4 methylation associated with transcriptional activation is critical for the maintenance of cell fates+ T cells+CD8+ thymocytes in vitro, suggesting that Ash1l may regulate early thymic T cell developmentAsh1l gene also constitutes part of the Idd17 locus associated with increased susceptibility to autoimmune diabetes in NOD miceAsh1l is highly expressed in CD4in vitro. By contrast, a newly-identified long non-coding RNA (lncRNA), lnc-Smad3, recruits HDAC1 to the Smad3 promoter and selectively suppresses Smad3 but not Foxp3 expression. Interestingly, activation of the TGF-\u03b2/Smad3 axis suppresses lnc-Smad3 transcription, restoring accessibility of the Smad3 promoter to Ash1l. Ash1l-silenced mice are more susceptible to T cell-mediated colitis due to the impairment of Treg cell polarization. Lastly, ASH1L, FOXP3 and SMAD3 are downregulated in peripheral CD4+ T cells from patients with rheumatoid arthritis. Our results provide insights for the epigenetic control of Treg cell polarization during immune homoeostasis, and suggest a possible association between Ash1l and immune disorders.In this study, we show that Ash1l upregulates Smad3 expression by directly activating its promoter, and thereby promoting Foxp3 expression and induced Treg (iTreg) cell differentiation + T cells, which inspired us to further analyse the function of Ash1l in T cell development and differentiationAsh1l allele as compared with wild-type (WT) mice+ and CD8+ T cells in splenocytes from Ash1l-silenced mice was normal+ single-positive (CD4SP) and CD8SP thymocytes, activated CD44hiCD62Llo CD4+ T cells in the spleen and mesenteric lymph nodes (mLN) were also normal in Ash1l-silenced mice  cell-skewing conditions to analyse the function of Ash1l in differentiation of CD4+ T cell subsets. Notably, we observed significantly impaired iTreg cell differentiation, but increased Th1 and Th17 differentiation in Ash1l-silenced CD4+ T cells as compared with WT CD4+ T cells . Accordingly, Ash1l-silenced Foxp3+ iTreg cells showed lower suppressive functions, with reduced expression of costimulatory molecules CD25, CTLA-4 and GITR on iTreg cells  lentiviruses expressing 1\u2013880 amino acids (aa), 881\u20131,855 aa and 1,886\u20132,958 aa containing SET  domain of Ash1l, respectively. We previously showed that the single amino acid point mutation Ash1ll\u0394N (N2212I) had abolished H3K4 methyltransferase activity T cells . Collectin vivo. We first investigated the susceptibility of Ash1l-silenced mice to the induction of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Compared to WT mice, Ash1l-silenced mice developed more severe colitis upon TNBS induction, with a sharper loss of body weight, more exaggerated shortening of colon in appearance, and more severe colon inflammation as indicated by extensive infiltration of mononuclear cells, reduction of goblet cells and mucosa erosion in histology  of patients with rheumatoid arthritis as compared to healthy controls  located 165 kb upstream of Smad3 , and the occupancy of Pol II were downregulated at r region . This waSmad3 transcription. Since lncRNAs usually regulate neighbouring genes by recruiting specific chromatin remodelersSmad3 promoter, we transfected HepG2 cells with lnc-Smad3 expression vector or empty control vector, as well as Smad3 promoter luciferase-reporter construct under TGF-\u03b2 stimulation. The luciferase activity was lower in the presence of exogenous lnc-Smad3 than that with empty control ; CD45.1+ mice were from The Jackson Laboratory . All mice were maintained under specific pathogen-free conditions and used at 6\u20138 weeks of age. All animal experiments were approved by the Scientific Investigation Board of Second Military Medical University, Shanghai.Ash1l-silenced mice on FVB background were from Dr Tian Xu , and were backcrossed (12 polarizations) onto the C57BL/6J backgroundA total of 10 rheumatoid arthritis (RA) and 9 healthy control  patients were recruited from Peking Union Medical College Hospital and Peking University People's Hospital and their peripheral blood samples were prepared from patients after informed consent was providedAnti-mouse CD11c, CD8 and B220-coated magnetic beads were from Miltenyi Biotech. Recombinant mouse GM-CSF and IL-4 were from Peprotech EC. APC-conjugated anti-mouse Foxp3 (FJK-16s), GITR (DTA-1), IFN-\u03b3 (XMG1.2), CTLA-4 (UC10-4B9), FITC-conjugated anti-mouse CD8a (53-6.7), IL-4 (BVD6-24G2), IL-10 (JES5-16E3), and anti-IFN-\u03b3 (R4-6A2), anti-IL-4 (11B11) mAbs were from eBioscience. FITC-conjugated anti-mouse Foxp3 (MF23), CD25 (7D4), PE-Cy7-conjugated anti-mouse CD4 (RM4-5), APC-conjugated anti-mouse CD62L (MEL-14), and anti-CD3 (145-2C11), anti-CD28 (37-51) mAbs, Golgi Plug were from BD PharMingen. FITC-conjugated anti-mouse CD44 (IM7), APC-conjugated IL-17A (TC11-18H10), recombinant mouse IL-2, IL-6 and IL-12 were from BioLegend. Recombinant human TGF-\u03b2 was from R&D Systems. Antibody specific to Ash1l (sc-98301X) was from Santa Cruz. Antibody specific to HDAC1 (ab7028) was from Abcam. The antibodies for cell staining were used at recommended dilution rates  according to the manufacturers' instructions. TRIzol reagents were from Invitrogen. PrimeScript RT-PCR Kit (2641A) and SYBR Premix ExTaq Kit (RR420) were from Takara Bio Inc. RPMI1640 medium and FBS  were from PAA laboratories.g, 30\u2009min). CD4+ T cells were sorted from fresh PBMCs with PerCP-conjugated anti-human CD4 antibody  by FACS Aria II flow cytometer (BD Biosciences). The FACS sorting strategy was introduced in Human PBMCs were collected and purified by Ficoll-Paque PLUS  density gradient centrifugation. In brief, collected whole-blood samples were diluted and mixed with Hanks' balanced salt solution (HBSS). Then blood mixture was slowly added to layer over Ficoll. PBMCs were collected at the interface of the upper layer and the Ficoll after centrifugation  or thymus with mouse naive CD4+ T cell isolation kit  and cultured under neutral (Th0) conditions with anti-CD3 (1\u2009\u03bcg\u2009ml\u22121), soluble anti-CD28 mAbs (1\u2009\u03bcg\u2009ml\u22121) and 10% (vol/vol) FBS in RPMI1640, unless otherwise indicated. For T cell polarization, CD4+ T cells were cultured in the neutral medium described above and stimulated with polarizing cytokines as follows: for iTreg, anti-IFN-\u03b3 (10\u2009\u03bcg\u2009ml\u22121), anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), IL-2 (5\u2009ng\u2009ml\u22121) and recombinant human TGF-\u03b2 (10\u2009ng\u2009ml\u22121); for Th1, anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), IL-2 (5\u2009ng\u2009ml\u22121) and IL-12 (10\u2009ng\u2009ml\u22121); and for Th17, anti-IFN-\u03b3 (10\u2009\u03bcg\u2009ml\u22121), anti-IL-4 (10\u2009\u03bcg\u2009ml\u22121), human IL-6 (10\u2009ng\u2009ml\u22121) and recombinant human TGF-\u03b2 (1\u2009ng\u2009ml\u22121). Differentiated iTreg cells were further enriched with mouse CD25 positive selection kit .Naive CD44 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled naive CD4+ effector T cells from CD45.1 mice were cultured with 1 \u00d7 104 CD11c+ DC used as antigen-presenting cells in 96-well round-bottom plates. Overall, 2.5 \u00d7 104 differentiated and purified WT or Ash1l-silenced iTreg cells were added as suppressive cells. Cells were stimulated with anti-CD3 (1\u2009\u03bcg\u2009ml\u22121) for 3 days. T cell proliferation was determined by CFSE dilution with flow cytometry.A total of 5 \u00d7 10\u2212/\u2212 mice were injected intravenously with 4 \u00d7 105 CD4+CD25\u2212 cells from CD45.1 WT mice with or without 1 \u00d7 105 WT or Ash1l-silenced CD4+CD25+ differentiated iTreg cells. Weight changes were monitored weekly. Mice were killed at week 6 and subjected to examination of colon morphology and histology.Mice were presensitizated with 150\u2009\u03bcl of 1% (w/v) TNBS solution diluted in acetone/olive oil  on the back. On day 7, mice were further given 100\u2009\u03bcl of 2.5% (w/v) TNBS solution (diluted in 50% ethanol) or ethanol only intrarectally through a catheter inserted into the colon 4\u2009cm proximal to the anus. Beginning from the second sensitization, inflammatory bowel disease development was monitored and recorded by measuring the body weight daily. Mice were killed 3 days after the second sensitization and subjected to examination of colon morphology and histology, as well as Treg cells proportion. T cell adoptive transfer colitis was performed as follows. Rag1\u2212/\u2212 mice were injected intravenously with 4 \u00d7 105 CD4+CD25\u2212 cells from WT or Ash1l-silenced mice. Monitored the weight changes and scarified the mice at week 4. T cells were isolated and Foxp3 expression was analysed with flow cytometry.Rag1For cell surface staining, the single-cell suspensions were incubated with the antibody cocktails for 20\u2009min at 4\u2009\u00b0C. For intracellular staining, cells were fixed and permeabilized with 4% paraformaldehyde for 20\u2009min at 4\u2009\u00b0C followed by intracellular staining with specific anti-cytokine antibodies. Data were obtained on an LSR II and analysed with FACSDiva software (both from BD Biosciences). The FACS gating strategies were represented in SYBR RT-PCR kit (Takara) and LightCycler (Roche) were used for quantitative RT-PCR analysisCells were lysed in 0.1% NP40 ice-cold PBS with protease inhibitor cocktail  and Ribonucleoside Vanadyl Complex (10\u2009mM) (New England BioLabs), then after short centrifugation, the supernatant was collected as cytoplasmic fraction and the remainder with additional washing were considered as nuclear pellets.The activated Smad2/3 in nuclear extract or the whole-cell lysate was detected with Smad2/3 ELISA Kit according to the manufacture's protocol . In brief, 5\u2009\u03bcg nuclear extract or the whole-cell lysate was added in the 96-well clear plate pre-immobilized with the Smad2/3 consensus sequencing oligo, incubated and washed. Then the activated Smad2/3 binding to the oligo was detected with a specific antibody against Smad2/3 subunit and a HRP-conjugated secondary antibody. The optical density of each well was determined with a microplate reader at 450\u2009nm.+ T cells and then subcloned into the pcDNA3.1 eukaryotic expression vector (Invitrogen). Lnc-Smad3, Ash1l-fragment 1, 2, 3, and Ash1l-fragment3 mutant expression vectors were constructed by PCR-based amplification from cDNA of mouse CD4+ T cells and then subcloned into pCDH cDNA Cloning and Expression Lentivectors (System Biosciences). All constructs were confirmed by DNA sequencing. These plasmids transfected into HEK293T cell line (from American Type Culture Collection) with JetPEI reagents (Polyplus), and the protein coding capacity was tested by western blotting assay. The Smad3 promoter luciferase reporter was constructed via cloning the fragment of the Smad3 promoter  into the pGL3 luciferase-reporter vector (Promega) and the DNA sequence of the insert was verified.Recombinant vectors encoding mouse Smad3 (NM_016769.4) were constructed by PCR-based amplification from cDNA of mouse CD44 cells per well, 96-well plate, from American Type Culture Collection) were transiently transfected with the lnc-Smad3 expression vector, or empty control vector as well as the Smad3 promoter firefly luciferase-reporter construct and Renilla luciferase-reporter vector (Promega) with Fugene HD (Promega). Cells were stimulated with TGF-\u03b2 24\u2009h after transfection. Twenty-four hours after stimulation luciferase expression was determined by measuring luminescence with the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to renilla luciferase activity.HepG2 cells (3 \u00d7 10+ T cells were stimulated with plate-coated anti-CD3 (1\u2009\u03bcg\u2009ml\u22121), soluble anti-CD28 mAbs (1\u2009\u03bcg\u2009ml\u22121) and IL-2 (10\u2009ng\u2009ml\u22121) for 24\u2009h before knockdown or overexpression assay. For knockdown assay, the target sequence of lnc-Smad3 was 5\u2032-CATTGGACCATTTGATTCTTCCTAA-3\u2032 (bolded and underlined in the following lnc-Smad3 sequence); the control siRNA sequence was 5\u2032-CATCCAGTTTATTAGCTTCCGTTAA-3\u2032. siRNA was transfected into activated CD4+ naive T cells through the use of Lipofectamine 2000 according to the manufacture's protocol (Life Technologies). For overexpression experiments, virus solutions containing lentivirus and polybrene (8\u2009\u03bcg\u2009ml\u22121) were added to the activated CD4+ T cells, followed by centrifugation at 2,500\u2009r.p.m for 2\u2009h at room temperature. Virus supernatants were replaced with fresh culture medium at 6\u2009h after infection. Cells were cultured for an additional 18\u2009h and then sorted as enhanced green fluorescent protein (EGFP)-positive cells. Sorted cells were cultured for 3 days under iTreg cell conditions. Transduction efficiency was determined by EGFP expression and knockdown efficiency was measured by RT-PCR.Naive CD4CATTGGACCATTTGATTCTTCCTAAAGAAAGGCCAAACCACCTGTCAAACCCACGAGCAACACAGCAATGGCTAAGCTGAAGAAGCCAAGTTATATGTCTAGCTCCTCTGTGGGTGGCAGGATCCAGCTGTGGGGAGTCCAAGGTTCCTTGTGTTCTTCTGGCTTGACTATGATATTTCCAATTTGGAGGCACAAAGAATTGAGAACTTGGGGATCCACTGAGGTCTGAGGCATGGTGGTACCAGAAGAAATAAAGGGGCCATCAGCATGAAGCTGGTCTCCCATTGACAGCACCCTGACTTCTCCATCAGGTTCTATCAGCATGTCTACTGTGAGGTTGGTGACGCTGTCTGCAGGTGGGTATGCTTCAATCACGCCACCTGGTCAGGAAACCATGGAGTAGAGGAAAAGACACAGTCAAGGGAATGCTATGCTTATAGCAGGAGGAAATGCTCTGTGTTTTAGCAAGGGAACTTAGACTATCTCACACCTGTCATATCCTGGGAAACTTTGGGTGAGTAGTTCAACCTTTGGCCTCCTTTTCCTTATTTATAGAATGGAGGCATAATATCTACCTTGCCAAGCTTTTGTGAAGTTTGAGAAACTGAGTCTATGGTATTTAGCCTGGTACTTGGAACATGGGAGGGCTCAATGAACAGTAAGTAGTCTTTATTATGATCATAATTACAAATATTACAAATACTTTATAAAAGCCATACTTAGAACAACTCATTACGTTAATAAAGAATCCAAGGATTGTGGTC>mouse lnc-Smad3 transcript (GenBank Accession number: KY652933) CGGCGCGTGCGCACAGGACGGGACGGGAGGGCGGAGCGACTGCGCAGATCAGGAAAATTGTCACCTCTGGCCTCAGGGAAACTGAGGCTCTGAGCAGTTAAGAGGCCAACGATCCAGGTTTATGCTATCAGTGTCTGAGATACAATTAAGTCACCTTTTTGGGTGACATTTCCCTTGACTAGTACCTCAAGATTATGATCCAGGTGGACCATCCCTCCATTGCCTCCAGACATGTCTATGACGCTACCTAGGGATTGTGAAGATTTACCACCTGGTGGAAAATTAAAAAAAAAGACTTATTCTGCAAATTGACCAAGCTTAAGAGATACAGCACTGAGAATTCACCCACTACAGAGCTGGTAACCAGGCCTTCAGAGTAAGCTGTGATGTATAGCCATTCTCCTCAGCAGCCTGTTTGACTGAGGGATATAGGAATGACTGCCCTTACCCATGTTTTTGTTCAGATTTATGGTTCTAAATCTGATGGAAAATCTATCHIP analyses were performed with the following antibodies: RNA Polymerase II (Pol II), acetyl-Histone H3 (Lys27), trimethyl-Histone H3 (Lys4), Ash1l and HDAC1. Fold enrichment was quantified using quantitative RT-PCR and calculated as a percentage of Input chromatin (% input). Sequences of the primers for amplification of the Smad2, Smad3, Smad4, Foxp3 and lnc-Smad3 promoter regions are in \u22121, Promega) at 37\u2009\u00b0C for 30\u2009min and then stopped by EDTA (50\u2009mM). Genome DNA was extracted and subjected to quantitative RT-PCR. Data were presented as changed fold concluded with 2\u0394Ct, with relative to CD4+ T cells transduced with Lenti-CTR, set as 1.For chromatin accessibility analysis, nucleus were pretreated with DNase I .Total RNA extracted from mouse B220RNA immunoprecipitation (RIP) was performed with antibodies specific to mouse Ash1l, Smad3 and HDAC1 by using Magna RIP RNA-Binding Protein Immunoprecipitation Kit according to the manufacture's protocol .+T cells were fixed in 4% formaldehyde plus 10% acetic acid in PBS for 15\u2009min at room temperature, and then were permeabilized in PBS plus 0.2%\u20130.5% Triton X-100 and 5\u2009mM vanadyl ribonucleoside complex (10\u2009mM) (New England BioLabs) for 5\u2009min on ice, washed in PBS three times and rinsed once in 2 \u00d7 SSC bufferFluorescence-conjugated lnc-Smad3 probes were used for FISH assay. Naive CD4t test. P values of <0.05 were considered statistically significant .The statistical significance between two groups was determined by Student's All the data supporting the findings of this study are available within the article and its How to cite this article: Xia, M. et al. Ash1l and lnc-Smad3 coordinate Smad3 locus accessibility to modulate iTreg polarization and T cell autoimmunity. Nat. Commun.8, 15818 doi: 10.1038/ncomms15818 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "The Malarial parasite resides in the host RBC during its erythrocytic cycle. Plasmodium meets its entire nutritional requirement fromRBC. It scavenges the hemoglobin of RBCs to meet its amino acid requirement. The host hemoglobin is made of different chains and itis dependent on age. Hemoglobin F (HbF), which has two-alpha and two gamma chain persists in children upto six years, andhemoglobin A (HbA) made of two alpha and two beta chains dominates. Therefore, it is of interest to compare the compositionalfeatures of HbA with HbF. Isoleucine is present in hemoglobin of children (gamma chain of HbF) while it is absent in adulthemoglobin (HbA). The presence of Isoleucine (I) makes HbF ideally suitable for the growth of parasite, as it does not have to dependupon the exogenous supply of the isoleucine, which might be responsible for making children more vulnerable to malaria as comparedto adults. Malaria is one of the most prevalent diseases in developingcountries . MalariaMalaria affects all age groups. However, the children are affectedthe most. According to WHO report there were an estimated 438000 malaria deaths around the world in 2015, 69% of total deathsdue to malaria are known to occur in children aged from 6months to 5 years .Though the parasite culture in RBC containing adult Hb isroutinely performed  The studIn this study, we show the abundance of different amino acids inPlasmodium falciparum 3D7 and distribution of different aminoacids according to its essential nature in host. We have alsocompared the amino acid composition of different chains ofhaemoglobin to determine the difference that leads to preferenceof RBC containing HbF, which might explain the disease severityin children.The FASTA format of all protein sequence, 5,369 proteins, ofPlasmodium was downloaded from PlasmoDB. Composition ofprotein sequences was completed using ProtParaman tool.ProtParaman tool is an online tool, which is freely available anddetermines the sequence composition and predicts other physicalparameters . Total aComplete protein sequences of 5369 were downloaded fromPlasmoDB for Plasmodium falciparum 3D7. The amino acid compositions of total 5369 proteins are represented by pie chart. As The The protein sequence of the haemoglobin chain was downloadedfrom NCBI and the composition of amino acid was determinedusing ProtParaman tool  as descrThe fasta formats for different chain of the haemoglobins wereobtained from NCBI. The assertion number of different chains ofare as follows:MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYHMVHFTAEEKAAVTSLWSKMNVEEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKNMDNLKPAFAKLSELHCDKLHVDPENFKLLGNVMVIILATHFGKEFTPEVQAA WQKLVSAVAI ALAHKYHMVHLTPEEKTAVNALWGKVNVDAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDKLHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANALAHKYHMVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYHMVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYMSLTKTERTIIVSMWAKISTQADTIGTETLERLFLSHPQTKTYFPHFDLHPGSAQLRAHGSKVVAAVGDAVKSIDDIGGALSKLSELHAYILRVDPVNFKLLSHCLLVTLAARFPADFTAEAHAAWDKFLSVVSSVLTEKYRMultiple sequence analysis of different chain of Hemoglobin wasdone using Clustal omega. The results show similarity betweendifferent chains of haemoglobin. Further, we looked at thedistribution of amino acid in different haemoglobin chain . We obsePlasmodium parasite, scavenges the haemoglobin to meets itsamino acid requirements while lipids are obtained from RBCsmembrane. The distribution of amino acids in the proteins ofPlasmodium shows incorporation of all the 20 amino acids.However, the amino acids, which are essential and conditionallyessential to the host, are present in higher percentage than nonessentialamino acids.Among the essential amino acids leucine is required mostfollowed by isoleucine. Among the conditionally essential aminoacids N is required the most. N is present in parasite as repeatwhich might have role in immune evasion by antigenic variation.Other amino acids like arginine, which is essential for polyaminesynthesis is important for robust growth of the parasite.I make up to 9% of total amino acids in Plasmodium falciparum hasto be obtained exogenously from blood, as it is absent in adulthaemoglobin. Istvan et al. has shown that the absence of I affectsparasite growth and I analogue inhibits the parasite growth .The gamAs Plasmodium solely depends upon the degradation ofhaemoglobin for its amino acid requirements it meets most of thenutritional requirements from haemoglobin. As HbF contains allthe amino acids including isoleucine (absent in HbA) required bythe parasite hence, this might be aid growth of parasite in HbFcontaining RBC. Besides at the age of 9 up to, 18% of RBCcontains HbF. Hence, we hypothesise that presence of HbFwould aid parasite growth leading to disease severity in children.Authors declare no conflict of Interest"}
+{"text": "AbstractMastigodiaptomus Light, 1939, named Mastigodiaptomuscuneatussp. n. was found in a freshwater system in the City of Mazatl\u00e1n, in the northern region of Mexico. Morphologically, the females of this new species are distinguishable from those of its congeners by the following combination of features: the right distal corner of the genital double-somite and second urosomite have a wedge-shaped projection, the fourth urosomite has no dorsal projection and its integument is smooth. The males are distinct by the following features: the right caudal ramus has a wedge-shaped structure at the disto-ventral inner corner; the basis of the right fifth leg has one triangular and one rounded projection at the distal and proximal margins, respectively, plus one hyaline membrane on the caudal surface close to the inner margin; the aculeus length is almost the width of the right second exopod (Exp2); and the frontal and caudal surfaces of the right Exp2 are smooth. Furthermore, the analysis of the COI gene of Mastigodiaptomuscuneatus sp. n. has revealed that Mastigodiaptomusalbuquerquensis  is its nearest congener, with 18.64% of genetic distance. A key for the identification of the known species of the genus is provided.A new species of the genus  Diaptomidae G. O. Sars, 1903 is one of the most common families of freshwater copepods worldwide, some genera of this family have restricted distributional patterns and present endemic forms, as the genus Mastigodiaptomus Light, 1939. Recent studies of this genus in the Neotropical region have added new species of these diaptomids  , Mastigodiaptomustexensis , Mastigodiaptomusamatitlanensis , Mastigodiaptomusalbuquerquensis , Mastigodiaptomuspatzcuarensis , Mastigodiaptomusmontezumae , Mastigodiaptomusnesus Bowman, 1986, Mastigodiaptomusmaya Su\u00e1rez-Morales & El\u00edas-Guti\u00e9rrez, 2000, Mastigodiaptomusreidae Su\u00e1rez-Morales & El\u00edas-Guti\u00e9rrez, 2000, and Mastigodiaptomussuarezmoralesi Guti\u00e9rrez-Aguirre & Cervantes-Mart\u00ednez, 2013.aptomids  or the mr, 1938) . CurrentCyclopidae genus Mesocyclops; and Cyclopidae genus Eucyclops, particularly the Eucyclopsserrulatus species complex. Mastigodiaptomus genus using differences in the integument of prosomal wings and fifth legs of both sexes. Empirical evidence gathered from several species of freshwater crustaceans has shown that these morphological differences are consistent with reproductive isolation  Chetumal, M\u00e9xico and the Colecci\u00f3n Nacional de Crust\u00e1ceos (CNCR) del Instituto de Biolog\u00eda, Universidad Nacional Aut\u00f3noma de M\u00e9xico.The type material was deposited at the Colecci\u00f3n de Referencia de El Colegio de la Frontera Sur  were preserved after capture in 96% ethanol and prepared for barcoding following standard methods. DNA was extracted using the HOTSHOT method . A segmeBOLD) produced sequences of Mastigodiaptomusalbuquerquensis, Mastigodiaptomuspatzcuarensis, Mastigodiaptomuscf.albuquerquensis , Microcrustacean from Mexico (MCM), and Zooplankton II (ZPII) and compared with our sequence of Mastigodiaptomuscuneatus sp. n., this latter sequence is into the project MCM. In these project files, the electropherograms, sequence data, photographs, primers data, and collection details are available (on the Barcode of Life Data System http://www.boldsystems.org). Fifty-two COI gene sequences > 500 bp were used for the analysis, and BOLD Aligner and the ID Tree using the model Kimura 2 parameter   were used as an outgroup.These sequences were downloaded from the ter K2P;  were utiPageBreakTaxon classificationAnimaliaCalanoidaDiaptomidaehttp://zoobank.org/FADC3B97-FB6F-4559-B71B-EB6F76A3246FECOCH-Z-09339. Collected 28.VIII.2014. Collectors: A. Cervantes-Martinez, N. Hern\u00e1ndez L\u00f3pez, M. Bastidas, and J. Aguilar Rubio.One adult female dissected on one slide: PageBreakECOCH-Z-09340. Collected 28.VIII.2014. Same collectors.One adult male dissected on one slide: ECOCH-Z-09341. Collected 28.VIII.2014. Same collectors.Four adult females and five adult males preserved in 90% ethanol with a drop of glycerine. PageBreakCNCR-31861. Collected 28.VIII.2014. Same collectors.Two adult females and two adult males preserved in 90% ethanol: 23\u00b014'10\"N; 106\u00b026'18\"W.A lagoon called Laguna El Camar\u00f3n in Avenida Insurgentes, Mazatl\u00e1n, Sinaloa City, M\u00e9xico; The name of the species means \u201cwedged\u201d in Latin and refers to the chitinous protuberance present on the right disto-lateral corner of the first and second urosomites in females, and on the right caudal ramus on the ventral surface in males.Adult female: Cuticle surfaces of prosomal somites smooth dorsal and laterally Fig. . AntennuAdult male: The cuticle surfaces of prosomal somites are smooth dorsally and laterally Fig. . Right an = 6  with a movable seta at tip. Rectangular, nude coxa. Basis with 4 long setae. Enp two-segmented, Enp1 with 4 ely Fig. .PageBreakand 2-segmented endopodite: Enp1 and Enp2 with 5 and 4 setae, respectively. Basis rectangular with 4 setae; coxal endite quadrangular 4-setulated. Praecoxal arthrite with 15 spiniform setae, 11 anterior, 4 posterior.Maxillule Fig. : Coxal eMaxilla Fig. : First pMastigodiaptomusalbuquerquensis and Mastigodiaptomuspatzcuarensis (see Maxilliped (not figured): Same structure as described and illustrated for nsis see .Diaptomidae family  with a smooth hyaline membrane.PageBreakAntennule, antenna, mandible, maxillule, maxilla, maxilliped, and P1-P4 as described for female.Right wing of fifth pediger with 1 tiny dorsal spinule and 1 ventral spine Fig. ; left wiPageBreakUrosome: Urosomites nude dorsally and ventrally. First urosomite with thin spine on right side and fold on left side Fig. . Fourth PageBreakP5: Coxal segments with strong spines on caudal view; left and right basis with a lateral seta Fig. .Left basis with a triangular protuberance on distal margin of frontal surface Fig. . Both leRight basis basally and distally projected: basal projection rounded whereas distal projection triangle-shaped; one semi-triangular sclerotization on caudal surface of right basis Fig. . Right EMastigodiaptomuscuneatus sp. n. is shown below:The nucleotide sequence (607 bp) obtained for specimen MAGA-0156 , identified as GGAGCCTGGTCAGGCATAGTAGGAACAGGCCTTAGAATGATTATTCGGATGGAGTTAGGACAAGCCGGGTCTTTAATTGGAGATGACCAAATTTATAATGTAGTAGTTACTGCTCATGCTTTTGTTATAATTTTTTTTATGGTGATACCTATTTTAATTGGGGGGTTTGGTAATTGGCTTGTTCCGTTAATATTAGGTGCAGCGGATATAGCTTTCCCTCGAATAAATAATATAAGATTTTGATTTTTATTGCCAGCTTTAGTCATATTGTTATCTAGGTCGCTTGTTGAAAGAGGGGCGGGAACAGGGTGAACTGTGTATCCCCCCCTGTCTAGCAACATTGCCCATGCTGGCAGGTCCGTTGATTTTGCTATTTTTTCGCTTCATTTAGCTGGGGTTAGGTCTATTTTGGGCGCAGTAAATTTTATTAGCACATTAGGAAATTTGCGGGCGTTTGGAATAATTTTAGATCGAATACCACTTTTTGCTTGAGCCGTTTTAATCACGGCTATCTTGTTATTGCTTTCTCTTCCTGTTTTAGCCGGGGCGATTACAATGCTTCTTACAGATCGGAACCTCAACTCAAGATTTTATGAT.K2P maximum distance between the surveyed species reaching 5.52%  appeared to be morphologically close to Mastigodiaptomusamatitlanensis . Similarities between these species in females include the presence of one protrusion on the second urosomite and the bulbose lateral margins of genital double-somite. The similarities in males are the short aculeus and the lack of hyaline membrane on the right Exp2 of the fifth leg.Mastigodiaptomuscuneatus sp. n. can be separated from Mastigodiaptomusamatitlanensis by the following features: the dorsal projection on the last pediger absent vs. present; the genital double-somite with vs. without a protrusion on the distal right side; on the genital double-somite, the right spine located at a higher level than the left spine vs. both left and right spines placed at same level; and the endopod of fifth leg long and 2-segmented vs. short and 1-segmented.However, Mastigodiaptomuscuneatus sp. n. in comparison to the rectangular basis with a transversal, distal, cuneiform lamella in Mastigodiaptomusamatitlanensis. In addition, the distal margins of the left and right endopods bear short setules in Mastigodiaptomuscuneatus sp. n. whereas in Mastigodiaptomusamatitlanensis these distal margins bear one slender seta. The aculeus on the right Exp2 is clearly straight in Mastigodiaptomuscuneatus sp. n. but short, distal and curved in Mastigodiaptomusamatitlanensis. The Exp2 is smooth in Mastigodiaptomuscuneatus sp. n. but in Mastigodiaptomusamatitlanensis an oblique ridge on the caudal surface is present. Finally, we assume that the wedge on the right caudal ramus present in Mastigodiaptomuscuneatus sp. n. is absent in Mastigodiaptomusamatitlanensis because there is no mention of a similar feature in The males of these species show more morphological differences in the fifth leg: the caudal surface of the right basis is bulbose with the oblique, medial, angulose and curved cuticular process in Mastigodiaptomuscuneatus sp. n. is genetically closest to Mastigodiaptomusalbuquerquensis s. str. and the species recorded in Mexico with one sclerotization  on the right basis of fifth leg of males such as Mastigodiaptomuspatzcuarensis. As previously discussed , which probably are cryptic species , Mastigodiaptomuspatzcuarensis, Mastigodiaptomusmontezumae , Mastigodiaptomusnesus (Caribbean and south eastern Mexico), and Mastigodiaptomustexensis (Texas and south eastern Mexico), whereas the species that are assumed to have restricted distribution or endemics are Mastigodiaptomusreidae, Mastigodiaptomusmaya, Mastigodiaptomuspurpureus, Mastigodiaptomusamatitlanensis, Mastigodiaptomussuarezmoralesi (see Mastigodiaptomuscuneatus sp. n.lesi see  and, proMastigodiaptomuscuneatus sp. n. was compared with the ten known Mastigodiaptomus species, particularly in the female and male urosomes, the male right antennule and fifth legs, and in the COI gene sequence. This report increases the number of recognized species of Mastigodiaptomus to eleven. Mastigodiaptomuscuneatus sp. n. appears to be part of the Mastigodiaptomusalbuquerquensis complex.Morphological and genetic differences were found when MalesFemales"}
+{"text": "Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA  Embryonic stem cells (ESC) are characterized by their ability of long-term self-renewal as well as their potential to differentiate into each cell type of the embryo proper. After the first isolation of embryonic stem cells from the mouse blastocyst , 2 the rRecent sequencing approaches have shown that the majority of the genome is transcribed . Among tTUNA was identified as sustainer of pluripotency but was also required for neural differentiation [Large-scale functional studies have identified numerous lncRNAs that play a regulatory role in the maintenance of pluripotency \u201310. It hlncR492 as an inhibitor of neural differentiation, which exerts its function by interacting with the mRNA binding protein HuR and activating Wnt signaling, thereby blocking ectodermal differentiation in a lineage specific manner.To identify specific neural differentiation regulating lncRNAs, we performed a large-scale loss-of-function RNAi screen in a Sox1-GFP ESC reporter line. We discovered the lncRNA Austin Smith and Konstantinos Anastassiadis kindly provided the Sox1-GFP and Oct4-GFP cell lines. The Foxa2-GFP and T-GFP cell lines were generated in the BAC TransgeneOmics project  and kind\u2122) supplemented with 1x N2, 1x B27, 1x NEAA, 1x Penicillin/Streptomycin, 50 \u03bcM 2-mercaptoethanol , 3 \u03bcM CHIR99021, 1 \u03bcM PD0325901  and LIF (generated in house). For differentiation towards the ectoderm cells were cultured in N2B27 containing medium without the two inhibitors and LIF. For differentiation into endoderm (Foxa2-GFP ESC) and mesoderm (T-GFP ESC), the two inhibitors and LIF were replaced by 30 \u03bcg/ml ActivinA (MPI protein facility) or 10 \u03bcg/ml BMP4 (R&D Systems), respectively. The cells were seeded on gelatin-coated dishes with a density of 15000 cells/cm2 and grown for 4 to 5 days before they were harvested for experiments.Sox1-GFP, Foxa2-GFP, T-GFP ESC and R1/E ESC lines were cultured on gelatin-coated plates in DMEM  supplemented with 10% Pansera ES FBS (PAN Biotech), 1x NEAA, 1x Penicillin/Streptomycin, 50\u03bcM 2-mercaptoethanol  and LIF (MPI-CBG protein facility).Oct4-GFP ESC were additionally cultured on gelatin-coated plates in DMEM  or the corresponding non-labeled amino acids, respectively. All other supplements were maintained as described. Cells were grown for five passages, lysed and checked for successful label incorporation by MS.For SILAC-labeling the standard DMEM was replaced with SILAC-DMEM medium  supplemented with 40 mg/mL EsiRNAs were synthesized as described previously . For theNheI and XhoI restriction sites in the flanking regions. After sequencing, the PCR products were cloned into the pCGIT destination vector under the CAG promoter . pCGIT without insert was used as control (EV\u2014empty vector).The cDNAs for lncR492 and HuR were amplified by PCR introducing ESCs were transiently transfected with the overexpression or control plasmids using Lipofectamine 2000 (ThermoFisher Scientific). 50 ng of plasmid DNA were combined with 0.2 \u03bcl Lipfectamine 2000 and incubated in 50 \u03bcl OptiMEM for 10 minutes. The transfection mix was added to one well of a 96-well plate. 4500 cells per well in 200 \u03bcl N2B27 medium supplemented with 2i+LIF were seeded on top. After 24h differentiation was initiated as described above.18 primer. Standard RT-PCR reactions were performed with a MyTaq\u2122 Red DNA Polymerase (Bioline). Quantitative RT-RCRs (qRT-PCR) were run with the SYBR Green qPCR kit (Abgene) on an CFX96 Touch\u2122 Real-Time PCR Detection System (Bio-Rad). Measured transcript levels were normalized to Gapdh. Samples were run in duplicates. Primers used are listed below.Total RNA was isolated by using the RNeasy Mini kit (QIAGEN). For each RT reaction 1 \u03bcg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)\u2122 Eukaryotic mRNA Isolation Kit, EpiCentre). Subsequent 500 ng of RNA were reverse transcribed as described above.For endonuclease treatment 5 \u03bcg RNA were treated with a 5\u2019-phosphate-dependent exonuclease according to manufactor\u2019s instructions (mRNA-ONLYThe forward and reverse primer sequences (written 5\u2019 to 3\u2019) were as follows:Gapdh, ACTCCACTCACGGCAAATTC, GGATGCAGGGATGATGTTCTlncR492, GCTGCTGTTTCACACCCAAG, TGACTAGGCGATCCTGACCASox1, CCTTGCTAGAAGTTGCGGTC, TCACTCAGGGCTGAACTGTGSrrm4, TCTCGTCGAAGTCCCAGCTA, CACTGGTTATCCTCCGAGCCOct4, AGAGGGAACCTCCTCTGAGC, TGATTGGCGATGTGAGTGATBrachyury (T), GAACAGCTCTCCAACCTATG, AGACTGGGATACTGGCTAGAGFoxa2, GCTGCAGACACTTCCTACTAC, GGACACAGACAGGTGAGACTPax6, CACCAGACTCACCTGACACC, TCACTCCGCTGTGACTGTTCNestin, GCAGGAGAAGCAGGGTCT, AGGTGCTGGTCCTCTGGTNanog, GGAAGCAGAAGATGCGGACT, ATGCGTTCACCAGATAGCCCMalat1, GTTTGTGATTGGAGCCGAG, AAGGGAGGGGAGAGAGAACAU1 snRNA, GCGCGATTTGGCAAGATGA, TTCTTCCCGGGTTTCTGCTCStar solution, exposed to Amersham Hyperfilm , which was developed using an OPTIMAX (PROTEC).The sequences of the gene-specific Northern blot probes are listed below (written 5\u2019 to 3\u2019). Two probes per gene were mixed together. Northern blot was performed according to the DIG Northern Starter Kit\u2019s (Roche) manual. Briefly, RNA was separated on a pre-cast 1% agarose gel at 35V for 3.5h in 1x MOPS buffer. The separated RNA was blotted on a nitrocellulose membrane  and cross-linked by UV-light. The membrane was prehybridized with DIG easy Hyb for 30 minutes at 68\u00b0C, followed by an hybridization step with a DIG-labeled, gene-specific RNA probe mix diluted in DIG easy Hyb over night at 68\u00b0C. Unspecific binding was eliminated by several stringency washes. Thereafter the membrane was blocked, incubated with a DIG-specific antibody and washed. Finally, the membrane was incubated with CDP-lncR492 probe 1UCAGAACCCAGCACACUGUCAGCCUCAGAGCAUACAAUCUUUUGAGUGAGUGAGCUAGAUUUGACAACGAUGCUUGAUGCUUGCGAGCAUAUGAGGGGCUCGCAGCCUCUUCCUAGGCACUACUGUCUCCCUCCGGAGACGCCUCCUGGCCUCCUUGAUUAUGAACACCUUUGCUAAUGCUCACAGUCUUCUUGUUCUCAUGGCCCUUGGAGGAUAUGUGCAGUGGUGAACACAGCUlncR492 probe 2AUGGCUUUUUCCUCGCUACAGUUCAGAGUCGCCACAAUCACAGGGGGCCGGGGAAGAUCACCAGCAAUCAGUGUUCAAACGGCCCAAAGAGAUUGUUCUGAGUCUCCUUGCCACUCCGCUGAUGGAGUAGGGCCUUCUAAUUGGCCCUUCUGUCUCUCCUGCCUCGCCCUUCCAAUCUUUUGCCUUCUUGGCAGCUAGGUUCAUGCUUAUAGACCAUUUUUCGGUGAGUCAUUGCCUUGAUUUAAAACCCUUCGGCUUCCACUUGAGACUUGAGCCAUCCAGGCUCCCUGCCAUGGAGGGAGAGGGCAGUCAUGAUCUGGUUUUGGCCAUCCAUCUCUCUCCUCUAUCCUGUUCCCUGAGUCCUUGUUCUGAGCAUCUACCUGGGGCAUCCUUCCUUCCCACUAACUCCUGUAUCAUGUCCCUGGACCACUCCCCACCUGACUGGUCCUCUGUGUUUUCAGAGUAGCUGAUUUCUUUUGCACUGCCUAGCGCCACUGGGAACAGACAUCGUCUGUGAGCUUUGGGAAGGGapdh probe 1CAGCGAACUUUAUUGAUGGUAUUCAAGAGAGUAGGGAGGGCUCCCUAGGCCCCUCCUGUUAUUAUGGGGGUCUGGGAUGGAAAUUGUGAGGGAGAUGCUCAGUGUUGGGGGCCGAGUUGGGAUAGGGCCUCUCUUGCUCAGUGUCCUUGCUGGGGUGGGUGGUCCAGGGUUUCUUACUCCUUGGAGGCCAUGUAGGCCAUGAGGUCCACCACCCUGUUGCUGUAGCCGUAUUCAUUGUCAUACCAGGAAAUGAGCUUGACAAAGUUGUCAUUGAGAGCAAUGCCAGCCCCGGCAUCGAAGGUGGAAGAGUGGGAGUUGCUGUUGAAGUCGCAGGAGACAACCUGGUCCUCAGUGUAGCCCAAGAUGCCCUUCAGUGGGCCCUCAGAUGCCUGCUUCACCACCUUCUUGAGapdh probe 2AAGCAGUUGGUGGUGCAGGAUGCAUUGCUGACAAUCUUGAGUGAGUUGUCAUAUUUCUCGUGGUUCACACCCAUCACAAACAUGGGGGCAUCGGCAGAAGGGGCGGAGAUGAUGACCCUUUUGGCUCCACCCUUCAAGUGGGCCCCGGCCUUCUCCAUGGUGGUGAAGACACCAGUAGACUCCACGACAUACUCAGCACCGGCCUCACCCCAUUUGAUGUUAGUGGGGUCUCGCUCCUGGAAGAUGGUGAUGGGCUUCCCGUUGAUGACAAGCUUCCCAUUCUCGGCCUUGACUGUGCCGUUGAAUUUGCCGUGAGUGGAGUCAUACUGGAACAUGUAGACCAUGUAGUUGAGGUCAAUGAAGGGGUCGUUGAUGGCAACAAUCUCCACU6 cells were washed with ice-cold PBS. Cells were resuspended in ice-cold, protease inhibitor (Roche) containing HD-buffer  and incubated on ice for 10 minutes. IGEPAL was added to a final concentration of 0.5%. Quickly citric acid (100 mM) was added to a final concentration of 1 mM. The cells were vortexed vigorously. The nuclei were separated from the cytoplasmic fraction by gentle centrifugation. Total RNA was isolated with the PARIS Kit (Ambion). 1 \u03bcg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.Approx. 10x10lncR492 probe 2 was used as FISH probe.Sox1-GFP ESC were transfected with esiRNA using Lipofectamine2000 (Thermo Fisher Scientific) and seeded on gelatin-coated chambered slides (Ibidi). After 48 h RNA-FISH was performed as described earlier . The seqFor immunofluorescent stainings cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. All following steps were performed at room temperature. After washing with PBS cells were blocked and premeabilized with 10% FCS in staining buffer (0.3%TritonX-100 in PBS) for 30 minutes. The primary antibody rabbit-anti-Tubb3  was diluted 1:1000 in staining buffer and incubated for 1 h. After washing the cells were incubated with donkey-anti-rabbit-Cy3 secondary antibody (Jackson ImmunoResearch Laboratories) and DAPI (Sigma) for 30 minutes. Finally the cells were imaged using a Zeiss Axiovert 200M microscope equipped with an ApoTome (Zeiss). Images are projections of the maximum intensity. Total numbers of nuclei as well as Tubb3+ cells were counted manually.To create the 450 bp RNA baits, forward primers containing the T7 promoter sequence and reverse primers with the S1 aptamer sequence were used in a PCR amplification reaction on the pCAGGS plasmid. A fragment of the human PTPN13 mRNA of equal length (450 bp) was used as a control bait. The primer sequences are written 5\u2019 to 3\u2019.CGTTAATACGACTCACTATAGGCTCGAGATCCAGCTGGGCACAGGC.AK016992 (1\u2013450 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAACTCTGCTGATCGGATCTGTCCTC.AK016992 (1\u2013450 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGGGCTCCCGGACCTTCCCAAAG.AK016992 (451\u2013900 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCAGATTGTTCTGAGTCTCCTTGCCAC.AK016992 (451\u2013900 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCTTTGGGCCGTTTGAACACTG.AK016992 (901\u20131350 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCTCTAGACGGGTACAATGCCTTC.AK016992 (901\u20131350 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGAGGGCCATGAGAACAAGAAG.AK016992 (220\u2013670 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCGTTTTGGCCATCCATCTCTCTC.AK016992 (220\u2013670 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGGCAGATCATGACTGCCCTCTCC.AK016992 (671\u20131120 bp) + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCGCTTGGGTGTGAAACAGCA.AK016992 (671\u20131120 bp) + BioApt reverse primer, CGTTAATACGACTCACTATAGG CAATATATTTTCTGCTATCAAGTC.PTPN13 + T7 forward primer, CATGGCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGTCGGTCCCATGGATCCCTTTATTAAAATATTGGAAAACATTTTTG.PTPN13 + BioApt reverse primer, in vitro transcription according to the manufacturer\u2019s protocol (Fermentas). Successful transcription was monitored by agarose gel electrophoresis and RNA concentration was quantified by A280 absorbance on a Nanodrop system (Peqlab). 25 \u03bcg of each S1-tagged RNA was coupled to paramagnetic streptavidin C1 beads  in RNA binding buffer , 0.5% IGEPAL CA-620) and incubated on a rotation wheel for 30 min at 4\u00b0C. RNA-bound beads were washed 3 times with RNA washing buffer , 0.5% IGEPAL CA-620), prior to incubation with 400 \u03bcg nuclear extract, with 20 \u03bcg competitor yeast tRNA (Invitrogen) added, for 30 min at 4\u00b0C with gentle agitation. After mild washing, SILAC heavy and light fractions were combined 1:1 and samples were boiled in 1x LDS buffer (Invitrogen) and separated on a 4\u201312% NuPAGE Novex Bis-Tris precast gel (Life Technologies) at 180 V in 1x MOPS.The PCR products were used for 17703201) using two layers of C18material (Empore).Coomassie stained gels were cut in one slice and destained with 50% EtOH/25 mM ammonium bicarbonate (ABC). The resulting gel pieces were dehydrated with 100% acetonitrile (ACN) and dried for 5 min in a concentrator (Eppendorf). Samples were incubated with reduction buffer (10 mM DTT/50 mM ABC) for 30 min at 56\u00b0C and further alkylated for 30 min in the dark with iodoacetamide (50 mM IAA/50 mM ABC). Gel pieces were completely dehydrated with ACN and covered in trypsin solution (1 \u03bcg trypsin per sample). Proteins were digested over night at 37\u00b0C and peptides were extracted twice by incubation with extraction buffer (3% TFA and 30% ACN) for 15 min. The gel pieces were dehydrated with 100% ACN and the extracted volume reduced to aproximately 150 \u03bcl in a concentrator (Eppendorf). Extracted peptides were desalted in StageTips (PMID:17721543). MS full scans were obtained in the orbitrap at 70,000 resolution with a maximal injection time of 20 ms, while MS/MS scan resolution was set to 17,500 resolution and maximal injection for 120 ms. Unassigned and charge state 1 were excluded from MS/MS selection and peptide match was preferred. Raw files were processed with MaxQuant (version 1.5.2.8.) (PMID:19029910) and searched against human UNIPROT annotated protein database provided with MaxQuant  using the Andromeda search engine (PMID:21254760). Carbamidomethylation was set as a fixed modification, while acetyl (N-term protein) and oxidation (Met) were considered as variable modifications. Trypsin (specific) was selected as enzyme specificity with maximal two miscleavages for MaxQuant analysis. Proteins were quantified with at least 2 ratio counts based on unmodified unique and razor peptides. Known contaminants and reverse hits were removed before plotting the protein ratios of the forward and reverse experiments in R (version 3.2.2).Eluted peptides were injected via an autosampler into an uHPLC  and loaded on a 25 cm capillary  packed in-house with Reprosil C18-AQ 1.9 \u03bcm resin (Dr. Maisch) for reverse-phase chromatography. The EASY-nLC 1000 HPLC system was directly mounted to a Q Exactive Plus mass spectrometer (Thermo). Peptides were eluted from the column with a 90 min optimized gradient from 2 to 40% ACN with 0.1% formic acid at a flow rate of 200 nL/min. Chromatography was stabilized with a column oven set-up operating at 40\u00b0C (Sonation). The heated capillary temperature was set to 250\u00b0C. Spray voltage ranged from 2.2\u20132.4 kV. The mass spectrometer was operated in data-dependent acquisition mode with one MS full scan and up to ten triggered MS/MS scans using HCD fragmentation (PMID:RNA pull-down experiments were performed 3 times\u2014once for mass spectrometry and twice for western blot to confirm the enrichment of HuR.\u00ae Reporter Assay (Promega) according to the manufacture\u2019s instruction and the EnVision Multilabel Reader (PerkinElmer).Cells were co-transfected with Super 8x TOPFlash or Super 8x FOPFlash luciferase plasmid together with either target-specific esiRNA or overexpression plasmid as well as pCMV renilla plasmid to correct for transfection efficiency. Cells were cultured in N2B27+2i+LIF. 48 h after transfection cells were lysed and bioluminescence was analysed using the Dual-LuciferaseTotal cell lysates were harvest 72h after transfection using RIPA lysis buffer. 20 \u03bcg of each protein sample were loaded and analysed by western blot using a mouse anti-HuR monoclonal antibody , rabbit anti-Tubb3  and mouse anti-Gapdh  as primary antibodies. As secondary antibodies HRP-conjugated species-specific antibodies  were used. For quantitative analysis species-specific secondary antibodies from LI-COR Biosciences have been used. Blots were scanned and analysed with Odyssey.t-test.The results are shown as mean \u00b1 standard deviation (SD) as indicated in the figure legends. Statistical significance was calculated using the unpaired Student\u2019s Sox1 is one of the earliest transcription factors marking the neural ectoderm  and Sox1lncR492 (accession no. AK016992) had the strongest effect (Z-score (1st esiRNA) = 4.0 and Z-score (2nd esiRNA) = 5.4; lncR492. Both esiRNAs achieved a knock-down efficiency of >60%  tail -tailed and capped , 6. cDNA(A) tail . Further capping . To furtock-down . Consistfraction .lncR492 as a non-coding nuclear transcript, which might play a role in regulating ectodermal cell fate commitment.Thus, the esiRNA-based screen in differentiating ESCs identified lncR492 acts as an inhibitor of ectodermal differentiation. To clarify its role and specificity in lineage commitment, we first tested its expression in pluripotent and differentiating ESCs. These results revealed that lncR492 has its highest expression in the pluripotent state. Interestingly, together with Oct4, lncR492 is down-regulated during neural differentiation , Brachyury-GFP+  and Foxa2-GFP+ (endoderm) cells after differentiation. In accordance with the reporter, the lineage specific genes were up-regulated when the cells were cultured under differentiation conditions in N2B27 alone (Sox1-GFP) or supplemented with 30 \u03bcg/ml Activin A (Foxa2-GFP) or 10 \u03bcg/ml BMP4 -positive cells in the lncR492 knock-down condition compared to cells treated with a control esiRNA as seen by immunofluorescent staining  and obtained similar results , which was most enriched in pull downs with fragment 1  was identified as well as an 11 bp long sequence in the 3\u2019 region (HuR BS2) of the transcript  mRNAs  and RNA-anscript . The posern blot . ESC difock-down . Importantiation . Likewisntiation . In accopression . Howeverpression . Given tpression . Hence, lncR492 gene expression. Interestingly, knock-down and overexpression of HuR resulted in a respective decrease and increase of lncR492, whereas Srrm4 transcript levels were not affected Click here for additional data file.S2 FigLncR492 expression in differentiated ESCs. Therefore T-GFP and Foxa2-GFP reporter cells were differentiated N2B27-containing medium supplemented with 10 \u03bcg/ml BMP4 or 30\u03bcg/ml ActivinA for 4 days, respectively. Sox1-GFP ESCs were differentiated for 4 days in medium containing the serum replacement N2B27 only. GFP+ cells were sorted by FACS, RNA was isolated and lncR492 as well as lineage specific gene expression was analysed by qRT-PCR. Expression was normalized to undifferentiated ESC. Data presents mean \u00b1 SD of three independent experiments.(A) lncR492 and Srrm4 during the time course of differentiation. Data presents mean \u00b1 SD of three independent experiments.(B) QRT-PCR analysis of lncR492 knock-down or overexpression in T-GFP ESCs. Bar graph represents the quantification of three independent western blot experiments. Data presents the mean \u00b1 SD.(C) Western blot analysis of Tubb3 after lncR492 knock-down or overexpression. Cells were harvested after 4 days of differentiation in N2B27. Data presents the mean \u00b1 SD of three independent experiments.(D) Gene expression analysis of the T-GFP reporter ESC (R1/E) by qRT-PCR after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in N2B27+2i+LIF medium. Data represents mean \u00b1 SD of four independent experiments.(E) FACS analysis of GFP expression after lncR492 knock-down and overexpression in Oct4-GFP ESC cultured in medium supplemented with FCS+LIF. Knock-down of Rad21 was used as a positive control. Data represents mean \u00b1 SD of four independent experiments.(F) FACS analysis of GFP expression after * p<0.05; ** p<0.01; *** p<0.001; n.s.\u2013not significant.(TIF)Click here for additional data file.S3 FigSrrm4 expression after lncR492 knock-down and overexpression measured by qRT-PCR. Data represents mean \u00b1 SD of three independent experiments.(A) (B) Summary table of proteins detected by mass spectrometry analysis. The lncRNA transcript was split into five overlapping fragments of 450 bp length each. The top ten putative interaction proteins for each lncRNA fragment are listed according to their abundance.lncR492. Putative binding sides for HuR are highlighted in red based on the consensus sequence NNUUNNUUU.(C) Nucleic acid sequence (mRNA) of (D) Western blot of HuR knock-down and overexpression. Gapdh was used as loading control. EV\u2014empty vector.lncR492 and HuR knock-down or HuR overexpression. Cells were differentiated for 4 days in N2B27 supplemented with 30 ng/ml ActivinA. Data presents mean \u00b1 SD of three independent experiments.(E) FACS analysis of Foxa2-GFP expression after (F) FACS analysis of Oct4-GFP expression 48h after HuR knock-down and overexpression. Oct4-GFP cells were cultured in N2B27+2i+LIF medium. Data presents mean \u00b1 SD of three independent experiments.* p<0.05; ** p<0.01; *** p<0.001; n.s.\u2013not significant.(TIF)Click here for additional data file.S1 TableZ-scores of the primary and the validation screen are shown for each replicate. Hits of the primary screen with an average Z-score >3 are highlighted in green (increasing the number of Sox1-GFP positive cells) and hits with an average Z-score < -3 are highlighted in orange (decreasing the number of Sox1-GFP positive cells). In the validation screen a Z-score > 2 or <-2 are considered as hit and highlighted in green.(XLSX)Click here for additional data file.S2 TableIdentified proteins for each fragment used in the pull-down experiment are shown.(XLSX)Click here for additional data file."}
+{"text": "Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3\u0394 S. pombe cells, while the dph3\u0394 mutant was sensitive. The msh3-ATG mutation, but not dph3\u0394 or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the The online version of this article (doi:10.1007/s00294-017-0711-x) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae    by Trm9\u2013Trm112 and to 5-carbamoylmethyl-uridine (ncm5U34) by an unknown enzymatic activity  and methyl methanesulfonate (MMS). Msh3 is a eukaryotic homologue of bacterial MutS. MutS is a DNA mismatch binding protein that initiates removal of mismatched and unpaired nucleotides, which were incorporated into the nascent strand during replication  that drug sensitivity was indeed due to an impaired dph3 function. Thus, Dph3 plays a role in response to DNA damage and replication stress, likely through modifications of tRNA and/or eEF2, which allow efficient biosynthesis of DNA damage response proteins.In this study, we discovered that replacement of the open reading frame of S. pombe msh3 and dph3 genes share an intergenic region of only 268 base pairs (bp) between the two ATG start codons . Data obtained by functional genomics further revealed that the intergenic region constitutes the 5\u2032 untranslated regions (5\u2032 UTR) of the msh3 and dph3 mRNAs, with divergent and likely overlapping promoters  from here on  of base\u2013base mismatches and loops of one to four nucleotides , demonstrating that the bands produced with the reverse-transcribed samples reflect amplification of cDNA derived from mRNA. In addition, PCR on a cDNA sample of a dph3::loxP-ura4-loxM strain yielded no bands , demonstrating that the amplified DNA was dph3 specific. We then used dph3q-F and seven reverse primers for PCR that prime at different positions, covering most of the dph3 mRNA. We could not detect any relevant differences between wild type and the msh3::kanMX mutant  colonies, indicative for a switching defect. The h90dph3::loxP-ura4-loxM and h90dph3-ATGmut mutants formed iodine positive colonies, although less homogeneously stained than wild type  colonies were not formed. Thus, replacement of dph3 by the ura4 marker did not disrupt the Msh3 function in mating-type switching. The phenotype of reduced sporulation of dph3 mutants is unrelated to Msh3 functions, as dph3-ATGmut does not have an integrated cassette that may interfere with msh3 expression.We noticed that colonies of the ies Fig.\u00a0a. Both dres Fig.\u00a0b. On thedph3 (kti11), like the other dph mutated genes in S. cerevisiae causes resistance to diphtheria toxin and sordarin due to the lack of the diphthamide modification of eEF2  mutants and unlike the other dph mutants  for lysine have low protein levels in elp3 mutants  after 3\u00a0days of incubation at 30\u00a0\u00b0C. Drugs used were HU (Formedium), MMS (ACROS Organics) and sordarin (Santa Cruz). Spot tests were carried out at least twice, with the exception of the experiments including the plates with 100\u2013150\u00a0\u03bcg/mL sordarin. To determine spore formation efficiency 10\u00a0\u03bcL of 1:10 dilutions in H2O of stationary phase cultures were spotted on MEA and incubated for 2\u00a0days at 30\u00a0\u00b0C. Means and standard deviations were calculated from six independent experiments, where 200 units (asci and zygotes) were counted for each of them. S. pombe strains are listed in Supplemental Table S1. The S. cerevisiae strain RCY2459 (MATa ho::LYS2 ura3 lys2 leu2::hisG in SK1 background) was a kind gift of Rita Cha .Yeast media were YEA (yeast extract agar), YEL (yeast extract liquid), MEA  and MMA  as described  was obtained by transformation of the 972 wild type strain (h\u2212) with a PCR product obtained with primers D3kf 5\u2032-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGCCAGCTGAAGCTTCGTAC-3\u2032 and Dk3r 5\u2032-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGGCCACTAGTGGATCTGATA-3\u2032 using plasmid pFA6a-kanMX  was constructed by transformation of OL2137 (h\u2212ura4-D18) with a PCR fragment obtained with primers D3kf and Dk3r (see above) and pFA6a-hphMX6  derived from transformation of EH238 (smt-0 leu1-32 ura4-D18) with a PCR fragment obtained with primers msh3_pAW1_For 5\u2032-TTTTTAAAACGTCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACCGGATCCCCGGGTTAATTAA-3\u2032 and msh3_pAW1_Rev 5\u2032-AGAACTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCGAATTCGAGCTCGTTTAAAC-3\u2032 using pAW1  is a transformant of OL2137 with a PCR fragment obtained with primers dph3_pAW1_For2 5\u2032-CTTAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAAAGCTTAGCTACAAATCCCAC-3\u2032 and dph3_pAW1_Rev2 5\u2032-TATCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAAAGCTTGTGATATTGACGAAAC-3\u2032 and pAW1 as template.DE4 , a derivative of pAW8 6 tag followed by three glycine-encoding codons, added between the natural ATG start codon and the second codon of msh3. Nucleotides underlined in msh3_pAW8_Rev 5\u2032 changed the two last codons of msh3 from GAA ATC (encoding the amino acids glutamic acid and isoleucine) to GAG CTC (encoding glutamic acid and leucine), thereby introducing a SacI restriction site, allowing further manipulation if desired.Plasmid pAW8-msh3 was constructed by in vitro Cre recombination between the h\u2212dph3-ATGmut ura4-D18) was constructed by transformation of DE4 with a PCR fragment obtained with primers dph3_ATGmut_For 5\u2032-TAGCTTGTAGTTTTCATTATGGGCGGTTCCCACATATAAAACAAATTTTTGGTGGAGTGGCCACGCACCTTCTGCCAGTAGTGCATTGAAGCGGCAAA-TGATCATTTTACGACGAAATCG-3\u2032 and dph3_Rev 5\u2032-TCACGATGTAAAGAGTAGCCCTCCTATCCTTCGTAATTTCAAGACATTTTGAGAATAAATAGAAGTAAAAAACCAAATAAGAAATTATAGGGAAAAATTATGCTGCAATGATTATAGGTG-3\u2032 and as template genomic DNA of strain RO144 (smt-0). \u201c-\u201din primer dph3_ATGmut_For indicates deletion of an A of the wild type sequence, which causes an in frame TGA stop codon instead of the ATG start codon. By this procedure, the lox sites flanking dph3 in DE4 were replaced in DE5. Sequencing confirmed the correct mutation in DE5 and an additional T155A point mutation, which is in intron II of the dph3 gene.Strain DE5 (smt-0 msh3-ATGmut leu1-32 ura4-D18) originated from the transformation of KK83 with a PCR fragment obtained from a mutagenized pAW8-msh3 plasmid as template. This plasmid contains the msh3 gene, with the 31st codon mutagenized from ATG to ATC by site directed mutagenesis with a QuikChange lightning site-directed mutagenesis kit (Agilent Technologies) using primers msh3_M31I_S 5\u2032-GGAGCAATATCAGAAGATATCGTTGCCCTCAGTGGTCCAG-3\u2032 and msh3_M31I_AS 5\u2032-CTGGACCACTGAGGGCAACGATATCTTCTGATATTGCTCC-3\u2032. Nucleotides deviating from the wild-type sequence are underlined. Primers for amplifying the 5\u2032 part of the open reading frame of msh3 with ATG mutations at codons 1 and 4 were msh3-ATGmut_For2 5\u2032-TCCGCCTATATTCATTTAGAAACTATAAATGCGTTACAAATCTATGTTGAGCAAATTTGATTGTACAGTTTTTATTTTCGCTTTATTACTCGAGAGGATAGAGTTATAACATTACTCATG-3\u2032 and msh3_Rev2 5\u2032-CTTAAAACTGATGTATAGTTGCTTAAATTTATTTATACAGTAAAATCTTATGTGTTGAGTATCCTAATAAAGTAGTTAATTCAGTAATGCTCTCTCTCAGATTTCTTCGAAAGCGGTAAG-3\u2032. Primer msh3-ATGmut_For2 contained base substitutions (underlined), which changed the ATG start codon of msh3 to TCG and the fourth codon from ATG to TAG. After integration into the genome, the mutated msh3 gene was amplified by PCR and sequencing confirmed the three desired ATG mutations. In addition, we found A555G, C696T and T1112C mutations. The primers used for PCR to construct an ATG mutated msh3 strain were designed in such a way that the (His)6 tag, the glycine linker and the lox sites of plasmid pAW8-msh3-M31I were not present in the genome of the resulting DE7 strain.DE7  including DNase I treatment. 2\u00a0\u00b5g of RNA were reverse transcribed with oligo (dT)18 primers using a Tetro cDNA Synthesis kit (Bioline). The resulting cDNA was subjected to 22\u201335 cycles of 30\u00a0s at 94\u00a0\u00b0C, 30\u00a0s at 57\u00a0\u00b0C and 30\u00a0s at 72\u00a0\u00b0C and a final extension for 10\u00a0min at 72\u00a0\u00b0C. For amplification of dph3 cDNA the forward primer dph3q-F 5\u2032-AGATTTCACGTTTGACGCCG-3\u2032  and the reverse primer dph3q-R 5\u2032-CTTGGGCAACGAGCAACATC-3\u2032 was used. In a pilot experiment , we used primers dph3_cPCR_For2 5\u2032-CTATGATGAAGATGAATTCATGGAAGTTG-3\u2032 and dph3_cPCR_Rev2 5\u2032-GAACAACAAACGTACTATGCCAATACAAAACC-3\u2032. Other reverse primers for amplification of dph3 cDNAs of various sizes were dph3q-R#2 5\u2032-TCACCCGGACAATCAAGCTG-3\u2032, dph3q-R#3 5\u2032-AGGTGCTGTAGAAGCATCGT-3\u2032, dph3q-R#4 5\u2032-GTAGCCCTCCTATCCTTCGT-3\u2032, dph3q-R#5 5\u2032-TGGAAAAGTCGTACGCTCAA-3\u2032, dph3q-R#6 5\u2032-AGGTGGGCTTTTAGTTTCGAGT-3\u2032, dph3q-R#7 5\u2032-ACGTTGAGCACAAGTACGAA-3\u2032 and dph3q-R#8 5\u2032-CCACTGTGGTATGTCGCACT-3\u2032. Primers for amplification of act1 cDNA were act1-For 5\u2032-AAGTACCCCATTGAGCACGG-3\u2032 and act1-Rev 5\u2032-CAGTCAACAAGCAAGGGTGC-3\u2032. PCR products were separated on 1.5% agarose gels in Tris\u2013Borate-EDTA buffer and images taken with a Gel Doc 2000 system (Bio-Rad). Band intensities of PCR products were quantified with the ImageJ software (NIH). dph3-specific bands were normalised to blanks to subtract background and to act1 cDNA.10\u00a0mL cultures were grown in YEL at 30\u00a0\u00b0C to a density of approximately 10S. pombe cells Supplementary material 2 (DOCX 15\u00a0kb)Below is the link to the electronic supplementary material."}
+{"text": "C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis.VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the  Highlighted Article:vpr-1 null mutants are sterile upon hatching, a defect rescued by the expression of MSPd from almost any tissue except for the somatic gonad itself. See also the companion paper by Schultz et al. The major sperm protein domain (MSPd) is an evolutionarily conserved immunoglobulin-like structure found in unicellular and multicellular eukaryotes . The nam2+ dynamics and other processes. In addition, the VAP MSPd is cleaved from the transmembrane domain and secreted in a cell type-specific fashion  A Lev et. Althoug fashion . The sec fashion . Hence, C. elegans. The C. elegans genome encodes numerous proteins that contain an MSPd  and mouse Vapb mutants  null mutation eliminates the first two vpr-1 exons, which encode the MSPd and part of the coiled-coil motif (vpr-1(tm1411) mutants derived from P0 vpr-1(tm1411)/hT2 heterozygous hermaphrodites exhibit limited fertility, with an average brood size of \u223c30 F2 progeny (vpr-1(tm1411) hermaphrodites lacking maternal (M\u2212) and zygotic (Z\u2212) vpr-1 expression (vpr-1(tm1411) mutant adults with maternal vpr-1 mRNA (M+ Z\u2212) contain functional sperm and oocytes /+ progeny produce functional gametes, similar to wild-type and M+ Z\u2212 vpr-1 mutant hermaphrodites  null mutants transgenically expressing vpr-1 from fosmid DNA contain \u223c10 sheath cells (vpr-1 M\u2212 Z\u2212 gonads contain severely reduced numbers of sheath (\u223c4) and spermathecal (\u223c6) cells  blast cells formed during L1 and early L2 C Hubbar. To inveransgene . In adulransgene E. We obscomplete E. Furtheskeleton . vpr-1 mth cells F. SS blath cells C. The adth cells C  cells H-J. The ntiation .vpr-1 is broadly expressed in most tissues (vpr-1 functions (i.e. in which cell type) to promote gonad development. Transgenic vpr-1(tm1411) null mutants were generated using a DNA fosmid containing the vpr-1 genomic locus and a plasmid containing the sur-5p::NLS-GFP lineage marker  worms rescued the gonad defects  or failed ovulation, two defects consistent with abnormal somatic gonad development or function  in the germ line using the pie-1 promoter (pie-1p::vpr-1g). Single-copy integrants on chromosome II were used to avoid germline silencing mechanisms. Two independently generated integrated transgenes completely rescued the vpr-1(tm1411) gonad and body wall muscle mitochondrial defects .Genetic mosaic worms lacking  fertile , consist defects A-D  gonad defects  background. A small percentage (<20%) of transgenic mutants exhibited the novel gonad phenotype described above. In addition to the neuronal promoters, overexpressing vpr-1g specifically in the intestine using the ges-1 promoter . These results show that vpr-1 overexpression in diverse cell types is sufficient to promote gonadogenesis.A caveat with integrated transgenes is that ectopic expression in unwanted tissues may result from surrounding regulatory sequences. To avoid this issue, we tested a panel of well-characterized neuron-specific promoters using high-copy extrachromosomal arrays . Overexppromoter  largely  defects B,F. Simirneurons , the unc neurons , and the neurons   null and clr-1(e1745ts) temperature-sensitive mutants have degenerating gonads , genetic mosaic and transgenic expression studies indicate that this degenerative defect is a consequence of hypodermal clr-1 loss, which causes massive fluid accumulation and larval lethality  (vpr-1(tm1411) clr-1 RNAi hermaphrodites and transgenic vpr-1(tm1411); clr-1(e1745ts) hermaphrodites expressing clr-1 in the hypodermis exhibit correct muscle mitochondrial localization, yet have arrested gonads . Therefore, the clr-1 muscle pathway is unlikely to influence gonadogenesis. The MSPd also antagonizes the VAB-1 Eph receptor, which is expressed throughout the nervous system (vab-1(dx31); vpr-1(tm1411) double-null mutants have arrested gonads , indicating that excess VAB-1 signaling does not cause arrested gonadogenesis. Collectively, the results are consistent with MSPd signaling being important for gonad development, but independent of excess CLR-1 or VAB-1 signaling individually.The VPR-1 MSPd has been shown to interact with two broadly expressed receptors, CLR-1 and VAB-1, as well as an unidentified receptor(s) . clr-1 esurvival . In adulsurvival . We consFig.\u00a0S1) . Adult vs system . vab-1; glp-1(ar202ts) double mutants. glp-1(ar202ts) is a temperature-sensitive (ts) gain-of-function mutation (vpr-1(tm1411) single-mutant, glp-1(ar202ts) single-mutant, and vpr-1(tm1411); glp-1(ar202ts) double-mutant hermaphrodites grown at the restrictive temperature (25\u00b0C) from L1 (vpr-1(tm1411); glp-1(ar202ts) gonads (glp-1(ar202ts) gonads (vpr-1(tm1411) gonads  gonads D lack th) gonads C and ins) gonads B. This rglp-1(bn18ts) is a temperature-sensitive loss-of-function mutation (glp-1(bn18ts) and vpr-1(tm1411) single mutants are small, but with an important difference. Germ cells in glp-1(bn18ts) gonads differentiate as sperm, whereas germ cells in vpr-1(tm1411) gonads fail to differentiate. If GLP-1 signaling is independent of vpr-1, then vpr-1(tm1411); glp-1(bn18ts) double-mutant gonads should contain sperm. To test this prediction, we grew single- and double-mutant hermaphrodites at 25\u00b0C from L1, stained them with DAPI, and performed fluorescence deconvolution microscopy. Characteristic highly condensed sperm chromosomes are not observed in vpr-1(tm1411) gonads (glp-1(bn18ts) single-mutant (vpr-1(tm1411); glp-1(bn18ts) double-mutant (vpr-1 mutants. These genetic data support the model that vpr-1 and glp-1 act in independent genetic pathways to promote germ cell expansion and differentiation.mutation . Gonads ) gonads B, but are-mutant E and vpre-mutant F gonads.vpr-1 activity we used the Q system, a drug-inducible binary gene expression system , which is added to plates, inhibits QS repressor activity, thereby activating gene expression. We used the glr-5 promoter to drive QF and QS expression in \u223c56 interneurons  mutants . Six lines grew robustly in the presence of QA. All seven lines exhibited minimal QA-independent vpr-1 expression, as indicated by gamete development in a small percentage of transgenic vpr-1(tm1411) hermaphrodites. The Q system was more tightly regulated than the heat shock promoter (data not shown). Providing QA produced functional VPR-1, as evidenced by rescue of the muscle mitochondrial defect . We selected line 3 for further characterization because it grew very slowly without QA but grew rapidly with QA. An advantage of this line is that sufficient numbers of transgenic vpr-1(tm1411) homozygotes could be generated for staging. Similar results were observed for lines that failed to grow without QA.SS blast cells form during L1 and early L2, but do not complete development until mid to late L4 . To deten system . The QF cription A. A tranrneurons A. Seven vpr-1 mutant gonads. Class 2 gonads contain visible oocytes or an expanded germ cell population, but are sterile with no fertilized eggs in the uterus. Class 3 gonads contain differentiated gametes and fertilized eggs. In the absence of QA, 57% of transgenic vpr-1 mutant gonads were class 1 and 19% were class 3  embryos is sufficient for gonad induction. As this maternal contribution is not renewable, we considered the possibility that vpr-1 is sufficient transiently, early in development. To investigate this idea further, we provided QA to transgenic vpr-1 mutant adults, let their progeny hatch on QA plates, and then moved the L2 worms to plates lacking QA. We found little difference in class 3 gonad percentage in these experiments compared with controls grown continuously on QA  or P4 (germ line) lineage caused gonads to arrest, although gonad development often appeared more advanced than that of gonads in M\u2212 Z\u2212 vpr-1 animals. Intestinal vpr-1 loss also caused sterility in some animals. Gonads from these mosaics produced oocytes but exhibited ovulation defects or other defects consistent with an abnormal somatic gonad. Our mosaics used fosmid DNA containing the entire vpr-1 genomic locus and likely most gene regulatory sequences. Although transgenes are typically silenced in the germ line due to their repetitive nature /hT2 [bli-4(e937) let-?(q782) qIs48] ; JK2868 qIs56 [lag-2p::GFP+ unc-119(+)]; WS2170 opIs110 [lim-7p::YFP::act-5+unc-119(+)]; OD58 unc-119(ed3) III; ltIs38 [pie-1p::GFP::PH(PLC1delta1)+unc-119(+)]; DG1575 tnIs6 [lim-7p::GFP+rol-6(su1006)]; CB4108 fog-2(q71); CZ337 vab-1(dx31); GC833 glp-1(ar202); DG2389 glp-1(bn18); XM1101 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; clr-1(e1745ts); XM1102 clr-1(e2530)/mIn1 [dpy-10(e128) mIs14]; XM1103 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; vab-1(dx31); XM1104 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(ar202); Xm1105 vpr-1(tm1411)/hT2 [bli-4(e937) let-?(q782) qIs48]; glp-1(bn18). E. coli . The folclr-1(e1745), glp-1(ar202) and glp-1(bn18) temperature-sensitive alleles were conducted at permissive (16\u00b0C) and restrictive (25\u00b0C) temperatures, as indicated. Strain construction was performed using PCR, sequencing and phenotypic analyses. vpr-1(tm1411) mutants are maternal effect sterile. Phenotypes were evaluated in vpr-1(tm1411) homozygous F2 progeny from vpr-1(tm1411)/hT2 heterozygotes (P0), unless otherwise indicated. vpr-1(tm1411) homozygous F1 progeny contain maternal vpr-1 mRNA. To investigate zygotic vpr-1 activity, progeny of fertile F1 vpr-1 mutants mated to wild-type males were examined. RNAi was performed by the feeding method        . This vpr-1 genomic fragment enhanced rescue of the vpr-1 mutant gonadogenesis defect relative to the vpr-1 cDNA fused to the unc-54 3\u2032 UTR. PCR was used to amplify sequences from genomic DNA. The unc-119p::vpr-1g, unc-25p::vpr-1g, unc-17p::vpr-1g, glr-5p::vpr-1g and ges-1p::vpr-1g constructs were generated using PCR and restriction enzymes in a TOPO vector backbone. The rol-6p::vpr-1g and myo-3p::vpr-1g constructs were generated using Gibson assembly (New England Biolabs) in a pGEM backbone. Primers are shown below. The pie-1p::vpr-1g construct was generated by Knudra Transgenics. The pie-1 promoter sequence included 1095 bp upstream of the translational start site. The vpr-1 DNA sequence included exons and introns, as well as 745 bp of the 3\u2032 UTR.Pan-neuronal (2000 bp) , GABA mo1893 bp) , choline2003 bp) , head in0003 bp) , intesti2003 bp) , body wa2385 bp)  and hypo2000 bp)  promoterclr-1 genomic locus, Gibson assembly was used to construct a plasmid containing clr-1 2\u2005kb left homology arm::tdTomato::clr-1 3\u2032 UTR::C. briggsae unc-119::2\u2005kb right homology arm. The single guide RNA (sgRNA) plasmid was derived from Addgene plasmid 46169. Cas9 targeting sequence was 5\u2032-ACTATATCTCTAAGACATAT-3\u2032. PCR was used to amplify the entire sgRNA backbone, except for 20 bp from unc-119. DNA fusions were constructed using Gibson assembly. The rol-6p::clr-1 construct was made with clr-1 genomic DNA. 2\u2005kb upstream of the rol-6 start codon was amplified by PCR. All constructs were confirmed by sequencing (UAB Heflin Center for Genomics Sciences). Primers  were: vpr-1 F1 sacII, GGGGACAACTTTCCGCGGAAAAAAATGTCTGAAAAGCACAGTCTTCTG; vpr-1 R1 kpnI, GGGGACTGCTTTGGTACCCCGAGATAATACGGCGAAAA; glr-5 F1 BHI NtI, GGGGACAACTTTGGATCCGCGGCCGCGTCACAATTTTCGGGTGTCGTAG; glr-5 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCGATGCTTATTATTCACATGTTTCAAACC; unc-17 F1 BHI NEI, GGGGACAACTTTGGATCCAGCGGCCGCTTCACACAATTAAGAATTTTAAGATTTGGG; unc-17 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTCTCTCTCTCCCCCTGGAATATT; ges-1 F1 BHI NtI, GGGGACAACTTTGGATCCAGCGGCCGCAAACTCCGAACTATGATGACGAA; ges-1 R1 NeI ScII, GGGGACTGCTTTCCGCGGGCTAGCCTGAATTCAAAGATAAGATATGTAATAGATTTTT; unc-25 F1 BHI NtI, GGGGACAACTGGATCCAGCGGCCGCGAGAAATAAGAAATAATTGTATAATTTTTTTTTC; unc-25 R1 ScII NeI, GGGGACTGCTTTCCGCGGGCTAGCTTTTGGCGGTGAACTGAGCTTTT; topo F1 KpnI, GGGGACAACTTTGGTACCCCTGAATGGCGAATGGAC; topo pR1 BHI, GGGGACTGCTTTGGATCCAGCTCACTCAAAGGCGGTAA; vpr-1 F1 NheI, GGGGACAACTTTGCTAGCAAAAAAATGTCTGAAAAGCACAGTCTTCTG; glr-5 R1 ScII NeI, GGGGACTGCTTTGCTAGCCCGCGGGATGCTTATTATTCACATGTTTCAAACC; F1 rol-6 pgem5, AGGTCGACCATATGGGAGAGCTAGAAAAACGATGGATTGAGTTATCTGG; R1 vpr-1 rol-6, AGACTGTGCTTTTCAGACATCTGGAAATTTTCAGTTAGATCTAAAGATATATCC; F2 rol-6 vpr-1, GATCTAACTGAAAATTTCCAGATGTCTGAAAAGCACAGTCTTCTG; R2 pgem vpr-1, CTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC; F1 pmyo-3 pgem5, GGTCGACCATATGGGAGAGCTGGCTATAATAAGTTCTTGAATAAAATAATTTTCCC; R1 vpr-1 pmyo-3, GCAGAAGACTGTGCTTTTCAGACATTTCTAGATGGATCTAGTGGTCGTGG; F2 pmyo-3 vpr-1, CCACGACCACTAGATCCATCTAGAAATGTCTGAAAAGCACAGTCTTCTGC; R2 pmyo-3 pgem5, GCTATGCATCCAACGCGTTGGGAACCATAAACATCAAATTTTATTGTACCATATAC.To create the Cas9 DNA template for tdTomato insertion into the Microscopy images were taken by a motorized Zeiss Axioskop equipped with epifluorescence and AxioVision software version 4.8. For DAPI staining, worms were fixed in 10% neutral buffered formalin (Sigma-Aldrich), mixed with 0.5\u2005\u00b5g/ml DAPI, and incubated for 24\u2005h at 4\u00b0C. Worms were then washed five to eight times with sterile water and mounted for microscopy. To image gonads, axial scans were performed and out-of-focus light was removed with deconvolution software (AxioVision).C. elegans, plasmids (5-60\u2005ng/\u03bcl) were injected into wild-type or vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. The myo-3p::mitoGFP or sur-5p::NLS-GFP constructs were used for selection  by Knudra Transgenics. Integrated transgenes were crossed into the vpr-1(tm1411) background and maintained as transgenic vpr-1 mutant homozygotes.To generate transgenic election . Multiplvpr-1 genomic locus together with 10\u2005ng/\u00b5l pTG96 (sur-5p::NLS-GFP) plasmid into vpr-1(tm1411)/hT2 hermaphrodite gonads  null hermaphrodites (vpr-1(tm1411) worms were screened from three independent lines. +vpr-1 loss in the AB, P1, P2, P3, P4, E, EMS and other lineages was scored as previously described  worms. Progeny were screened for rescue of the unc-119 movement defect and loss of myo3p::mitoGFP. Individual worms were isolated repeatedly to ensure 100% segregation. PCR and sequencing were used to confirm tdTomato insertion. The clr-1::tdTomato Cas9 line did not exhibit the fluid accumulation phenotype caused by reduced clr-1 function . Primers were : XW08 F1, CGGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATC; XW08 R1, GTTCTACGACACCCGAAAATTGTGACGCATGCAAGCTTGGCGTAATC; XW08 glr-5 F1, GATTACGCCAAGCTTGCATGCGTCACAATTTTCGGGTGTCGTAGAAC; XW08 glr-5 R1, GATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACCG; XW09 F1, GGTTTGAAACATGTGAATAATAAGCATCATGGGCGCGCCTCTAGAGGATCC; XW09 R1, CTACGACACCCGAAAATTGTGACGGCCGGCCCAGTCAGTGCG; XW09 glr-5 F1, CGCACTGACTGGGCCGGCCGTCACAATTTTCGGGTGTCGTAG; XW09 glr-5 R1, GGATCCTCTAGAGGCGCGCCCATGATGCTTATTATTCACATGTTTCAAACC; XW12 F1, GGGAAACTGCTTCAACGCATCATGAGTAAAGGAGAAGAACTTTTCACTG; XW12 R1, GCAGAAGACTGTGCTTTTCAGACATTTTTTCTACCGGTACCGTCGAC; VPR-1 F1, GTCGACGGTACCGGTAGAAAAAATGTCTGAAAAGCACAGTCTTCTGC; VPR-1 R1, AGGTGAAAGTAGGATGAGACAGCAACCATAAACATCAAATTTTATTGTACCATATACA; SL2 F1, TGTATATGGTACAATAAAATTTGATGTTTATGGTTGCTGTCTCATCCTACTTTCACCT; SL2 R1,CAGTGAAAAGTTCTTCTCCTTTACTCATGATGCGTTGAAGCAGTTTCCC.Q system plasmids ang Shen . Gibson C. elegans, glr-5p::QF-SL2::mCherry::3\u2032UTR-unc-54 (10\u2005ng/\u03bcl), glr-5p::QS-SL2::mCherry::3\u2032UTR-unc-54 (50\u2005ng/\u03bcl), QUASp::\u0394pes10::vpr-1-SL2::GFP::3\u2032UTR-unc-54 (10\u2005ng/\u03bcl) and myo-3p ::mito::GFP (30\u2005ng/\u03bcl) plasmids were injected into vpr-1(tm1411)/hT2 young adult hermaphrodite gonads. Transgenic lines were selected based on mCherry and myo3p::mito::GFP expression. Seven independent lines were created. Six responded well to quinic acid (QA) treatment. For treatment, 300\u2005\u00b5l 300\u2005mg/ml pH 6.5 QA (Sigma-Aldrich) was mixed with 40\u2005\u00b5l M9 buffer and added to NGM plates seeded with NA22 bacteria. All lines exhibited very low vpr-1 expression in the absence of QA, partially rescuing the vpr-1 mutant gonadogenesis defect in a small percentage of transgenic worms. Increasing the glr-5p::QS dosage in the transgenic arrays appeared to limit QA-independent vpr-1 expression.To generate transgenic t-tests were computed using Excel 2013 (Microsoft) without the assumption of equal variance.Two-tailed Student's"}
+{"text": "Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. Published 21, April 2016D. melanogaster, sequence #13 in Supplementary file 1, was incorrect due to a mistaken copy-and-paste while preparing the manuscript. The correct D. melanogaster sequence is now entered in the corrected The protein sequence of EMRE from This error did not affect any results or conclusions of the paper.Incorrect sequence:MTSKTVFQNAFKTFLDFAINSLPSTQGGLNITATAPGGVGQRPFTNKAGVLKLIFVSASSLYIGGLIAHKGASYLEENEIFVPTDEDDDDCorrected sequence:MIVPRLALPISLALQRVSRRVAEHPHNLRILQRHMSSVYFRSGAIKPKPEEMPFGLLAIFCAVIPGLFVGATISKNVANFLEENDLFVPADDDDDEDThe article has been corrected accordingly."}
+{"text": "The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-\u03b2 signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping. In previous work, we isolated a key gene involved in the cryptogenic cerebrovascular disorder moyamoya diseasein vivo.Here, we report the first example of isoform-specific bias in substrate preference of USP15. Biochemical evaluation of the association between USP15 and mysterin revealed that mysterin is predominantly recognized by the long isoform of USP15. Moreover, the USP15 long isoform potently deubiquitylated mysterin and maintained its basal expression level, indicating that alternative exon skipping biases the deubiquitylation substrate preference of USP15. In addition, we performed an extensive LC-MS/MS analysis of USP15-binding proteins and identified multiple isoform-specific binding proteins, suggesting that the two isoforms of USP15 play roles in distinct proteome networks and have overlapping but independent cellular activities To elucidate the cellular function of the moyamoya disease-associated protein mysterin, we sought to identify mysterin-binding proteins, reasoning that such proteins would include cofactors and/or substrates of mysterin. To this end, we transiently overexpressed mysterin harboring a FLAG tag at its C-terminus (mysterin-FLAG) in HEK293T human embryonic kidney cells. We precipitated mysterin-FLAG from cell lysates using anti-FLAG antibody and performed LC-MS/MS analysis on total co-precipitates without an isolation process such as SDS-PAGE. Four positive hits, including USP15, LYAR, hnRNPK, and IP3 receptor isoform 3 (IP3R3) in addition to mysterin itself, were reproducibly identified in four trials. In a replication study, we transiently expressed each candidate mysterin-binding protein in HEK293T cells, immunoprecipitated using specific antibodies, and then immunoblotted for mysterin. In this analysis, USP15 and LYAR were detectably physically associated with mysterin, whereas the other two proteins were not . However23We obtained cDNAs encoding both isoforms of USP15 from HEK293 cells by reverse transcription\u2013polymerase chain reaction (RT-PCR). To our surprise, the isoform of USP15 with longer linker region (hereafter USP15L) had significantly stronger affinity for mysterin than the other isoform with the shorter linker . Mysterin-3FLAG was precipitated using FLAG affinity beads, and its ubiquitylation state was examined by immunoblotting using anti-ubiquitin antibody. Overexpression of USP15L significantly decreased the polyubiquitin chain signal associated with mysterin-3FLAG, whereas overexpression of USP15S did not, suggesting that USP15L has the stronger deubiquitylation activity against mysterin . The pol0.5% SDS . Mutatio0.5% SDS , but did0.5% SDS , supportPrevious studies demonstrated that USP15 removes K48-linked polyubiquitin chains and thereby protects its substrates against proteasomal protein degradation triggered by K48-linked polyubiquitylation5How does the alternative variation in the linker region bias the affinity of USP15 for mysterin? Although the substrate-binding domain of USP15 has not been definitely identified, one simple model is that the linker region directly mediates mysterin binding. We evaluated the roles of structural regionss and domains in USP15, including the long linker region, in the mysterin-binding process using five truncated mutants of USP15, schematically represented in To further characterize the functional divergence of the two major isoforms of USP15, we performed an interactome analysis on each isoform, based on the expectation that each interactome will include substrates and/or cofactors specific for each isoform. We transiently overexpressed the long and short isoforms of USP15, C-terminally tagged with three tandem FLAG epitopes, in HEK293T cells; precipitated them from cell lysate using anti-FLAG antibody; and performed LC-MS/MS analysis on total co-precipitates without isolation. Positive hits reproducibly obtained in all four trials are shown in 927Alternative skipping of exon 7 in USP15 resulted in deletion of a short element consisting of 29 amino acids in the inter-region linker of USP15 , which s5The exon 7-encoded short element in the linker region of USP15 was necessary but not sufficient for the physical association with mysterin. Likewise, the DUSP domain and the C-terminal region were also essential but not sufficient. The C-region harbors the enzymatic core responsible for deubiquitylation activity12299279Sixma and colleagues recently reported that the DUSP domain of USP4 promotes the deubiquitylation cycle by discharging hydrolyzed free ubiquitin from the enzymatic core of USP4 through inter-domain interactionsVariation in the linker region is likely to have been maintained under natural selection. Phylogenic study suggested that USP4, 11, and 15 arose from a single ancestral USP, conserved between fungi and metazoans, following the emergence of vertebrates4in vivo. Despite the obvious phenotypes in mysterin knockdown/knockout zebrafish, the cellular and molecular functions of mysterin remain controversial. Recent studies proposed roles in insulin production in pancreatic \u03b2 cellsWe initiated this study based on our interest in the function and/or regulation of the moyamoya disease susceptibility factor mysterin. The resultant data suggest that USP15L stabilizes mysterin via removal of K48-linked polyubiquitin chains. These observations were obtained under conventional cell culture conditions, suggesting that USP15L maintains the basal expression level of this ubiquitin ligase, which was previously shown to have autoubiquitylation  activity19NheI/NotI sites (Myc) or NheI/HindIII sites (3FLAG) into the mammalian expression vector pcDNA3.1+ . Restriction enzymes were purchased from New England Biolab  or Fermentas . DNA ligase was purchased from Takara . The enzymatic mutant of USP15L (15Lmt), which harbors alanines instead of the cysteines at positions 269 and 812, and that of USP15S (15Smt), which harbors alanine instead of the cysteine at positions 269, were generated by mutagenesis PCR using the QuikChange Site-Directed Mutagenesis kit  using primers USP15C298AF (GCCTCTGTGGCCTAAGTAACTTGGGAAATACGGCTTTCATGAACTCAGCTATTCAGTGTTTGAGCAACACACC), USP15C298AR (GGTGTGTTGCTCAAACACTGAATAGCTGAGTTCATGAAAGCCGTATTTCCCAAGTTACTTAGGCCACAGAGGC), USP15C812AF (GCTGAAGATCCCTGGTATTGTCCGAATGCTAAAGAACATCAGCAAGCCACAAAGAAATTGG), and USP15C812AR (CCAATTTCTTTGTGGCTTGCTGATGTTCTTTAGCATTCGGACAATACCAGGGATCTTCAGC). Deletion mutants were generated using seven primers USP15FNheI (described above), USP15F2NheI (ATGCGCTAGCATGCCATCATATACCGCTTATAAGAAC), USP15FdN2NheI (ATGCGCTAGCCGCCATGGGTCCTTCTACTCCTAAGTCC), USP15FdN3NheI (ATGCGCTAGCCGCCATGGTAAAGCACTGCAAAGTAGAA), USP15R13FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAAGACAGTAATTTGAGTTTTTCACATT), USP15R23FLAGHindIII (ATGCAAGCTTCTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCAATAATCACGTGCTCTGTATCTTCTGTCC), and USP15R33FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). \u0394C1, \u0394C2, \u0394N1, \u0394N2, and \u0394N3 deletion mutants were generated by PCR using USP15FNheI and USP15R13FLAGHindIII, USP15FNheI and USP15R23FLAGHindIII, USP15F2NheI and USP15R33FLAGHindIII, USP15FdN2NheI and USP15R33FLAGHindIII, and USP15FdN3NheI and USP15R33FLAGHindIII, respectively.Full-length cDNAs for USP15L (NM_001252078.1) and USP15S (NM_006313.2) were obtained simultaneously by RT\u2013PCR using the OneStep RT-PCR kit  with template RNA extracted from HEK293 cells using the RNeasy Mini Kit (QIAGEN). USP15L or S with Myc or three tandem FLAG (3FLAG) epitope tag at the C-terminus was generated by PCR using forward primer USP15FNheI (ATGCGCTAGCACCATGGCGGAAGGCGGAGCGGC) and reverse primers USP15RMycNotI (ATGCGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGTTAGTGTGCATACAG) or USP15R3FLAGHindIII (ATGCAAGCTTTTACTTATCGTCGTCATCCTTGTAGTCGATGTCATGGTCTTTGTAGTCACCGTCATGATCCTTGTAATCGTTAGTGTGCATACAGTTTTC). These PCR products, harboring exogenous restriction sites and epitope tag at the 3\u2032 terminus, were subcloned using KpnI, NotI, SalI, and BamHI. Exogenous NheI and XbaI sites and the Myc epitope sequence were introduced into the 5\u2032 and 3\u2032 termini by PCR using forward primer CloNheIIP3R3F1 (ATGCGCTAGCCGCCATGAGTGAAATGTCCAGCTTTCTTCACATCGG) and reverse primer CloXbaIIP3R3R53FLAG (TACGTCTAGATCAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCGCGGCTAATGCAGTTCTGG). The PCR product was subcloned into pcDNA3.1+ using the NheI/XbaI sites. Full-length cDNA of LYAR was obtained by RT-PCR using forward primer KpnILYARCloF (ATGCGGTACCGCCACCATGGTATTTTTTACATGC) and reverse primer NotI3FLAGLYARCloR (TACGGCGGCCGCTTAGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCTTTCACAAGCTTGACTTTG), and then subcloned into pcDNA3.1+ using the KpnI/NotI sites.Mysterin-FLAG and -3FLAG were generated in our previous studies19HEK293 (used only for cloning of USP15) and HEK293T cells were maintained in DMEM  containing 10% fetal bovine serum, 100\u2009U/mL penicillin, and 100\u2009\u03bcg/mL streptomycin. Plasmids were transfected into HEK293T cells using Lipofectamine LTX (Life Technologies) or PEI MAX . The amount of DNA was adjusted to obtain equivalent expression levels of the introduced proteins in each experiment. For detection of ubiquitylation, cells were treated with 1\u2009\u03bcM epoxomicin  for 8\u2009h (or other indicated period) before harvest.Antibodies used for immunoprecipitation (IP), immunoblotting (IB), or immunostaining (IS) in this study include anti-FLAG antibody , anti-Myc antibody , andi-HA antibody , anti-ubiquitin antibody , anti-K48 linked ubiquitin antibody , and anti-GAPDH antibody . Anti-human mysterin mouse monoclonal antibody (1C9) was generated with the method previously reportedMysterin-FLAG or USP15L/S-3FLAG was expressed in HEK293T cells, and associated proteins were recovered from cell extracts by immunoprecipitation with anti-FLAG antibody. The associated protein complexes with mysterin-FLAG were immediately digested with lysyl endopeptidase . Protein complexes associated with USP15L/S-FLAG were reduced with 5\u2009mM TCEP, and then alkylated with 10\u2009mM monoiodoacetoamide. The alkylated protein complexes were digested with Lys-C and trypsin . The resultant peptides were analyzed using a direct nano-flow LC-MS/MS system, as described previouslyg, and the supernatant was incubated for 1\u2009hour with anti-FLAG M2 agarose beads (Sigma Aldrich) or anti-Myc antibody and protein G beads . At this point, aliquots of supernatant were resolved with Laemmli\u2019s sample buffer containing 1\u2009mM DTT as a control . The beads were washed three times with digitonin-containing HEPES buffer and resolved with Laemmli\u2019s sample buffer containing 1\u2009mM DTT.Protein-encoding or empty vector was transiently transfected into HEK293T cells. The cells were harvested 24 or 48\u2009hours after the transfection and lysed with HEPES buffer  containing 1% NP40 (for ubiquitylation assay) or digitonin . The lysate was centrifuged at 13,000\u2009Samples were separated with SDS-PAGE using 7.5% or 3\u201310% acrylamide gel  and transferred to nitrocellulose  or PVDF  membrane for 1.5\u2009hours at 75\u2009V at 4\u2009\u00b0C. Membrane was blocked using Blocking One  for 1\u2009hour at room temperature, and then incubated sequentially with primary and secondary antibodies diluted in Can Get Signal . Detection was performed via the chemiluminescence or the alkaline phosphatase method.2HPO4 and 1.47\u2009mM KH2PO4, pH 7.4). After incubation with Blocking One  for 4\u2009hours at room temperature, the cells were incubated sequentially with primary and fluorescence-conjugated second antibodies; each of the antibody incubations were followed by PBS washes. The nucleus was stained using Hoechst 33342. Fluorescence images were obtained by confocal microscopy .HEK293T cells expressing USP15L-Myc or S-Myc together with mysterin-3FLAG were immobilized with 4% paraformaldehyde  for 10\u2009min at room temperature 24\u2009hours after transfection. The cells were permeabilized with 100\u2009ng/mL digitonin for 5\u2009minutes, and then washed with phosphate-buffered saline .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or \u2018CATAC\u2019. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of  Drosophila melanogaster, a heterodimer consisting of CLOCK (CLK) and CYCLE (CYC), acts as the core transcription factor , Per-Arnt-Sim (PAS) domain transcription factors (TFs) for transcriptional activation . In the n factor ,3. CLK/Cn factor \u20136. The dn factor ,8.Drosophila, studies of the promoter regions of known clock genes, period (per) and timeless (tim), found that E-box-dependent enhancers are necessary for circadian transcriptional modulation. The per promoter has, arguably, the best studied circadian enhancer motif to date. The enhancer is a 69-bp sequence upstream of the transcription start site (TSS) that activates circadian gene expression  boxes, which are a variant of the consensus E-box sequence  and transcription factor binding sites (TFBSs) have provided more tools to determine the elements responsible for transcriptional regulation . As a continuation of this work, we, hereby, identified a second novel and independent 29-bp motif. The present study describes spatiotemporal expression information contributed by the latter motif, which was named Clock-Associated Transcriptional Activation Cassette or \u2018CATAC\u2019.Increasing knowledge of the gulation ,11. In pgulation , we identim, vrille, Pdp1 and cwo (stich1) in 12 Drosophila species . This identified a conserved set of CATAC sequences (Drosophila melanogaster genome (BDGP R5/dm3) is included in the The bioinformatics model used to predict the \u2018CATAC\u2019 element is identical to that previously described for the discovery of the E1\u2013E2 element . Brieflyequences . In ordeequences , we traiPdp1 or Slob promoters: Slob 4xCATAC enhancer, 5\u0384pGGCCGATAACGCGGCGTATGCGCAATGTCGAAGCATATTACGCATACGCCCCATCCGC; 5\u0384pGTTTGCTGTGGCGGATGGGGCGTATGCGTAATATGCTTCGACATTGCGCATACGCCGCGTTATC; 5\u0384pCACAGCAAACGCTGCGTATGCGTAATACTTTGTGCACACGTTGCGTATGAGTAATGTCCT; 5\u0384pAATTAGGACATTACTCATACGCAACGTGTGCACAAAGTATTACGCATACGCAGC, Slob mt4xCATAC enhancer, 5\u0384pGGCCGATATCCCGGCCTTTGCCCTATGTCGAAGCATATAAGGCAAGGCCCGAACCGC; 5\u0384pGATTGCTGTGGCGGTTCGGGCCTTTGCCTTATATGCTTCGACATAGGGCAAAGGCCGGGATATC; 5\u0384pCACAGCAATCCCTGCCTTTGCCT TATACTTTGTGCACTCCTTGCCTTTGACTTATGTCCT; 5\u0384pAATTAGGACATAAGTCAAAGGCAAGGAGTGCACAAAGTATAAGGCAAAGGCAGG, Pdp1 4xCATAC enhancer, 5\u0384pGGCCAGCACATTACGCATACGTCACGTGTTGCAAAAATAATACGTATACGACGCGTGTC; 5\u0384pTGGTACAGAAGACACGCGTCGTATACGTATTATTTTTGCAACACGTGACGTATGCGTAATGTGCT; 5\u0384pTTCTGTACCATGCGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCCCGTGGGC C; 5\u0384pAATTGGCCCACGGGGCGTATGGGTAACGTATTAACAGATTGCTCATACGCCGCA, Pdp1 mt4xCATAC enhancer, 5\u0384pGGCCAGCACATAAGGCAAAGGTCAGGAGTTGCAAAAATAAAAGGTAAAGGACGGGAGTC; 5\u0384pAGGTACAGAAGACTCCCGTCCTTTACCTTTTATTTTTGCAACTCCTGACCTTTGCCTTATGTGCT; 5\u0384pTTCTGTACCTTC CGGCCTTTGACCTATCTGTTAATACGTA AGCCAAAGGCCCGGAGGGCC; 5\u0384pA ATTGGCCCTCCGGGCCTTTGGCTTACGTATTAACAGATAGGTCAAAGGCCGGA, Pdp1 4xCATAC with Slob-like E-boxes enhancer, 5\u0384pGGCCAGCACATTACGCATACGTAACGTGTTGCAAAAATAATACGTATACGAAGCGTTTC; 5\u0384pTCGTACAGA AGAAACGCTTCGTATACGTATTATTTTTGCAACACGTTACGTATGCGTAATGT GCT; 5\u0384pTTCTGTACGATGGGGCGTATGAGCAATCTGTTAATACGTTACCCATACGCCGCGTTGGC C; 5\u0384pAATTGGCCAACGCGGCGTATGGGTAACGTATTAACAG ATTGCTCATACGCCCCA. Oligos were annealed to match their order and orientation in the native Pdp1 or Slob promoters. Flanking EagI and EcoRI sequences allowed for the 4xCATAC enhancers to be inserted upstream of the hsp70 basal promoter in the pPTGAL vector (luc) were created by blunt-end cloning to replace a 3193 bp fragment containing Gal4 flanked by PstI sites with a 1955 bp HindIII\u2013BamHI fragment containing the luciferase gene from pGL3-Basic (Promega) (luc can be requested online (http://www.addgene.org/Herman_Wijnen/). For each reporter construct, several independent lines were generated.Synthetic oligonucleotides were designed to match four separate, native occurrences of the CATAC element from either the L vector  (obtainePromega) . Copies 1118w,\u00a0UAS-CD8::GFP, and 2eya mutations were obtained from the Bloomington Drosophila Stock Center. The 01, per01cyc and tim-luc alleles have been described previously  and relative amplitude error (RAE) by an iterative, coupled fast Fourier transform-non-linear least squares (FFT-NLLS) multicomponent cosine analysis . RAE is Gal4 driven expression of membrane-tethered green fluorescent protein were dissected in Ringer's solution . Flies were harvested onto dry ice at time points CT0, CT6, CT12, CT18 and CT24, and adult heads were dissected on a chilled platform and transferred to guanidinium thiocyanate buffer. Four separate groups of flies (\u223c50 each) were used for each experimental condition. Total RNA was obtained from the heads using the RNAqueous4PCR kit (Ambion). Sample concentration and purity was analysed using a NanoDrop spectrophotometer. Samples exhibited OD 260/280 ratios between 1.8 and 2.1. Concentrations were adjusted to 25 ng/\u03bcl in 10 mM Tris\u2013HCl 0.1 mM EDTA pH 8.0 buffer and samples were frozen at \u201380\u00b0C in aliquots until further use. The RNA samples were then analyzed with the SensiFAST SYBR No-ROX One-Step qPCR Kit (Bioline) using experimental primer pairs designed to specifically amplify fragments of the circadian Pdp1 or Slob transcripts, the transgenic luciferase transcript or the EF1\u03b2 control transcript  relative to EF1\u03b2 were determined using the comparative Cycle threshold (Ct) method . ript see . All pri,23. riptcis-acting elements associated with clock gene expression, we combined MEME  for 12 species of Drosophila. As a result, a 29-bp motif designated the Clock-Associated Transcriptional Activation Cassette or \u2018CATAC,\u2019 was identified as over-represented in these promoters  is provn = 97 genes) encoding transcripts predicted to exhibit circadian oscillations (Slob (Slowpoke binding protein)  predicted to be induced upon activation of the circadian regulator CLK  as well llations \u201328 (Suppllations . A promiprotein) . With th genomes .Based upon the sequence alignment, conservation, and subsequent CLK/CYC target gene enrichment analyses, CATAC was hypothesized to be a regulatory element involved in mediating spatiotemporal expression of clock-regulated genes.Pdp1 and Slob, that not only exhibit strong CLK/CYC-associated circadian oscillations, but also have an unusually high number of CATAC motifs. The Pdp1 gene encodes a clock component (PDP1-\u03b5) that impacts molecular circadian oscillations, particularly in the clock neurons, as well as circadian behaviour  as well as the CRCGTG consensus for the E-box-like element within CATAC  which allowed measurement of the transcriptional reporter constructs at relatively high frequency in vivo. Native Pdp1 and Slob 4xCATAC both showed strong luciferase expression  and both mt4xCATAC constructs shared characteristically low level expression  in which the orientation of Slob 4xCATAC in the vector was inverted and \u2018Pdp1 4xCATAC with Slob-like E-boxes\u2019 (PSE) in which the four E-boxes of Pdp1 4xCATAC were mutated such that their sequence fidelity matched that of the four E-boxes in Slob 4xCATAC , was significantly stronger than those for mt4xCATAC and empty vector controls, respectively  and Slob (ZT 3\u20135). Heads showed luciferase expression in not only the compound eyes, but also the proboscis and antennae; each of which has been shown to possess a circadian oscillator  genetic background , by and large there was little effect of the 01per mutation on their expression levels  construct. PSE is a modified version of Pdp1 4xCATAC, which has E-box sequences that were mutated such that their deviation from the CACGTG consensus matched that of the four E-boxes in the Slob 4xCATAC enhancer , constructs. Thus, the considerable variation in quality of E-box sequences between these constructs did not obviously impact reporter gene rhythms. The sequence immediately flanking an E-box element has also been implicated in affecting regulation  is known to bind E-boxes in clock-controlled genes (For all the similarities that CATAC shares with E-box motifs, CATAC is not simply another E-box element. Normally, an E-box regulated transcriptional element exhibits low reporter expression in a in per01 ,41. CATAed genes ,43 and mPdp1 and Slob. Of course, this conclusion comes with the caveat that the multimerized constructs do not represent the complexity of the native genomic environment. Mutational analysis of CATAC sequences in their natural context will constitute an important next step in the functional analysis of this cis-regulatory element. Nevertheless, the identification of CATAC contributes to our knowledge of circadian transcriptional elements, and may be used to further characterize the regulatory regions of clock and clock-regulated genes.Thus, we have discovered a novel circadian regulatory element that, although it possesses an E-box-like motif, exhibits non-E-box-like responses. Through use of multimerized CATAC element, we concluded that CATAC is capable of contributing to the rhythmicity of clock genes, such as Supplementary DataClick here for additional data file."}
+{"text": "Open Biol. 7, 170139. (Published online 21 November 2017). (doi:10.1098/rsob.170139).A correction is required to the sequence of sgRNA_FD2 in .The correct sequence is as follows:sgRNA_FD2:TTCCAGAAGCCGCCTTTGCCCGGGTTTTAGAGCTAGAAATAGCAAG-3\u20325\u2032-TAATACGACTCACTATAAll the authors gave their consent to this correction."}
+{"text": "Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite.Examining essential biochemical pathways in Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2.Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. Despite the efficacy of artemisinin-based combination therapy (ACT) in the treatment of malaria, an estimated 214 million cases and 438,000 malaria deaths occurred globally in 2015  was added to each well on the plate. The plate was shaken for 10\u00a0min to ensure mixing and the absorbance at 595\u00a0nm was obtained using a CLARIOstar plate reader. .PF3D7_0525900) was purchased (with codon optimization for Escherichia coli expression) from GeneArt\u2122 Life Technologies. The codon optimized DNA sequence was as follows (the codon encoding V24 is highlighted in bold); ATGAGCAAACCGAAAATGATCGGTCCGTATGAAGTTGTGAAAAGCATTGGTCGTGGTAGCTTTGGTATTGTTACCGCAGTTAAAGATGAAAATGAAAAAATCTTTGTGATTAAAGAACTGGATATTAGCTGCATGAATAACAAAGAAAAAATGAATGTGGTGAATGAAATTCGTGCCCTGATTAAAATGAGCGTGCATCCGTTTATTGTGCGCTATAAAGAAGCCTTTGTTGAAGATTGCGTTCTGTATGTTGCGATGGATTATTGCATTAATGGTGATCTGGGCAAAGTGATCAAAAAACACAAAGAACTGGAAACCCCGATTCCTGAGAAAAAAATCAAACGTTGGCTGCTGCAGATTATTATGGCCATCAAATTCATTCATGATAAAAAACTGATCCATCGTGATCTGAAATGCAATAACATCTTCCTGGATGAAAAAGAACGTGCCAAAATTGGTGATTTTGGTCTGGCCAAATTTATCGAACAGACAGAACAGACCAATACCCTGTGTGGCACCATTGGTTATATGGCACCGGAAATTTGCAAAAATATCAATTACAGCTTCCCTGCCGATATTTGGAGCCTGGGTATTATTCTGTATGAACTGATTAGCCTGAAACCGCCTTTTAAAAGCAATAACAGCAATATGCTGAGCGTGGCCCAGAAAATTTGTGAAGATGAACCGGATCCGCTGCCGGATAGCTTTAGCAAAGATCTGATTAATCTGTGCTATTGGATGCTGAAAAAAGATTGGAAAGATCGTCCGACCATCTACGATATTATCAGCACCGATTATATCCAGGATGAACTGCAGCTGTTTAAACGTGAAATGCTGCAAGAACGTAACAGCCAGATT.The synthetic full-length coding sequence for Pfnek-2  and reverse (5\u2032-tctttaactgcggtacaaataccaaagctaccacgaccaatgc-3\u2032).E. coli BL21-Codon Plus(DE3)-RIPL strain (Agilent Technologies). Cells were treated with 1\u00a0mM IPTG when the optical density of the cell culture at 600\u00a0nm reached 0.6, and protein expression was induced overnight at 18\u00a0\u00b0C. For purification, cells were lysed in buffer  [1 tablet per 10\u00a0ml (cOmplete\u2122 ULTRA Tablets by Roche)] by sonication for 10 cycles of 10\u00a0s bursts at 10\u00a0mA with a 30\u00a0s rest between each cycle. During sonication the cells were kept on ice. Lysates were then centrifuged at 20,000g for 30\u00a0min. The resulting supernatants were loaded onto a 0.5\u00a0ml pre-equilibrated Ni\u2013NTA Superflow resin (Qiagen) column, washed with buffer  and proteins were eluted with the same buffer containing 400\u00a0mM Imidazole. Eluted proteins were dialysed against 50\u00a0mM bicine, pH 8.0, 150\u00a0mM NaCl, 1% glycerol and 1\u00a0mM DTT at 4\u00a0\u00b0C overnight. Dialysed Pfnek-2 protein was aliquoted and stored at \u221280\u00a0\u00b0C.Full-length Pfnek-2 as a 6-HIS-tagged protein was expressed in 2, 4\u00a0mM imidazole, 1\u00a0mM DTT) in the presence of 5\u00a0\u00b5g MBP. The reaction was started by the addition of 5\u00a0\u00b5l 200\u00a0\u00b5M ATP/32P-ATP (0.0185\u00a0MBq per reaction)\u2014total volume 20\u00a0\u00b5l. Following incubation of 30\u00a0min at 37\u00a0\u00b0C, the reactions were stopped by adding 10\u00a0\u00b5l 2\u00d7 Laemmli buffer and proteins were separated by SDS\u2013PAGE on 12% gels and stained with Coomassie blue. Dried gels were exposed to X-ray film.Recombinant His-tagged WT or V24C mutant Pfnek-2 were assayed for protein kinase activity using myelin basic protein (MBP) as a substrate. 1.8\u00a0\u00b5g of either Pfnek-2 WT or Pfnek-2 V24C were pre-incubated at room temperature for 40\u00a0min with/without NCL-00016066  in Pfnek-2 buffer  was dissolved in DMSO to 10\u00a0mM and frozen in aliquots of 10\u00a0\u00b5l at \u221280\u00a0\u00b0C. These aliquots were kept in the dark and a fresh aliquot diluted prior to each kinase assay. All samples in each kinase assay were pre-incubated with NCL-00016066 for 40\u00a0min at room temperature and the reaction initiated by the addition of ATP/2, 1% glycerol, 4\u00a0mM imidazole, 1\u00a0mM DTT at room temperature for 30\u00a0min. 50\u00a0\u00b5l Ni\u2013NTA Superflow resin (Qiagen) was added and the samples made up to 500\u00a0\u00b5l in buffer  and rotated for 1\u00a0h at room temperature. The beads were washed 3\u00d7 in 500\u00a0\u00b5l buffer for 5\u00a0min rotation and supernatants discarded. The in vitro kinase assay protocol described above was followed and the enzyme immobilized on beads (washed and supernatant removed) is assumed to be 5\u00a0\u00b5l for the purpose of the kinase assay. During incubation at 37\u00a0\u00b0C the beads were gently re-suspended every 10\u00a0min.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described and incubated (1:1) without/with 20\u00a0\u00b5M NCL-00016066 diluted in 10\u00a0mM bicine, pH 8.0, 40\u00a0mM NaCl, 10\u00a0mM MgCl2, 4\u00a0mM imidazole, 1\u00a0mM DTT) at room temperature prior to mass spectrometry analysis.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described except glycerol was absent from all buffers. The absence of glycerol had no effect on the activity of the enzyme and was only added to previous preparations to stabilize the enzyme. Glycerol however interferes with the mass spectrometry and so was omitted here. Both wild type and mutant (V24C) Pfnek-2 were incubated (1:1) without/with 100\u00a0\u00b5M NCL-00016066 diluted in buffer  and molecular weight calculations made using ESIprot  for 1\u00a0h. The gel was stained with four parts colloidal blue to one part methanol and destained with 10% acetic acid in 25% (v/v). Protein bands were excised from the gel and cut into small pieces and washed three times with 200\u00a0\u03bcl of buffer B [20\u00a0ml 400\u00a0mM ammonium bicarbonate (3.16\u00a0g/100\u00a0ml) with 100% acetonitrile in ratio 1:1]. The gel pieces were then washed twice with 200\u00a0\u03bcl acetonitrile (100%) and gel pieces air dried.Recombinant Pfnek-2 (4\u00a0\u00b5l of 13\u00a0mg/ml stock solution of enzyme) was incubated with/without 1\u00a0mM ATP in Pfnek-2 buffer  followed by a further addition of acetonitrile for 10\u00a0min at room temperature followed by aspiration of the fluid and air drying. Trypsin solution (1\u00a0\u03bcg in 50\u00a0\u03bcl of 50\u00a0mM TEAB buffer per sample) was added and incubated at 37\u00a0\u00b0C overnight. The gel pieces were washed in 50\u00a0mM TEAB buffer for 5\u201310\u00a0min on a rotating platform. The digests and washes were combined and dried completely in a SpeedVac. The samples were then enriched for phosphopeptides as described below.g, and the flow-through (unbound peptides) collected and frozen. The IMAC beads were washed twice with 200\u00a0\u00b5l of IMAC load/wash buffer, once with 200\u00a0\u00b5l of HPLC grade water and eluted twice with 100\u00a0\u00b5l of solution containing 22\u00a0\u00b5l ammonia solution , 300\u00a0\u00b5l acetonitrile to a total volume of 1\u00a0ml with HPLC grade water. Eluates were concentrated to a small volume (15\u201320\u00a0\u00b5l) in a Speedvac centrifuge and submitted for mass spectrometry analysis.PHOS-Select iron affinity gel (Sigma) was equilibrated with 5\u00a0\u00d7\u00a01\u00a0ml IMAC load/wash buffer . 500\u00a0\u00b5l IMAC load/wash buffer was added to completely dry samples and 50\u00a0\u00b5l of 50% PHOS-Select slurry was added followed by rotating at room temperature for 1\u00a0h. Following incubation with IMAC beads, the samples were transferred onto Mobicol \u2018Classic\u2019 spin columns (2B Scientific Ltd), centrifuged for 30\u00a0s at 1000\u00d7This was carried out as previously described  with one50 values of 23.95\u00a0\u00b1\u00a02.50 and 35.63\u00a0\u00b1\u00a03.03\u00a0\u00b5M as measured by SYBR Green I and LDH parasite death assays, respectively  were resolved by SDS PAGE and appeared as a single band at approximately 38\u00a0kDa  value, 6.30\u00a0\u00b1\u00a00.25 (n\u00a0=\u00a03)]  by NCL-00016066, the mutant kinase was pre-incubated with NCL-00016066 (20\u00a0\u00b5M) prior to immobilization on Ni\u2013NTA beads, and unbound inhibitor were removed by extensive washing. As a control, Pfnek-2 was similarly incubated with high concentrations of NCL-00016066 (20\u00a0\u00b5M), which gave partial inhibition of kinase activity, which was reversed following washout Fig.\u00a0a. In conIn an effort to further characterize the nature of the inhibition of the mutant Pfnek-2 with NCL-00016066, wild type Pfnek-2 or Pfnek-2(V24C) were pre-incubated with or without 100\u00a0\u00b5M NCL-00016066 and the intact protein subjected to analysis by mass spectrometry.m/z ratio of Pfnek-2 (red trace Fig.\u00a0m/z ratio upon addition of NCL-00016066 (Fig.\u00a0Pfnek-2 without inhibitor (black trace Fig.\u00a0Table\u00a0Mass spectrometry analysis of the intact protein as described above indicated that Pfnek-2 undergoes multiple autophosphorylation events. Analysis of tryptic phospho-peptides unequivocally identified three autophosphorylation sites, Ser20, Ser215 and Ser219 Fig.\u00a0. InteresThe aim of the current study was to engineer Pfnek2 in a manner that would confer sensitivity of the kinase to a kinase inhibitor that otherwise would show low levels of activity to wild type Pfnek2. This was achieved by mutating Val24 in the wild type Pfnek-2 to cysteine. This mutation confers susceptibility to the kinase inhibitor NCL-00016066, and that the nature of this inhibition is likely through an irreversible covalent link of NCL-00016066 with the modified cysteine.Plasmodium berghei showed that parasites lacking the enzyme Pfnek-2 are able to undergo gametocytogenesis, gametogenesis and fertilization, but do not develop into ookinetes; this is likely due to a defect in meiosis, as pbnek-2\u2212 parasites appear to be unable to implement the pre-meiotic DNA replication that brings the DNA content to 4C in wild-type parasites, but remains 2C in the mutant parasites [Pfnek-2 is expressed predominantly in female gametocytes . Transmiarasites . The demarasites  suggestsStructural analysis of the Hsnek2 in complex with NCL-00016066 demonstrated that this inhibitor acts in an irreversible manner by covalent linkage to cysteine 22 in the glycine rich loop. Interestingly, Pfnek-2 does not contain this cysteine, or any other cysteine, in this loop. Demonstrated here is the weak inhibitor activity of NCL-00016066 against wild-type Pfnek-2 likely due to a lack of a cysteine in this position. Consistent with this notion is that NCL-00016066 showed potent inhibitory activity if a cysteine residue is introduced into the glycine-rich loop of Pfnek-2. Furthermore, this inhibition appeared to be irreversible, and mass spectrometry studies indicated that this is likely due to covalent modification of the variant kinase at the substituted cysteine.Recent studies have deployed a chemical genetic approach to the study of PfPKG where parasites expressing a mutant form of the kinase that is resistant to inhibition by a selective PfPKG inhibitor was used to identify cellular targets and physiological roles of the kinase . The engIn this way, the data presented here opens the door to investigating the essential role that Pfnek-2 plays in the transmission of malaria from the host to the mosquito vector and in doing so might inform on the mode of action of potential transmission-blocking drugs targeting this kinase."}
+{"text": "RIP (ribonucleoprotein immunoprecipitation) and RNA pull-down assays showed that Meg3 was co-immunoprecipitated with ATG3. In addition, Meg3 protected ATG3 mRNA from degradation following treatment with actinomycin D. Overall, our results suggest that the lncRNA Meg3 acts as a tumor suppressor in EOC by regulating ATG3 activity and inducing autophagy.Maternally expressed gene 3 (Meg3), a long non-coding RNA, has been reported to be associated with the pathogenesis of multiple malignancies. However, little is known regarding the role of Meg3 in epithelial ovarian cancer (EOC). In this study, we found that the expression of Meg3 was lower in epithelial ovarian carcinoma, and has potential to be considered as a biomarker for ovarian cancer. After transfecting the ovarian cancer cell lines OVCAR3 and A2780 with Meg3, phenotypic changes and autophagy-related molecules were examined. Upregulation of Meg3 inhibited cell proliferation, plate colony formation, induced cell cycle arrest in G2 phases, and promoted apoptosis. Observation of autophagosomes was performed by transmission electron microscopy. The expression levels of LC3-II, ATG3, LAMP1 were elevated, while SQSTM1/p62 expression declined. Upregulated expression of Meg3 also suppressed tumorigenesis  Epithelial ovarian carcinoma (EOC) remains one of the most common gynecologic malignancies, with 21,290 new cases diagnosed and 14,000 deaths reported in the United States in 2015 [Long non-coding RNAs (lncRNAs) are a family of non-protein-coding RNAs greater than 200 nucleotides in length . LncRNAsMeg3 mRNA expression was lower in EOC tissue as compared to normal ovarian tissues and benign ovarian carcinomas. In metastatic omentum tumors, expression was found to be significantly lower as compared to all above  assays were performed to confirm the functional interaction between MEG3 and autophagic protein ATG3. RNA from RIP assays with an antibody against ATG3 was used for qPCR analysis, which demonstrated an enrichment of the lncRNA Meg3 Figure . Next, wCells transfected with Meg3 were treated with actinomycin D to study the stability of ATG3 mRNA. Our results showed that ATG3 mRNA in cells transfected with Meg3 showed a slower rate of decay compared with control cells Figure .qRT-PCR and Western blot analysis were used to measure LC3, SQSTM1/P62, LAMP1 mRNA and protein expression after transfecting si-RNA to silence expression of ATG3 in OVCAR3 cells. Both mRNA and protein expression of LC3 Figure  and LAMPRecently, a series of studies have focused on the molecular mechanism of pathogenesis and progression of tumors, and the action of lncRNAs has attracted intense research interest. Studies have revealed that lncRNAs are involved in various processes including development, cell proliferation, metastasis, fate decision, invasion and migration \u201315, whicTo investigate the action of Meg3 in EOC, we analyzed the mRNA expression of Meg3 in normal epithelial ovarian tissues, benign tumor tissues, primary ovarian carcinomas, and metastatic omentum tumors. We found that the expression of Meg3 was reduced significantly in primary tumors as compared to normal ovarian tissue and benign tumors. Furthermore, the expression of Meg3 in metastatic omentum tumors was even lower than in primary ovarian carcinoma. These results demonstrated that there is a close relationship between Meg3 and tumorigenesis, as well as the development of ovarian cancer.in vivo. In conclusion, Meg3 plays a suppressive role in EOC, which is consistent with the role of Meg3 in gastric cancer, cervical cancer, lung cancer and hepatocellular carcinoma.Transfection of ovarian cancer cell lines with Meg3 resulted in inhibition of cell proliferation, colony formation, migration and metastasis, and promotion of G2 phase arrest, as well as cell apoptosis, while after given Meg3-transfected cells with autophagy inhibitor 3MA, the cell viability and colony formation rate was improved as compared with Meg3-transfected cells. Nude mice xenograft assays showed that overexpression of Meg3 could reduce tumorigenesis of ovarian cancer cells in vivo and in vitro. Therefore, we suggest that Meg3 acts as a tumor suppressor in EOC, which may be achieved by initiating autophagy and causing type II cell death.Furthermore, autophagosomes observed under transmission electron microscopy indicated that Meg3 induced autophagy in EOC. Autophagy, in general, is a survival process. Autophagic (type II) cell death usually represents a failed attempt to overcome lethal stress, and disruption of this process promotes rather than inhibits cell death in many cases , 25. SevRecent studies have provided a significant perspective on the important role of lncRNAs in regulation of gene expression. Reports have revealed that lncRNAs may act as either scaffolds that contain distinct protein-interacting domains to bring specific protein into proximity of each other, and form unique functional complexes, or as guides to recruit proteins \u201332. UpreIn conclusion, the present study has shown for the first time that upregulated expression of Meg3 triggers autophagic flux through an interaction with ATG3 to suppress tumorigenesis and progression of EOC. Meg3 may serve as a potential biomarker of EOC. Our findings may provide a novel insight into the early diagnosis and treatment of ovarian cancer. And further research should delineate the role and regulation of the crosstalk between Meg3 and autophagy in pathogenesis and progression of EOC.EOC tissues (Ca), metastatic omentum tissues (Om), borderline tumor (Bo) tissues, benign tumor tissues (Be) and normal ovarian specimens (No) were obtained from patients who underwent surgical resection at the Department of Gynecology, the First Affiliated Hospital of China Medical University  among 2005.01-2015.12. Tumor specimens were reviewed by microscopy by two independent pathologists and staged according to the FIGO staging system. Samples were frozen in liquid nitrogen immediately and stored at \u2212 80\u00b0C until use. No patients underwent chemotherapy or radiotherapy treatment prior to surgery. Informed consent was obtained from all subjects. Ethics board approval was obtained from the China Medical University Ethics Committee, and all specimens were handled and anonymized in an ethical and legal manner.2. The medium was changed every one to two days based on the culture state. MEG3-specific pcDNA overexpression vector (pcDNA-MEG3), ATG3-specific siRNA (si-ATG3) and corresponding control including empty pcDNA, and si-scramble were completed by Shanghai Genechem Co., Ltd., China. These recombinants were transfected into OVCAR3 or A2780 cells using Lipofectamine 2000 reagent  according to the manufacturer's instructions. The sequence of Meg3 plasmid is CTCGAGCTAGCCCCTAGCGCAGACGGCGGAGAGCAGAGAGGGAGCGCGCCTTGGCTCGCTGGCCTTGGCGGCGGCTCCTCAGGAGAGCTGGGGCGCCCACGAGAGGATCCCTCACCCGGGTCTCTCCTCAGGGATGACATCATCCGTCCACCTCCTTGTCTTCAAGGACCACCTCCTCTCCATGCTGAGCTGCTGCCAAGGGGCCTGCTGCCCATCTACACCTCACGAGGGCACTAGGAGCACGGTTTCCTGGATCCCACCAACATACAAAGCAGCCACTCACTGACCCCCAGGACCAGGATGGCAAAGGATGAAGAGGACCGGAACTGACCAGCCAGCTGTCCCTCTTACCTAAAGACTTAAACCAATGCCCTAGTGAGGGGGCATTGGGCATTAAGCCCTGACCTTTGCTATGCTCATACTTTGACTCTATGAGTACTTTCCTATAAGTCTTTGCTTGTGTTCACCTGCTAGCAAACTGGAGTGTTTCCCTCCCCAAGGGGGTGTCAGTCTTTGTCGACTGACTCTGTCATCACCCTTATGATGTCCTGAATGGAAGGATCCCTTTGGGAAATTCTCAGGAGGGGGACCTGGGCCAAGGGCTTGGCCAGCATCCTGCTGGCAACTCCAAGGCCCTGGGTGGGCTTCTGGAATGAGCATGCTACTGAATCACCAAAGGCACGCCCGACCTCTCTGAAGATCTTCCTATCCTTTTCTGGGGGAATGGGGTCGATGAGAGCAACCTCCTAGGGTTGTTGTGAGAATTAAATGAGATAAAAGAGGCCTCAGGCAGGATCTGGCATAGAGGAGGTGATCAGCAAATGTTTGTTGAAAAGGTTTGACAGGTCAGTCCCTTCCCACCCCTCTTGCTTGTCTTACTTGTCTTATTTATTCTCCAACAGCACTCCAGGCAGCCCTTGTCCACGGGCTCTCCTTGCATCAGCCAAGCTTCTTGAAAGGCCTGTCTACACTTGCTGTCTTCCTTCCTCACCTCCAATTTCCTCTTCAACCCACTGCTTCCTGACTCGCTCTACTCCGTGGAAGCACGCTCACAAAGGCACGTGGGCCGTGGCCCGGCTGGGTCGGCTGAAGAACTGCGGATGGAAGCTGCGGAAGAGGCCCTGATGGGGCCCACCATCCCGGACCCAAGTCTTCTTCCTGGCGGGCCTCTCGTCTCCTTCCTGGTTTGGGCGGAAGCCATCACCTGGATGCCTACGTGGGAAGGGACCTCGAATGTGGGACCCCAGCCCCTCTCCAGCTCGAAATCCCTCCACAGCCACGGGGACACCCTGCACCTATTCCCACGGGACAGGCTGGACCCAGAGACTCTGGACCCGGGGCCTCCCCTTGAGTAGAGACCCGCCCTCTGACTGATGGACGCCGCTGACCTGGGGTCAGACCCGTGGGCTGGACCCCTGCCCACCCCGCAGGAACCCTGAGGCCTAGGGGAGCTGTTGAGCCTTCAGTGTCTGCATGTGGGAAGTGGGCTCCTTCACCTACCTCACAGGGCTGTTGTGAGGGGCGCTGTGATGCGGTTCCAAAGCACAGGGCTTGGCGCACCCCACTGTGCTCTCAATAAATGTGTTTCCTGTCTTAACAAAAAGGATCC. The sequence of the siRNA targeting ATG3 was as follows Sense: CAUUGAGACUGUUGCAGAAdtdt; Anti-sense:UUCUGCAACAGUCUCAAUGdtdt.The human ovarian carcinoma cell lines OVCAR3 and A2780 were obtained from the Tumor Cell Bank of the Chinese Academy of Medical Science . OVCAR3 cells were cultured in RPMI 1640  and A2780 cells were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin (100 U/mL). The cells were cultured in an incubator at 37\u00b0C and an atmosphere of 5% COThree thousand cells were seeded into each well of 96-well plate in 100 \u03bcL medium. At the time points of 0 h, 24 h, 48 h and 72 h, 20 \u03bcL of 5 mg/mL MTT reagent was added and plates incubated for 4 h. The medium was then removed and replaced with 150 \u03bcL DMSO. Absorbance at 490 nm was measured using a microplate spectrophotometer .3 cells in 100 \u03bcL medium were seeded into each well of the E-Plate. Following incubation at room temperature for 30 min, the E-Plates containing cells were placed on the RTCA SP/MP station positioned in a cell culture incubator. The CI values were measured automatically every 15 minutes to form a continuous proliferation curve.For real-time cell proliferation assays, 50 \u03bcL medium was added to each well of a 96-well E-Plate for background measurement. Subsequently, 5 \u00d7 10After washing with PBS, cells were treated with trypsin to be collected and washed, then fixed with 70% ice-cold ethanol at -20\u00b0C for at least 12 h. Cells were washed and re-suspended by centrifugation then stained with PI following the manufacturer's protocol  for cell cycle analysis by flow cytometry.Cells were trypsinized, counted, suspended and seeded into six-well plates at a density of 500 cells per well, in regular culture medium, then cultured at 5% CO2 and 37\u00b0C for 2 weeks until visible clones appeared. The medium was discarded and the cells were carefully washed twice with PBS. After being fixed with methanol for 15 min, the cells were stained with Giemsa's solution for 15 min before washing with tap water and air-drying. The clone formation rate was calculated with the formula: Plate clone formation efficiency = (number of clones/number of cells inoculated) \u00d7 100%. All the experiments were repeated 3 times and the average values were reported.Cells were collected and centrifuged for 5 min and washed twice with cold PBS. Cells were then resuspended in 100 \u03bcL 1\u00d7buffer with 5 \u03bcL 7AAD and 5 \u03bcL PE-labeled annexin V (KeyGen) per sample in the dark. An additional 400 \u03bcL of 1\u00d7 buffer was added and the rate of apoptosis for each samples determined by flow cytometry within 1 h.TRIzol  was used to extract the total RNA from EOC cell lines and tissues. Isolated RNA was transcribed into cDNA using an avian myeloblastosis virus reverse transcriptase and random primers . Amplification of the cDNA was performed by real-time quantitative PCR using the SYBR Premix Ex Taq\u2122 II kit . The expression level of each target gene was normalised to 18s mRNA. The data analysis was calculated according to the sample threshold cycle (Ct) value from three independent experiments, primers for RT-PCR could found in The complete proteome from ovarian cancer cells was extracted in RIPA buffer, preventing protease-mediated degradation of samples. Protein concentration was determined for each sample, and 40 \u03bcg of the denatured proteome was resolved by 15% SDS-polyacrylamide gel electrophoesis and then electro-transferred to Hybond membranes . After blocking the membranes with 5% fat-free milk at room temperature for 2 h, membranes were incubated with the primary antibodies primary antibodies targeting ATG3, LAMP1 , LC3 , and SQSTM1  at 4\u00b0C overnight. Membranes were washed three times with TBST then secondary antibodies (anti-rabbit), at dilutions of 1:5000 were added. Following incubation for 2 h at room temperature, the protein bands were visualized by enhanced chemiluminescence (ECL) following the manufacturer's instruction . GAPDH  served as the loading control.The interaction between LncRNA MEG3 and ATG3 protein was examined using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo fisher) according to the manufacturer's protocols. Biotin-labeled MEG3 or antisense RNA was co-incubated with protein extract of OVCAR3 cells and magnetic bead. The generated bead-RNA-Protein compound was collected by low-speed centrifugation. After washed with Handee spin columns, bead compound was boiled in SDS buffer, and the retrieved protein was detected by western blot with expressional level of GAPDH as control.The Magna RIP RNA-Binding Protein Immunoprecipitation Kit  was used for RIP assays following the manufacturer's protocol. Briefly, OVCAR3 cells were collected and lysed using RIP lysis buffer. Whole-cell extracts were incubated with RIP buffer containing magnetic beads conjugated to human anti-ATG3 antibody or the control IgG. Proteinase K was added to the samples to digest the protein, and the immunoprecipitated RNA isolated. Purified RNA was used for qRT-PCR analysis. Expressional level of GAPDH was as control.Control and transfected cells were treated with actD (dissolved in 100% ethanol) at a final concentration of 1-5 \u03bcM. The actD was added to cells 0 h, 1 h, 2 h, 4 h, 6 h or 12 h prior to RNA extraction with TRIzol reagent. Subsequently, qRT-PCR was used to analyze changes in RNA levels.7  were injected into the right flanks of the mice. The tumour volume was directly measured following inoculation and weight calculated using the formula: (length \u00d7 width2)/2.All animal studies were approved by the China Medical University Animal Care and Use Committee, and all experimentation on animals was performed in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four-week-old female BALB/c nude mice with weights of approximately 20 g were obtained from Vital River Laboratories  and housed in a pathogen\u2013free and temperature-controlled environment. Two hundred microliters of PBS containing approximately 1 \u00d7 102O2 to endogenous peroxidase activity. Next, the sections were heated in target retrieval solution (Dako) for 15 min in a microwave oven  to retrieve the antigen. Non-specific binding was blocked by incubation with 10% goat serum for 2 h at room temperature. Slides were then incubated overnight at 4\u00b0C with anti-ATG3 primary antibody. An appropriate secondary antibody was added and incubated for 20 min at 37\u00b0C, and binding was visualized with 3,39-diaminobenzidine tetrahydrochloride (DAB). After each treatment, slides were washed three times with TBST for 5 min. Three independent observers  randomly selected and counted 100 cells from five representative fields from each section. Any discrepancies were checked by three observers until a consensus was reached. Positive expression was graded as follows: 0 = negative; 1 = 1%-50%; 2 = 51%-74%; 3 \u2265 75%. The staining intensity was graded as follows: 1 = weak; 2 = intermediate; 3 = strong. The two grades were multiplied to obtain a final score: - = 0; + = 1-2; ++ = 3-4; +++ = 6-9).Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and then incubated for 20 min in 3% HTransfected or control OVCAR3 cell slides were fixed with 4% formaldehyde, blocked using 5% skimmed milk in 0.01 M PBS, pH 7.2 (PBS-M). Primary antibodies targeting LC3, SQSTM1 (P62) or ATG3 , were diluted 1:500 in PBS and added to the slides. After incubation overnight at 4\u00b0C, slides were washed once with 0.2% Tween 20 in PBS and twice with PBS, and then incubated for 30 min at room temperature with anti-rabbit IgG\u2013TRITC . The slides were washed three times with PBS, then fixed with glycerol/PBS , and observed and photographed using a laser confocal microscope .Data were analyzed using SPSS 17.0 software , and are presented as mean \u00b1 SD. Two groups were compared using an unpaired, two-tailed Student's t-test. Spearman rank correlation was used to test the association between Meg3 expression and FIGO stages. The ROC characteristics curve was used to calculate the sensitivity and specificity values of Meg3. and P < 0.05 was considered to be statistically significant."}
+{"text": "Syringomyelia (SM) is a common condition affecting brachycephalic toy breed dogs and is characterized by the development of fluid-filled cavities within the spinal cord. It is often concurrent with a complex developmental malformation of the skull and craniocervical vertebrae called Chiari-like malformation (CM) characterized by a conformational change and overcrowding of the brain and cervical spinal cord particularly at the craniocervical junction. CM and SM have a polygenic mode of inheritance with variable penetrance.PCDH17 on CFA22 and ZWINT on CFA26. PCDH17 codes for a cell adhesion molecule expressed specifically in the brain and spinal cord. ZWINT plays a role in chromosome segregation and its expression is increased with the onset of neuropathic pain. Targeted genomic sequencing of these regions identified respectively 37 and 339 SNPs with significantly associated P values. Genotyping of tagSNPs selected from these 2 candidate loci in an extended cohort of 461 CKCS  identified 2 SNPs on CFA22 that were significantly associated to SM strengthening the candidacy of this locus in SM development.We identified six cranial T1-weighted sagittal MRI measurements that were associated to maximum transverse diameter of the syrinx cavity. Increased syrinx transverse diameter has been correlated previously with increased likelihood of behavioral signs of pain. We next conducted a whole genome association study of these traits in 65 Cavalier King Charles Spaniel (CKCS) dogs . Two loci on CFA22 and CFA26 were found to be significantly associated to two traits associated with a reduced volume and altered orientation of the caudal cranial fossa. Their reconstructed haplotypes defined two associated regions that harbor only two genes: PCDH17 and ZWINT, significantly associated to two traits associated with syrinx transverse diameter. The locus on CFA22 was significantly associated to SM secondary to CM in the CKCS dog breed strengthening its candidacy for this disease. This study will provide an entry point for identification of the genetic factors predisposing to this condition and its underlying pathogenic mechanisms.We identified 2 loci on CFA22 and CFA26 that contained only 2 genes, The online version of this article (10.1186/s12863-018-0605-z) contains supplementary material, which is available to authorized users. Canine syringomyelia (SM) is a painful condition where fluid-containing cavities (syrinx or syringes) develop within the parenchyma of the spinal cord and which progress over time , 2. DepeIn the CKCS, risk of SM has been shown to be associated with increased brachycephaly with rostrocaudal doming i.e. a heightened cranium that slopes caudally  and reduBMP3 (BONE MORPHOGENETIC PROTEIN 3) variations to skull diversity GGAATGGGAAGTGAGATGAACAGCATAGGAGATTTGAAAGGTAGCAAACAATCCAGGAGACCTACAGGCCCGTGATCAATGACTTATAAGATGATATTAAGGAAACATTATATATGATACTATATCCTCCTTTGAGAGTCTGTATGCATT and TTAATCTTCAAAACGGCCCAATGAGGATATCCCCATTTTGCAGATGAAGAATGAAGGAACGTTGAAGTTCAGTGATTTGATCCAGAATAAGTGCAGTGAGACTTCAGATCCAGCTATGGGGTTTGCCAAATTCAATCTCTGCCCTTCCTC[A/C]CTATTCTTAACCGCCAAATATTATTTATATTTGTAAGAATGCAGTTTTAAAGGTTGAAATTTTCAACTTCTCACACAGAGCAGATAGCTGGAGACGAAGATGGTAGGACTGCTCTCTCATTGCCTGCATTGTGCTCTCTGAGTAGTGAAA respectively. Two SNPs were also amplified on CFA26 at position 32,757,080 (rs23302138) and 32,797,595 with the probes TGTCCTCCTGGCTTCTGAGGGGGTGGGTGCGGGGCCTGGAGGCCCAGAGGGGAACAGGATGTGGCCACAGGATGGAGAGCTGACTTGTGCACAGGGGCCTGTGTGGGTCAGTCTGTGTCCCCGGCACCCCTGAAGCTGCAGGTGTCTCAG[T/C]AGAGCCCCTCAGTGGGTAACTCTGCCCCCAATTCCCTCCTTGGAGACTGCATCTCCTCCTGTGCCTCCTGCAAGTCGCTGTCAGCTTCCCTCCCCTGAGGTCTGACGCCTCCTGCAGGAAGTTCTCTGGGATTGGATCTCAAAATGGTGC and CATGAGTTGGAAGGGCAGTTAAGGGCAGAAGGACTTAGAGGCGGAGAGCATAGAGAAGGAAAAGGCACGATGGTGTGTTTGATTATCTCCCCCTCTCCATTCTCATGGTGCCACCTATCCTAATTCCAGTTCGTATTATCATAGGTCTCA[C/T]TCCACCAATAACGTCTCAATCACACACACCATGTCCTGTCTTCTCGTTGGTCTGTGCTATGATCTGTGTGGTTCTTCTCTTTCCCAGGAGGCCAGATCTGTATTTTGCTGATTACAATCTACTCTTTAATTCTGGATTGAATTGCTAACT respectively. Genotyping was performed using the TaqMan assay (Applied Biosystems) on 393 CKCS dogs .Tagging SNPs were selected to maximize coverage of each locus. They were identified using the tagger routine in the Haploview V4.2 software with a maximum rP values obtained from the association were corrected together for multiple testing using a storey\u2019s q value method [Initial GWAS analysis was done in 2 phases: association of the quantitative traits  to disease, followed by association of SNPs to these quantitative traits. Association of the quantitative traits to the disease was done using a linear regression with a type III sum of squares in R V3.0.1  and age e method . Haplotye method  and assoe method  with ageL4\u2009+\u2009L7 all showed a significant association to STD size (Pbonferroni\u2009<\u20090.0019) and were therefore further investigated . This resulted in the identification of a group of 13 SNPs on CFA15  associated to ratio F-d/BC under a FDR of 0.05  and two SNPs on CFA26 , which were significantly associated to L4\u2009+\u2009L7 under a FDR of 0.05 (both P\u2009=\u20090.03754)  and two on CFA26 (P\u2009=\u20090.01067 and 0.00231), that were significantly associated to ratio F-d/BC  surrounding the SNPs significantly associated to PCDH17 (PROTO-CADHERIN 17) on CFA22 and ZWINT (ZW10 INTERACTING KINETOCHORE PROTEIN) on CFA26. In order to identify potential SM -predisposing mutations, both CFA22 and CFA26 candidate regions were submitted to targeted next generation sequencing. The reads of the sequences obtained from the 65 CKCS were aligned on CanFam 3.1 resulting in 5608 SNPs on CFA22 and 10,814 SNPs on CFA26. Except for one SNP (rs2305483), that was identified in the coding region of PCDH17 and that was non-conserved and synonymous, all identified SNPs resided in intergenic and deep intronic regions. Using improved coverage of the region, linkage disequilibrium blocks were reevaluated based on the significant SNPs in the regions. A total of 37 and 339 SNPs were defined as significantly associated to F-d/BC and L4\u2009+\u2009L7 respectively. Based on the hypothesis that causative SNPs would be significantly associated to their respective trait, these SNPs refined the regions of interest to 13,785,828-14,183,295\u00a0bp  on CFA22 and 32,721,057\u00a0bp to 33,094,292\u00a0bp  on CFA26.Each of the SM- associated regions harbored only one coding gene: P value\u2009=\u20090.7637 and 0.7614), the 2 selected TagSNPs on CFA22 at position 13,933,606\u00a0bp and 13,804,718\u00a0bp reached significance . Bonferroni corrected P value of the TagSNP at position 13,933,606\u00a0bp on CFA22, was still significant at a P value of 0.0104. Hence, we successfully identified a region on CFA22 associated to ratio F-d/BC and SM in the CKCS dogs.To investigate the potential association between the identified SNPs and SM, a cohort of 461 CKCS including 187 unaffected and 274 SM-affected  were genotyped with two TagSNPs from each of the associated regions on CFA22 and CFA26. While the 2 selected TagSNPs on CFA26 at position 32,797,595\u00a0bp and 32,757,080\u00a0bp did not show any significant association to SM  significantly associated to STD size. Genetic investigation of these traits identified significant association with SNPs on CFA15 with F-d/BC and on CFA26 with L4\u2009+\u2009L7. Both these traits represented a combination that demonstrated a reduction in the overall size of the caudal cranial fossa and rearrangement of the neural parenchyma  identified a significant association of the candidate locus on CFA22 and SM. Hence, this candidate locus that was suggestive of association to the ratio F-d/BC in CKCS and to the line BC in a previous association study in GB dogs was found to be significantly associated to SM, strengthening its candidacy for SM. On the other hand, the tagSNPs on CFA26 that was significantly associated to PCDH17 and ZWINT (respectively). Targeted next generation sequencing of both CFA22 and CFA26 candidate loci identified a total of 37 and 339 significantly associated SNPs with ratio F-d/BC and L4\u2009+\u2009L7 respectively. No mutation in the coding region of either gene was detected, except for one synonymous mutation in PCDH17. We hypothesize that predisposing mutations in these two regions are most likely regulatory that would affect RNA expression of either PCDH17 or ZWINT or other unannotated transcripts. Alternatively, these regulatory mutations could have a long-range expression effect on transcripts residing outside the 2 candidate regions. RNA-sequencing or quantitative RT-PCR studies in affected tissues from dogs carrying the associated haplotypes are needed to test this hypothesis.The associated loci on CFA22 and CFA26 harboured each only one gene: PCDH17 (PROTOCADHERIN 17) belongs to the family of protocadherins that are involved in the adhesion and sorting of cells during tissue morphogenesis. It is expressed specifically in several regions of the developing and adult brain and spinal cord [PCDH17 in primary cortical neurons is associated with significantly decreased dendritic spine density and abnormal dendritic morphology [ZWINT (ZW10-INTERACTOR) has an important role in kinetochore assembly and proper chromosome segregation [ZWINT in pain development and in SM associated with CM.nal cord \u201341. It rnal cord . Overexprphology  and it iregation . In ratsregation \u201346. We tCM with SM in the dog is very similar to a condition in humans called Chiari malformation I (CMI) with a reported frequency of 1 in 1280. As in dogs, the prevalence of SM secondary to CMI in humans is high reaching 65%\u201385% , 48. GenL4\u2009+\u2009L7 respectively. Genotyping of a larger cohort of CKCS dogs confirmed association of the locus on CFA22 with SM in this breed. Each of these 2 haplotypes harbored only one gene: PCDH17 on CFA22 that codes for a cell adhesion molecule specifically expressed in the brain and spinal cord and ZWINT that plays a role in proper chromosome segregation and whose expression is increased with the onset of neuropathic pain. Additional molecular genetic studies in larger CKCS cohorts from various affected brachycephalic breeds and in cell and animal models are needed to further investigate the role of the 2 associated loci and the genes they harbor in the pathogenesis of SM secondary to CM. Our study represents an essential step towards a better understanding of the complex genetics of this devastating condition and development of breeding strategies that aim at eliminating it from the affected dog breeds. It also provides an important model for studying CMI/SM in humans.In this study, we have used a genome-wide association study to decipher the genetics of SM secondary to CM in the CKCS breed. We identified 6 cranial T1-weighed sagittal MRI measurements that were associated with the syrinx transverse diameter. We next identified 2 haplotypes on CFA22 and CFA26 that were significantly associated to ratio F-d/BC and Additional file 1:Table S1. Characteristics and measurements of 96 CKCS of the cohort. This table includes the gender, age, clinical status and all MRI cranial measurements taken on the 96 CKCS dogs included in this study. (XLSX 41\u00a0kb)Additional file 2:Table S2. SNPs suggestive of association to SM in the CKCS breed. This table enlists all SNPs suggestive of association with FDR corrected scores between 0.05 and 0.1. These SNPs were identified following a GWAS using a mixed linear model with age as a covariate on the previously identified traits . (DOCX 15\u00a0kb)Additional file 3:Figure S1.P value distribution inside the CFA15  associated region. This region spans 1.7\u00a0Mb surrounding SNPs associated to ratio F-d/BC and was identified using Haploview V4.2. (PDF 54\u00a0kb)"}
+{"text": "Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutS\u03b1- and MutL\u03b1-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutS\u03b1 (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutL\u03b1 (Mlh1-Pms1), which facilitates downstream steps. In addition, MutS\u03b2 (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of  Repetitive DNA elements are widespread in genomes. They are located in centromeres, telomeres, rDNA genes, transposons, and intergenic regions . The newMSH2 and MLH1 causes microsatellite instability and a predisposition to colon and other types of cancer  and MutS\u03b2  mutants exhibit instability of mono- and dinucleotide repeats and generate duplications of sequences flanked by repeats  complex has single-stranded 3\u2032-exonuclease and endonuclease activities as well as structural functions in recombination processes . After 5nd MutS\u03b2 . S. cere repeats  and repaSchizosaccharomyces pombe encodes the MutS homologs Msh1, Msh2, Msh3, and Msh6, the MutL homologs Mlh1 and Pms1, and the exonuclease Exo1. Based on homology with S. cerevisiae Msh1, S. pombe Msh1 likely acts in MMR of mitochondrial DNA. S. pombe Msh2, Msh6, Mlh1, and Pms1 are indispensable for repair of base-base mismatches and small loops with one or two nucleotides , yeast extract agar (YEA), and yeast extract liquid (YEL), and general genetic methods were used as described ; OL937 \u2212 mlh1h::kanMX his3-D1 ura4-D18 (\u2212 exo1h::ura4 ura4-D18 (\u2212 rad2h::ura4 ade6-704 leu1-32 ura4-D18 (smt-0 rad50::kanMX ura4-D18 (+ rad51h::kanMX ade6 leu1-32 ura4-D18 (The \u2212 msh2h::hphMX ura4-D18 and KK37 \u2212 msh6h::hphMX ura4-D18 originated from transformations of OL2137 (\u2212 ura4-D18h), with DNA fragments obtained by fusion PCRs. pFA6a-hphMX (smt-0) was used to amplify either 450 bp of the 3\u2032 UTR of the msh2 locus or 500 bp of the 5\u2032 UTR of the msh6 locus. Primers for the msh2 disruption were msh2_For 5\u2032-GAGGTTTTTTATTTATCCTTTTTGAGGACTTAACTGTGGCAAGGAGTTTCTTCTCCTGTTTTATACATTTCGCGTTCGCGCTTTAGAACATTCAATCAATCGGATCCCCGGGTTAATTAA; msh2_Rev 5\u2032-TTTCCTCGTTTTAGTAAAAAATTATTTTATTCATAAAATGCGCTTCCAAAAAACATGTACCTTGGTTGAATTCTTTCAATTAGTACCTTGCTCACATTCTGAATTCGAGCTCGTTTAAAC; msh2_For3 5\u2032-TTGAAAGAATTCAACCAAGG; and msh2_Rev3 5\u2032-GCTAAAACAAAATTATGCCG. Primers for the msh6 disruption were msh6_For 5\u2032-TATATATGTTATTTTGTGCTCTCATGTTAGCTTTGTTTACTATTAGAATGCTGCTTTTTGTAAATAACTGAACTTAGCCAAAACCAACACTTGTTCCAGTCGGATCCCCGGGTTAATTAA; msh6_Rev 5\u2032-ATAACGTAAGTAAATGGTAAATAAAAGCAAGCTTCCGCTTGCCAGCAAACGAAAGATATTGCTTTGAATAGTCATAAAACTGATAGAGTGTTGACAGTTAGAATTCGAGCTCGTTTAAAC; msh6_For2 5\u2032-CTCATCTTACCTAAACTCTC; and msh6_Rev2 5\u2032-GAACAAGTGTTGGTTTTGGC.Strains KK42 DraIII-HindIII fragment of the ade6 gene containing a (GACC)7 repeat near the DraIII site . The fragment was obtained by PCR with primers ade6-GACC7 5\u2032-TCCCACTTGGTGACCGACCGACCGACCGACCGACCGACCTTTATGTTGAAAAGTTCGTTC-3\u2032 and ade6-H 5\u2032-GGGCAAGCTTCAATGGTGTA-3\u2032, and subsequent digestion with DraIII and HindIII. PCR was performed under standard conditions using pCG162 as template (3 run immediately 3\u2032 to the (GACC)7 repeat was deleted. The 1.7-kb XhoI-EcoRI fragments of the plasmids containing either (GACC)7 or (GACC)7\u0394T were transformed into the S. pombe strain AM1 7 his3-D1 leu1-32 ura4-D18h and \u2013 ade6-(GACC)7h\u0394T his3-D1 leu1-32 ura4-D18. A strain with a (GACC)8 repeat was isolated during a fluctuation test with an h+msh2::7his3 ade6-(GACC) mutant.In pAN-K, a pUC18 derivative, the kanamycin resistance gene was replaced by a 340-bp \u2212 ade6h::ura4 ura4-D18) was constructed by transformation of strain OL2137 (\u2212 ura4-D18h) with a PCR fragment obtained with primers ade6_d_ura4_F 5\u2032-TCCTTTTGTACTGAAAAGTAAAACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTagcttagctacaaatcccac and ade6_d_ura4_R 5\u2032-GAATGGTCTCAGTTGTAGGATAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATagcttgtgatattgacgaaac, and pAW1 as template  in ade6 were deleted and replaced by ura4 . After initial denaturation for 1 min at 94\u00b0, we applied two cycles with 30 sec at 94\u00b0, 30 sec at 45\u00b0, and 30 sec at 72\u00b0, followed by five cycles with 30 sec at 94\u00b0, 30 sec at 55\u00b0, and 30 sec at 72\u00b0. Reaction samples were transformed into strain DE1 using the method of 6ade6-(CTGCC). Primers Hepta-F 5\u2032-ACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCC ATCGTCCTTTATG and Hepta-R 5\u2032-AAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC). Primers HeptadT-F 5\u2032-AACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCCATCGTCCTTATG and HeptadT-R 5\u2032-TAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC)\u0394T. Here, a single T immediately downstream of the repeats has been deleted \u0394T, repeat tract changes were also analyzed by inspection of the color of Ade+ revertants 7\u0394T, (CTGCC)6, and (ATCGTCC)7, insertions of one repeat unit and deletions of two repeat units are the major events detectable. The opposite is the case with (GACC)8 and (ATCGTCC)7\u0394T, where deletions of one repeat unit and insertions of two repeat units are the principal events that can be detected, although in the case of (ATCGTCC)7\u0394T, we identified exclusive deletions as described below.We have previously reported that in-frame nucleotide insertions and codon changes in a region around nucleotide 1397 of the  mutants . Inserti7of ade6-(GACC)\u0394T, deletions and insertion can be distinguished by the color of revertants \u0394T repeat in the various genetic backgrounds revealed deletions and sequencing of 23 7(GACC)\u0394T revertants revealed insertions (7(GACC)\u0394T repeat can be easily determined from a large number of revertants. Analyzing revertants of the other ade6 repeats did not allow a distinction by color, probably because Ade+ originating from deletions and insertions were both not fully functional. In these cases, PCR products of independent Ade+ revertants were subjected to sequencing to identify the number of repeats.In the case vertants . Deletiosertions . All revmsh2, msh3, msh6, mlh1, or exo1. In wild type, 8ade6-(GACC) and 7ade6-(GACC)\u0394T reverted to Ade+ with 1.2 \u00d7 10\u22125 and 2.1 \u00d7 10\u22125 reversions per cell division, respectively (8ade6-(GACC) reversion rates increased 39\u201344-fold in msh2, msh6, and mlh1 mutants and slightly decreased in msh3 and exo1 mutants. Similarly, 7ade6-(GACC)\u0394T reversion rates increased 17\u201322-fold in msh2, msh6, and mlh1 mutants and decreased 3\u20135-fold in msh3 and exo1 mutants. None of the differences between msh3, exo1, and wild type was statistically significant.We first analyzed stability of GACC tetra-nucleotide repeats in wild type, and in mutants deleted for either ectively . The ade7ade6-(GACC)\u0394T can be determined by colony color as described above and in Materials and Methods, and as illustrated in msh3, and exo1 strains, reversions mainly occurred by deletions  revealed that in wild type, all 10 had two repeat insertions 6 repeat appeared to be more stable in the msh3 mutant. It reverted mostly through gain of one repeat unit in all strain backgrounds, with the possible exception of mlh1  to Ade+ . Rates w of mlh1 . We conc5ade6-(ATCGTCC)\u0394T and 5ade6-(ATCGTCC) reverted to Ade+ at rates of 7.2 \u00d7 10\u22126 and 7.5 \u00d7 10\u22126, respectively \u0394T (not significant) and 2.8\u20132.9-fold for 5ade6-(ATCGTCC) (significant) (5ade6-(ATCGTCC)\u0394T revertants contained four hepta-nucleotide repeats and thus originated from deletion of one repeat (ade6 (+. 5ade6-(ATCGTCC) reverted mainly by insertion of one repeat and less frequently by deletion of two repeats without any significant differences between the spectra of wild type and any of the mutants  . All seqe repeat . The facat 5 repeat 5 repeat instability in msh3 and exo1 is not due to a defect in MMR. We therefore wanted to analyze the genetic context of msh3 and exo1 defects in microsatellite stability. To do this, we measured reversion rates of the hepta-nucleotide repeat in FEN1rad2, rad50, and rad51 mutants and in various double mutants 5 repeat when msh3 is mutated.The 5 repeat . Since d mutants . FEN1 anon rates . The msh mutants . Like wieat unit . In the S. cerevisiae, msh3 and msh6 mutants show little to moderate increases of mutation rates in mono- and dinucleotide repeats 7 and (GACC)8 tetra-, (CTGCC)6 penta-, and (ATCGTCC)5 hepta-nucleotide repeats 7\u0394T, were the predominant reversion events in msh2 and msh6 mutants, slippage of one repeat can occur in the template and in the nascent strand during replication, and both types of events are corrected by MMR mediated by MutS\u03b1 and MutL\u03b1.The assay with the (GACC)vertants . We founsertions . Thus, t6 in msh2, msh6, mlh1, and exo1 mutants were similar to that of wild type, but decreased in msh3 5\u0394T and (ATCGTCC)5 (5\u0394T and mainly by insertion of one repeat in (ATCGTCC)5. We conclude that loops in penta- and hepta-nucleotide repeats are not substrates of MutS\u03b1 and MutL\u03b1 in S. pombe.Mutation rates of the penta-nucleotide repeat (CTGCC) in msh3 . All stre repeat . InactivTCGTCC)5 . Like wimsh3 mutants had no significant defects in repair of base-base mismatches and of loops with one unpaired nucleotide in a T6 repeat and in nonrepetitive DNA (msh3 mutations caused some instability of a (GT)8 dinucleotide repeat, which was mostly evident by a reversion spectrum different to wild type (msh3 mainly reverted by two-nucleotide deletions in the (GT)8 repeat. However, this was clearly less frequent than in msh2, msh6, and pms1 mutants 8 repeat t strand . Rates wS. pombe Msh6, as part of MutS\u03b1, recognizes base-base mismatches and loops with one to four unpaired nucleotides, while Msh3 does not play a significant role in MMR, but rather maintains repeat stability independently of MMR. Consequently, S. pombe MMR cannot repair loops with five or more nucleotides, in contrast to human MMR  (S. pombe microsatellites. It is therefore critical for humans, but not for S. pombe, to have an MMR system that can deal with larger loops.We conclude from our studies that"}
+{"text": "Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5. Mouse preimplantation embryonic development culminates in the blastocyst stage. A blastocyst consists of three cell lineages: epiblast, trophectoderm, and primitive endoderm (PrE). The epiblast develops into most of the embryo proper, the amnion, and the extraembryonic mesoderm of the yolk sac; the trophectoderm gives rise ultimately to the fetal portion of the placenta; and the primitive endoderm forms the two extraembryonic endoderm lineages \u2013 the visceral endoderm (VE) and the parietal endoderm (PE) of the yolk sac134in vitro model to identify patterning activities of the extraembryonic endoderm such as factors involved in cardiac induction17Stem cell lines have been derived from these three cell lineages91114Gata4 or Gata6 . We find that this and other pre-XEN cell lines are immunoreactive for XEN cell markers GATA4, GATA6, SOX7, SOX17, and DAB2, but negative for ES cell markers OCT4 and NANOG, and negative for TS cell marker CDX2.Xist locus on the X-chromosome; Sox17 and Gata6 are XEN-cell markers; and Cdx2 is a marker for trophoblast stem cells. We removed the ectoplacental cone of the embryos as much as possible, and transferred each embryo separately into a well of 4-well dish coated with 0.1% gelatin and covered with MEF in TS cell medium including 25\u2009ng/ml FGF4 and 1\u2009\u03bcg/ml heparin (referred to as F4H). One day later, the embryos had attached to the surface and started to form an outgrowth. The embryos had formed a large outgrowth after 5 days. We used TrypLE Express to disaggregate the outgrowths and passaged cells into a well of a 4-well dish. After cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish. After they reached 70\u201380% confluency again, cells were passaged into a well of a 6-well dish, and we then obtained stable post-XEN cell lines. The intrinsic red fluorescence of mTomato produced from the Gata6 promoter in the transgene was sufficiently high to detect it in the whole embryo and outgrowth, but not in the established post-XEN cell line at day 60  by immunofluorescence for GATA4 and counterstaining with DAPI. Sets of fluorescence images were captured for each line visualizing the intrinsic fluorescence of GFP, DAPI, and GATA4 immunoreactivity . The imaWe asked if post-XEN cells can differentiate into a VE identity by incubation with BMP438Dab2, Gata4, Gata6, Pdgfra, Sox7, and Sox17, versus low or no expression of ES cell-specific genes such as Nanog, Pou5f1/Oct4, Sox2, and Nr0b1. There is no expression of EpiSC-specific genes such as Cer1 and Fgf5 (data not shown). Thus, the NanoString gene expression analysis confirms and extends the immunofluorescence profiles.We applied the NanoString multiplex platform for gene expression4041in vivo. We injected into blastocysts  cells of two R26-tauGFP41+ pre-XEN cell lines (X-ICM-4 and X-ICM-5) and one PDGFRa-GFP+ pre-XEN cell line (X47) that we had derived from isolated ICMs or blastocysts, and cells from four R26-tauGFP41+ post-XEN cell lines  and one PDGFRa-GFP+ post-XEN cell line (X-E6.5-Z0663-9) that we had derived from E5.5 embryos or disaggregated E6.5 embryos  that are at the origin of the post-XEN cell lines.Sox17 locus, we cannot detect GFP expression in embryos and cell lines derived from them.) The Xist1loxGFP strainThe PDGFRa-GFP strainAdvanced RPMI-1640  was supplemented with 20% (vol/vol) FBS , 2\u2009mM GlutaMAX Supplement (Gibco #35050), 1% penicillin/streptomycin , 0.1\u2009mM \u03b2-mercaptoethanol (Gibco #21985-023), 1\u2009mM sodium pyruvate (Gibco #11360-039), supplemented with 25\u2009ng/ml FGF4 (Peprotech #100-31) and 1\u2009\u03bcg/ml heparin (Sigma #H3149).ES cell lines were maintained on MEF-coated, pregelatinized tissue culture dishes  in DMEM  supplemented with 15% FBS (HyClone #SH30071.03), 2\u2009mM GlutaMAX Supplement, 1% penicillin/streptomycin, 1% \u03b2-mercaptoethanol , 0.1\u2009mM nonessential amino acids (Gibco #11140-035), 1\u2009mM sodium pyruvate, and 1000\u2009IU/ml leukemia inhibitory factor (LIF) (Millipore #ESG1107).Embryos were collected at the 2\u20138 cell stage by flushing oviducts using M2 medium (Sigma #M7167), and cultured in KSOM medium (Millipore #MR-106-D) to the blastocyst stage. The zona pellucida of blastocysts was then removed using acid Tyrode solution (Sigma #T1788). Blastocysts were transferred separately into a well of a 96-well dish  coated with 0.1% gelatin  and covered with MEF in ES medium supplemented with LIF and 1\u2009\u03bcM PD0325901 (Axon #1408) and 3\u2009\u03bcM CHIR99021 (Axon #1386), a combination of chemicals that is typically referred to as \u201c2i\u201d. After 4 days TrypLE Express (Gibco #12604-013) was used to disaggregate the embryonic outgrowths, and cells were passaged to a well of 24-well dish to derive ES cell lines.Embryos were collected at the 2\u20138 cell stage embryos and cultured in in KSOM medium to the blastocyst stage. The zona pellucida of blastocysts was removed using acid Tyrode solution. Blastocysts were transferred separately into a well of a 4 well-dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in ES medium supplemented with LIF. The XEN lines were derived as describedThe ectoplacental cone was removed with forceps or needles. A whole embryo was placed in a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. After the embryos formed a large outgrowth, TrypLE Express was used to disaggregate the outgrowths and passage cells into a 4-well dish. When cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish. If the outgrowth of an E5.5 embryo grew well and XEN cells thrived, we continued to culture cells in TS medium including F4H. But if the outgrowth grew slowly and XEN cells were surrounded by trophoblast-derived cells, we cultured cells in ES medium supplemented with LIF, culture conditions that would inhibit trophoblast-derived cells; when XEN cells started to become abundant, we switched to TS medium including F4H. To prepare XEN cells for RNA extraction, cells were cultured in dishes coated with 0.1% gelatin but without MEF in TS medium including F4H.The ectoplacental cone was removed with forceps or needles. A whole embryo was treated with 0.1\u2009mg/ml collagenase (Gibco #17104-019) and 0.01\u2009mg/ml deoxyribonuclease (Gibco #D5025) for 20\u201330\u2009min at room temperature, followed by 0.2\u2009mg/ml TrypLE for 5\u2009min at room temperature. The embryo was disaggregated into single cells using a glass pipette with a diameter of 50\u201360\u2009\u03bcm. Cells were transferred into a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. Three days later XEN colonies appeared. We picked these colonies, disaggregated them with a glass pipette or by TrypLE Express for 5\u2009min at 37\u2009\u00b0C, and passaged them into a well of a 4-well dish. When cells reached 70\u201380% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish.http://cellprofiler.org/ . The DAPI signal was used to segment individual cells by thresholding, de-clumping, and applying size and roundness filter in an effort to evaluated single cells that are not MEFs. After filtering, the GFP and GATA4 signals were evaluated in each cell, and the population characteristics for these two markers were quantified.Images representing Fields of View of post-XEN cell lines were imaged for intrinsic fluorescence of GFP, fluorescence of DAPI, and GATA4 immunoreactivity. Automated cell population characteristics were determined with CellProfiler Gelatin-coated plates were prepared by coating with 0.1% gelatin overnight at room temperature. XEN cells were cultured in TS medium with F4H and with or without 10 ng/mL BMP4  on gelatin-coated plates for four days.Cell lines X42, X44, X47 (PDGFRa-GFP\u2009\u00d7\u2009CAG::mRFP1); X35, X36 (B2D6F1\u2009\u00d7\u2009D4/EGFP); X97, X107 (D4/EGFP\u2009\u00d7\u2009DBA2/N); X-E6.5-81346-8 (ROSA-STOP-taulacZ x Sox17-Cre); X-E6.5-Z0617-2, X-E6.5-Z0617-5, X-E6.5-Z0617-8 (R26-tauGFP41 x Sox17-Cre); X-E6.5-78097-4 (Xist1loxGFP\u2009\u00d7\u2009DBA/2\u2009N); X-E6.5-82278-4 (Gata6-mTomato\u2009\u00d7\u2009Cdx2-GFP); X-E6.5-Z0663-12 (CD1\u2009\u00d7\u2009PDGFRa-GFP); and X-E5.5-6, X-E5.5-8, X-E5.5-9, X-E5.5-10, X-E5.5-13 (R26-tauGFP41\u2009\u00d7\u2009Sox17-Cre) were cultured in 4-well or 24-well dishes. Cells were fixed in 4% paraformaldehyde at 4\u2009\u00b0C overnight or room temperature for 30\u2009min, permeabilized with 0.1% Triton X-100 in 1\u00d7 PBS (1\u00d7 PBST) for 30\u2009min and blocked with 5% normal donkey serum  diluted in 1\u00d7 PBST (blocking solution) for 1\u2009hr. Primary antibodies were diluted at 1:50\u20131:200 in blocking solution and samples incubated at 4\u2009\u00b0C rotating overnight. After three 10-min washes in 1\u00d7 PBST 10\u2009min, samples were incubated for 1\u20131.5\u2009hr at room temperature in a 1:500 dilution of secondary antibody in blocking solution, then washed and covered with 1\u00d7 PBST containing DAPI. Images were taken with an AMG EVOS (Life Technologies), Zeiss LSM 710 confocal microscope, and Nikon SMZ25 stereofluorescence microscope.Primary antibodies from Santa Cruz Biotechnology were against GATA4 (#SC-1237), DAB2 (#SC-13982), OCT3-4 (#SC-5279), NANOG (#SC-376915), and CDX2 (#SC-166830). Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), SOX17 (#AF1924), and PDGFRa (#AF1062). Primary antibodies against E-cadherin (ECCD2) were from Invitrogen (#13-1900). Secondary antibodies from Jackson ImmunoResearch Laboratories were Cy5\u2122 AffiniPure Donkey anti-goat IgG (H+L) (#705-175-147), and Cy5\u2122 AffiniPure Donkey anti-rabbit IgG (H+L) (#711-175-152). Secondary antibodies from Invitrogen were Donkey anti-rabbit IgG (H+L) with Alexa Fluor 546 (#A10040), Goat anti-rat IgG (H+L) with Alexa Fluor 546 (#A11081), Donkey anti-mouse IgG with Alexa Fluor 546 (#A10036), Donkey anti-mouse IgG with Alexa Fluor 488 (#A21202), and Goat anti-chicken IgY (H+L) with Alexa Fluor 488 (#A11039).Actb and Gapdh. Normalized counts are displayed in a heatmap generated by the nSolver Analysis Software v2.5, using agglomerative clustering.Cells were cultured in 12-well plates with TS medium including F4H or ES medium supplemented with LIF. Dissociated cells were collected by trypsinization and centrifugation. Cell pellets were dispensed directly in RNAlater Stabilization Solution (Qiagen) and stored in \u221280\u2009\u00b0C for later use. Cell pellets were lysed in RLT Lysis Plus Buffer using a TissueLyser LT (Qiagen) at 40\u2009Hz for 2\u2009min. Total RNA extraction was performed using RNeasy Plus Micro kit (Qiagen) according to manufacturer\u2019s protocol. The custom NanoString CodeSet \u201cExtra\u201d was used. 100\u2009ng of total RNA samples were hybridized at 65\u2009\u00b0C for 18\u2009hr and processed with the nCounter Analysis System GEN1 (NanoString Technologies). The reporter counts were processed using nSolver Analysis Software v2.5 (NanoString). Two normalizations were performed to the counts, the first normalization to the generic positive controls, followed by normalization to the reference genes, Below are the nucleotide sequences of the NanoString probes; first line is the capture probe, second line the reporter probe.ActbACGATGGAGGGGCCGGACTCATCGTACTCCTGCTTGCGGGTGTAAAACGCAGCTCAGTAACAGTCCGCCTAGAAGCACTTGCGGTGCDab2GTCTCCTCGAGCATCAGGCACATCATCAATACCGATTAGCTTGGCCCAGCTGCTGCCATTCCCTTGAGTTTCATCATAGAATCCTGACTCATTTTDnmt3lGAGGCAGCGCATACTGCAGGATCCGGTGGAACTGGAACATGCCATGAATATCCAGAAGAAGGGCCGCTGACTCTCCTGGCDppa4CAAGTCTTTACAGTTGACTGCTGAACTGGTTATGACGCCCGTTGTGCTGGCACTACAACCCAGGGAAGAGGACATGCATGCGGAGGCTACAGGTATAAGCDppa5aCGCACGGCCCACAGCTCCAGGTTCAGGAAGTTTTAGTACCCTGCCAAGGAACCAGACTTCAGGGAAGACGAGATCAAGCTTATCCACCAEsrrbTTTCCAGAATGAACCGCTTCATCTTTAGGACATCCTGTCAACCCAAACCCGAGCAGGTAAAGCCGGAGGACTTGTCATGAAAGTGGCGTGTCCATFgf4ATTCTGGTAACAAAATTCCAAAGATACAGTCTTGTCCCTGGGCGCAGGAAACAGACCGACTCGGTAACAGTGGCAGATACAGAGCAGAAACATCAAACCCFoxa2CACAGACAGGTGAGACTGCTCCCTTGAGGCCTGAAGTCCCTTCCCTATTTAGAATGACAGATCACTGTGGCCCATCTATTTAGGGAFoxq1GCTGTCCTTACTCCGAGGTTTAGAGACTTTGAGCGGAAGACAAGCGTTTTGATTGTTGGGTGAAGTGAGGAGTGGAGTGATAGAAGTTGGTGCAGTFstAGGACTTTGTGATACACTTTCCCTCATAGGCTAATCCAATGGATCTGCCCCTTGGAATCCCATAGGCATTTTTTCCCGCCGCCACACTGGATATCTTCACFxyd3TGAGCCCGCCGACTCGGAGGCTGTACCAATCATAGTAGAAAGGATCATTTGCCACTCATAAGGACTATAATGCCCAGGGCACAGAGAATCCCTGCACAAAGapdhATACTTGGCAGGTTTCTCCAGGCGGCACGTCAGATCCACGACGCCTCAGATGCCTGCTTCACCACCTTCTTGATGTCATCGata4AGAGCCAGGTAACTGTCTGACTTAAGAGGGCTTGGCTTGGGCAAACAGTCTGTATTTTCTAGACAAAGGATCTGTGCTGGAGAAAGTCCCGata6ATCTGGACTGCTGGACAATATCAGACACAAGTGGTATGAGGCCTTCAGAGGGCACAGAAATCACGCATCGAAGGAATGTTATGTCTGCATTTTTGCTGCCKrt8TTCCCATCTCGGGTTTCAATCTTCTTCACAACCACAGCCTTGCAGTGGCCATTCACTTGGACACGACATCAGAAGACTCGGACACCAGCLama1CATTGGCTAAATCGGCATGGCGGTCATCCTTGATACAGACAGAACTCAGACATAACCTTTCCTACATGGACACTGACCTGGCCACTTTCLamb1CCAGGAAGGAATGCGGTCCTGAATGTACTGCCTTTCCACCACAATGACAAAAACTCCAAATAAGCCCCTTCAGGCACCCGGACGAACCCAGGTCCTGTNanogCATATTTCACCTGGTGGAGTCACAGAGTAGTTCAGGAATAATTCCAAGGCGAAGGAACCTGGCTTTGCCCTGACTTTAAGCCCAGATGTTGCGTAAGTCTNr0b1CACTTGAAAAAGAAACTCTTGATGGCCTGGACCGCAGCAGCTGGGAGCAACCTTTCAGATAGGCATACTCTTTGGTGTCAATGTTCAGACTCCAGPdgfraTATGGAGTAAGTCGCTCTCACACACTTACCACACCACCATGTTGGGAACAAAACATGAACAGGGGTATCTGGAAGCCATCTTGTATTGGAAGACCCTTCCPou5f1ACATGGTCTCCAGACTCCACCTCACACGGTTCTCAATGAGTGATCTGCTGTAGGGAGGGCTTCGGGCACTTCAGAAPth1rCCTGGGCACGGTGCAGCAGGAAAATCTGTTCCTCTTTGGTAAAGACATCGTGCTGTGTGCAGAACTTCCTTGAGCAGCTTGTCACATTGCGSox2CCCCGCCGCCCTCAGGTTTTCTCTGTACAAAAATAGTCCCCCAAAAAGAATGCGTAGTTTTTTTCCTCCAGATCTATACATGGTCCGATTCCCCCGCCCTSox7AGAAATCAGCACACCCCAACACTTTTGTGGACAGACGTTTTAGGTTTCTATCATCTTTTGCATCTGTAGATAAGAGTATGCTACAGCTCTGCTCTSox17GGCAGATACTGTTCGAATTCCGTGCGGTCCACCTCTGTCCCTGGTAGGGAAGACCCATCTCGGGCTTATACACAAAGTet3TTTTCAAAGAGCTGAATGAATGCACCAGGATTTTAGGATGGGCGTGTTTCGTCAGGCCCAACCTCTCAATGTCACAGAAATTAAAGCACCAACGTTCTCAAll animal studies were carried out in accordance with the German Animal Welfare Act, European Communities Council Directive 2010/63/EU, and institutional ethical and animal welfare guidelines of the Max Planck Institute of Biophysics and the Max Planck Research Unit for Neurogenetics. All experimental protocols were approved by the Regierungspr\u00e4sidium Darmstadt and the Veterin\u00e4ramt of the City of Frankfurt.How to cite this article: Lin, J. et al. Efficient derivation of extraembryonic endoderm stem cell lines from mouse postimplantation embryos. Sci. Rep.6, 39457; doi: 10.1038/srep39457 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm  \u03b2-globin gene locus can protect a gene promoter from DNA methylation. Protection from DNA methylation is separable from other insulator activities and is mapped to three transcription factor binding sites occupied by the zinc finger protein VEZF1, a novel chromatin barrier protein. VEZF1 is a candidate factor for the protection of promoters from DNA methylation. We found that VEZF1-specific binding sites are sufficient to mediate demethylation and protection of the APRT gene promoter from DNA methylation. We propose that barrier elements in vertebrates must be capable of preventing DNA methylation in addition to blocking the propagation of silencing histone modifications, as either process is sufficient to direct the establishment of an inactive chromatin state.DNA sequences known as chromatin insulator or barrier elements are considered key components of genome organization as they can establish boundaries between transcriptionally permissive and repressive chromatin domains. Here we address the hypothesis that barrier elements in vertebrates can protect genes from transcriptional silencing that is marked by DNA methylation. We have found that the HS4 insulator element from the  It has been proposed that genes and gene clusters are organized into chromatin domains that are maintained independent of their surroundings through the establishment of boundaries \u03b2-globin genes are clustered within a thirty kilobase domain of nuclease accessible chromatin, the 5\u2032 boundary of which is marked by a constitutive DNaseI hypersensitive site called HS4  and adult chicken red blood cells (an abundant source of nuclear protein for purification purposes). Complexes of similar mobility and intensity are observed between the two nuclear extracts and each of the FI, FIII and FV sites  and determined whether it interacts with the G-rich footprinted sites of the HS4 insulator, as it was previously reported that human VEZF1  interacts with similar G-rich sites FV sites . The comA-globin\u03b2 promoter that contains a (dG-dC)16 string. A nuclear factor called Beta Globin Protein 1 (BGP1) was previously characterized as interacting with this site in vitro, but was considered to indirectly assist in activation by directing nucleosome placement A promoter site with an identical specificity to that of erythrocyte nuclear protein(s) (A promoter site A site is competed by unlabelled FI which contains a (dG-dC)9 string 6 string .The (dG-dC) strings present in the VEZF1 sites at HS4 are reminiscent of a site in the chicken s FV see , which mA sites  analyses were performed to analyze the binding of VEZF1 at the chicken analyses . VEZF1 bxpressed . None ofin vivo, as all three VEZF1 binding sites are required for protection from DNA methylation. We found that this was not the case, as VEZF1 remains tightly bound at HS4 when any one of its binding sites is deleted (in vitro (data not shown). We have attempted to disrupt VEZF1 function at HS4 following knockdown by RNAi. We strived to achieve substantial knockdown of VEZF1 to disrupt its binding to the high affinity sites at HS4. Prolonged knockdown was also required as we have previously found that the de novo DNA methylation of these transgenes is a gradual process that takes many days to establish de novo DNA methylation of the HS4 element and no change in HS4's ability to protect a transgene from silencing during this period (data not shown). The inadequacy of RNAi to strip constitutive transcription factor binding from high affinity sites has also been observed for CTCF Vezf1 null ES cells, as we recently found that they are defective for de novo DNA methylation due to the requirement of Vezf1 for full transcriptional activity of the Dnmt3b gene in these cells We tested whether VEZF1 requires all three of its sites for binding to HS4  deleted . VEZF1 aAPRT gene promoter and its effects on methylation. SP1-like binding elements have been shown to be required to prevent methylation of the mouse and hamster APRT CpG island elements: two earlier papers have shown that deletion of the SP1-like elements is sufficient to induce methylation in these islands APRT CGI that contain three SP1-like elements are subject to demethylation upon integration into mouse ES cells APRT gene remaining unmethylated and expressed normally in Sp1 null ES cells and embryos APRT CGI elements (APRT CGI stably integrated into mouse ES cells (in vitro (CCCCCCTTTCCCC) that is reminiscent of the VEZF1-specific bipartite footprint III site found at the HS4 insulator element (CCCCCCGCATCCCC).To address whether VEZF1 elements also protect CpG island (CGI) promoters from DNA methylation, we investigated VEZF1 binding to the elements . We perfES cells . We founin vitro . Site 3 APRT CGI from methylation, we replaced each of the three SP1-like elements with the VEZF1-specific FIII element from the HS4 insulator  a promoter can be protected from DNA methylation even when it lacks active histone modifications and transcriptional activity, and 6) short DNA elements bound by VEZF1 mediate the demethylation and protection of a CpG island from DNA methylation.Here we have studied the paradigm HS4 element to address our hypothesis that a barrier element in vertebrates must be capable of defending a gene from silencing by DNA methylation and have identified a novel CpG island factor. We have presented six findings: 1) a vertebrate barrier element protects a gene promoter from DNA methylation-mediated silencing, 2) the essential transcription factor VEZF1 is a barrier/anti-methylation factor, 3) there is a modular division of labor at the compound HS4 insulator as VEZF1-mediated protection from methylation is separable from CTCF-mediated enhancer blocking and USF-mediated recruitment of active histone modifications, 4) the de novo DNA methylation.We have previously demonstrated that the HS4 insulator acts as a barrier to the spread of histone methylation marks associated with repressive chromatin bona fide activity that is protective against DNA methylation.It was previously shown that the transgenes used in this study become marked by dense promoter DNA methylation upon chromosomal position effect silencing de novo DNA methylation is key to our understanding of how VEZF1 binding at HS4 could protect a promoter from epigenetic silencing. Previous studies using the same transgene system studied here found that non-insulated transgenes, regardless of integration site, are consistently subject to promoter methylation upon chromosomal silencing, and that flanking with HS4 elements can shield transgenes from this methylation \u03b2-globin locus, the spreading of repressive histone modifications is observed upon perturbation of active histone modification recruitment at the HS4 barrier Determining the source of de novo DNA methylation arise via spreading from the chromosomal integration site in our transgene system, we would expect to see high levels of methylation at compromised mutant insulators either coincident with, or prior to promoter methylation. However, we observe that promoters become methylated prior to the insulators, which remain unmethylated or become partially methylated. The observed independence of methylation states between insulator and promoter argue against spreading and clearly show that there can be no single mechanism that controls the methylation state of both the insulator and promoter. It remains possible that VEZF1 elements at HS4 are acting as a barrier to the spreading of a DNA methylation mechanism, but that additional processes prevent the accumulation of methylation at HS4 itself. An alternative possibility is that DNA methylation does not result from spreading, and that the insulator directly interacts with the promoter to deliver VEZF1 co-factors that prevent promoter methylation. In this model, the promoter itself would have its own program to recruit de novo DNA methylation, and VEZF1 would act as a factor that mediates inhibition of methylation. This would distinguish the activity of VEZF1 from those of USF1/USF2, which bind elsewhere in the insulator element and recruit a number of enzymes that deliver active histone modifications to the reporter gene Should Igf2/H19 domain de novo DNA methylation of HS4. It has previously been shown that DNA binding proteins can prevent the methylation of their binding sites simply by steric hindrance of de novo DNA methyltransferases (DNMTs) The 275 bp \u201ccore\u201d HS4 element comprises a CpG island (CGI) that is free of DNA methylation regardless of neighboring gene expression de novo methylation during early development, and how they remain hypomethylated irrespective of transcriptional status. Recent epigenomic profiling studies have begun to reveal a significant portion of CGIs that are subject to varying degrees of tissue-specific methylation in human somatic tissues de novo methylation, which can be selectively inactivated during development and may become defective during cancer progression cis-regulatory elements and trans-acting factors that control CGI methylation status is key to unraveling these processes.All constitutively expressed genes and \u223c40% of genes with tissue-restricted expression have CGI promoters APRT gene CGI promoter. It was previously shown that SP1-like binding elements are required to prevent CGI methylation APRT CGI. A promoter-less APRT CGI fragment containing only site 3 remains protected from DNA methylation de novo DNA methylation. Thus, VEZF1 binding elements can protect a CGI from DNA methylation. We attempted to definitively address the requirement for VEZF1, but discovered that global de novo DNA methylation mechanisms are defective in Vezf1 null ES cells DHFR gene  were cultured and assayed for IL-2R expression by FACS as described previously Chicken 6C2 erythroleukemia cells carrying Gel mobility shift assays were performed as described previously in vitro translation using rabbit reticulocyte lysate (Promega).FI- and FIII-binding proteins were purified from adult chicken red blood nuclear protein extracts by ion exchange chromatography. Throughout the purification, eluate fractions were analyzed for FI- and FIII-binding activity with gel mobility shift assays. The binding specificity of partially purified proteins was checked by competition analysis after each purification step. FI- and FIII-binding activities co-fractionated following ion exchange chromatography with SP XL and Heparin sepharose . Phosphocellulose, Q and Sephacryl S300 columns were used in early purification attempts to resolve FI- and FIII-binding activities but they co-fractionated in each case (data not shown). FI- and FIII-binding activities both eluted in two distinct fractions of approximately 200 and 400 kDa following gel filtration (data not shown). The active fractions eluted from heparin sepharose were pooled then split into two and fractionated in parallel by FI or FIII DNA affinity as described previously 5\u2032CATGCCGCTCGAGCGGTTTTTTTTTTTTTTTTT was used in first strand cDNA synthesis with Superscript II reverse transcriptase (Invitrogen). The primers VEZF1_5\u2032, 5\u2032CCATGACCCATGGGCAGAGCCAAAGT and Adaptor 5\u2032CATGCCGCTCGAGCGG were used to amplify a full length chicken VEZF1 cDNA by PCR which was TA cloned into pCRII (Invitrogen). VEZF1 cDNA was sub-cloned into pCITE4b (Novagen) to generate p4bVEZFfull for the purpose of in vitro transcription. cDNA encoding chicken ZF5 was isolated by RT-PCR from 6C2 cell total RNA using primers designed from the published sequence (U51640). We found that the bases CpG 1306-7 in the published sequence were GpC in our clone, causing codon 436 to translate as alanine instead of arginine. We obtained the vectors pCDNA3-ZF5 and pEVRFO-ZF5  and we also found the CpG to GpC conflict with the published sequence. Full length chicken SP1 and SP3 cDNAs cloned in the pBluescript-based vectors pH-SP1 and pH-SP3-3 were a kind gift from Marc Castellazzi .Chicken VEZF1/BGP1 cDNA was cloned following RT-PCR from 6C2 cell total RNA based on an assumption of conservation of 5\u2032 cDNA sequence with human VEZF1/DB1. The oligonucleotide Adaptor-A E.coli . Peptides were produced in M15 [pREP4] E. coli followed by rapid lysis with B-PER reagent (Pierce). ZF5 peptide was soluble and purified on Ni-NTA agarose (Qiagen). VEZF1 peptide was insoluble and resulting inclusion bodies were prepared using B-PER reagent (Pierce), solubilized with 6M guanidium hydrochloride and immobilized on TALON Sepharose resin (Clontech) at pH 7. VEZF1 peptide was renatured in a stepwise manner with 6, 4, 3, 2, 1 and 0.5 M guanidium hydrochloride prior to elution. VEZF1 and ZF5 polyclonal IgG antibodies  were purified from rabbit serum using PROSEP-A media . Anti-full length chicken VEZF1 antibodies were raised previously Polyclonal antibodies were raised  against chicken VEZF1 (Ser376-Ala547) and chicken ZF5 peptides (Ser131-Lys248) produced in 7 cells/ml) and fixed with a final concentration of 0.25% formaldehyde at room temperature for 30 seconds. 6C2 cells were harvested in mid-exponential growth phase, divided into 30 ml aliquots containing 1\u00d7108 cells in fresh media and fixed with a final concentration of 0.8% formaldehyde at room temperature for 5 minutes. Reactions were stopped by adding glycine to a final concentration of 0.125 M. The cells were washed in PBS and resuspended in cell lysis buffer  to isolate nuclei. Chromatin was prepared following washing  and lysis  of nuclei. Crosslinked chromatin was fragmented by sonicaton (Misonix) for a total time of 10 minutes in regular 10 second pulses at 4\u00b0C. Debris was removed by centrifugation at 15000 g for 10 minutes and chromatin was diluted in 10 volumes of X-ChIP buffer . Agarose gel electrophoresis was used to confirm that chromatin fragments were \u223c500 bp in length on average.ChIP analysis of transcription factor binding in chicken cells was performed using formaldehyde crosslinked chromatin prepared from chicken 10 day embryonic erythrocytes isolated from fertilized White Leghorn eggs  or cultured 6C2 erythroleukemia cells. 10 day erythrocytes were washed and resuspended in 25 mls of PBS . Incubation with antibodies was performed overnight at 4\u00b0C on a rotating wheel. Between 10 and 30 \u00b5g of specific antibodies or 10 \u00b5g of normal rabbit IgG (Santa Cruz) were used per ChIP. Chromatin was precipitated with protein A agarose  for 4 hours at 4\u00b0C with rotation. Immunoprecipitated chromatin was collected, washed, eluted and crosslinks reversed. DNA was then extracted by phenol-chloroform and ethanol precipitated in the presence of 10 \u00b5g glycogen.Chromatin was pre-cleared with 100 \u00b5l of protein A agarose  and 50 \u00b5g of normal rabbit IgG (Santa Cruz) for 3 hours at 4\u00b0C on a rotating wheel. Aliquots of chromatin were taken to generate input DNA and protein for western analysis. Individual ChIPs were performed using chromatin from 1\u00d710\u03b2-globin locus as described previously Relative DNA enrichments were quantified by TaqMan real-time qPCR using the comparative Ct method relative to input DNA and normalized to the primer set 10.35 within the 16 kb condensed chromatin region upstream of the chicken GGAACAAGTTGGCAAGGTCCTAT10.35_For:\u2003TCTTCTGCCCTGCCCGTAT10.35_Rev:\u2003FAM-TGCAGTTCCCTGTTCATGTGCTTTTCG-TAMRA10.35_TM:\u2003TCCTGGAAGGTCCTGGAAG21.54_For:\u2003CGGGGGAGGGACGTAAT21.54_Rev:\u20036FAM-CCCAAAGCCCCCAGGGATGT-TAMRA21.54_TM:\u2003CTGTGGTCTCCTGCCTCACA39.8_For:\u2003AGGCTGGGTGCCCCTC39.8_Rev:\u2003FAM-CAATGCAGAGTGCTGTGGTTTGGAACTG-TAMRA39.8_TM:\u2003CACAGGAAACAGCTATGACATGATTPGI_5\u2032_For\u2003TCTGCCTTCTCCCTGATAACGPGI_5\u2032_Rev\u20036FAM-AATTCCTGCCCACACCCTCCTGC-TAMRAPGI_5\u2032_TM\u20039 cells treated with 1% formaldehyde for 5 minutes. Chromatin was prepared as described above, where fragments sizes ranged from 500\u2013700 bp. Semi-quantitative PCR was performed using the following primersChIP analysis of transcription factor binding in murine E14Tg2A.4 ES cell lines was performed using formaldehyde crosslinked chromatin prepared from 1\u00d710AAAGGCGTGCGGGAGCCAGAAATAPRT1/2_For: CCTTGGTAGGTGGGGAPRT1/2_Rev: CCCTGTTCCTGGGCTCCAPRT3_For: TGACTGGCCAGGAGGAPRT3_Rev: ChIP analysis from human embryonic kidney 293-T cell line SD5 that contains a stably integrated copy of the 275 bp HS4 core chicken insulator was performed as described for cultured chicken cells above. SD5 cells were crosslinked with 1.6% formaldehyde for 5 minutes. The following primers were used in SYBR quantitative PCR analysis:CGGGATCGCTTTCCTCTGAHS4_21.726_F: CCGTATCCCCCAGGTGTCTHS4_21.726_R: TCGCCTGCACAAATAGGGACP_DHFR_F: AGAACGCGCGGTCAAGTTTP_DHFR_R: ControlGACAGCAGCCGAACTTCGTTVEZF1_CDS_F: TGGTGCCCGAGGAAGATGVEZF1_CDS_R: APRT elements. The following elements were amplified from Hamster liver genomic DNA:AvaII and HpaII restriction sites in bold.Sp1-like motifs in red. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGGCCCCGCCCCGTCCCGCCCCCTCCCGGCCTCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCTCCGCCCCCCTTTCCCCGGCCACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAAvaII and HpaII restriction sites in bold.Mutated bases in blue were introduced by site directed mutagenesis. Overall CpG content increased from 46 to 48 following these mutations. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGACCCCCCGCATCCCCGACGCTACCCCCCGCATCCCCGATCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCCGCTCCCCCCGCATCCCCGACACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAHpaII site in bold.TCTAGATTGCTAGGAGTAGCACCTAAGATGAACTAGATGCTAAAAAATGCTGTATCTTTGGGGCACACGAGGGCATGCCTGGGCAGGCTTAGAGCCTGGTAGTCTCAGGGGCTGCACCAAAGTGTAATTCTTGTGCTAAATAACTTTCACTTACCAGTGCCAAGCACGGGCTTCAGAAACACCCTAGGGTCGCTGAATGTCCACCAGGGGAGTCAGACATGTCCAGAGGGTGAGAACCCCAGAGAATTCGGTAGCCCTGACATGTGCTACAATTACTGATGCCCACTTCCTACTGGTTCCTCCTGGCCATACCTCAGGAATTAGGGCATGCTTTCTGCCTGCTACAGTAGCTCATCCTCCCTGGAAGTGACCCCAGACATATACCCTGAACTGTAACCGATAAAGTGCGCCTGGGCAGATGTATTTGAGAGGTGGCAAAAGTAAACCATAGGTGTCCCCGAGCTAGATACAGAAGGCAGATAACATCCCCAAGGCTAAGCTGCTGCCCCAATAGCCATCAGCCTTCTAGTTATAGCTAGTAAGACCTAGTATTCCTGGTCAATACTATTCACTCAATCCTTACACCTCAGCCCTAACACGCCCCCTCTCTCATCCTAACAGGCCTAGACTCCAGGGGATTCTTGTTTGGCCCCTCCCTAGCTCAGGAGCTGGGCCTGGGCTGTGTGCTCATCCGGAAGCGAGGGAAGCTGCCAGGCCCCACAGTGTCAGCCTCCTATGCTCTCGAGTATGGCAAGGTAAGCAGGCAGTGGGTAGCTGTCTAGGAGTAAATGTGGGGGCTCAGAGAGGTTAAGTCATCAGGCCAGGTTTATACCACCAGGAAACATGGAGAAGCTAGGGGTGGTGGTTCTAGAAPRT CpG islands and an 870 bp non-island region of the APRT gene were cloned into pUC19. Each element was PCR amplified, half of which was subject to in vitro CpG methylation by M.SssI methyltransferase(New England Biolabs). In vitro methylation was validated by HpaII and MspI digestion. 1 \u00b5g of each APRT element was transfected into murine E14Tg2A.4 ES cells (BayGenomics) with 1 \u00b5g of the XbaI fragment of pREP7 (Invitrogen). Hygromycin resistant clones were grown in LIF without feeder co-culture. Genomic DNA was extracted after two weeks of culture. Probes for Southern blotting were generated using Ready-to-go dCTP beads .\u223c720 bp wild type and mutant Oligonucleotides were generated on an ABI 394 DNA synthesizer.The top strand sequences for each of the duplexes used for gel mobility shift analyses were:GACAGCCCCCCCCCAAAGCCCCCAGGGA5\u2032 GGAGCTCACGGG,FI wt CGCTCCCCCCGCATCCCCGAGCCGGggcgcgcct5\u2032 aggcgcgccCCGGTCCGG,FIII wt CCTGGGGGGATACGGGGAAAAAGCTTTAGG5\u2032 CCTGCAGACA,FV wt 5\u2032 ATTCGATCGGGGCGGGGCGAGC,Sp1 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGGGGGGGGGGGCGGGTGGTGGTGTGG,glo wt 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGGCGGGTGGTGGTGTGG,glo 7G 5\u2032 AATTGCAGAGCTGGGAATCGGGGGGCGGGTGGTGGTGTGG.glo 6G Asc I restriction sites used for cloning FIII sites in an earlier study are shown as lower case. All FI and FIII oligonucleotides were identical to the wt sequences above except for mutations indicated in DNA affinity columns were prepared using the following oligonucleotides5\u2032 gatcTCACGGGGACAGCCCCCCCCCAAAGCCCCCAFI-DA TOP 5\u2032 gatcTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAFI-DA BOTTOM 5\u2032 gatcGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAFIII-DA TOP 5\u2032 gatcTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCFIII-DA BOTTOM PCR primers used in bisulfite sequencing were5\u2032 GGTATTAGAGTAGATTGTATTGAGAGTGTAHS4 5\u2032 double forward\u20035\u2032 CATAACTATTTCCTATATAAATCCCCHS4 5\u2032 double reverse\u20035\u2032 AGAGTAGATTGTATTGAGAGTGTATTATAHS4 5\u2032 single forward\u20035\u2032 ACATCCCTAAAAACTTTAAAAAAAAHS4 5\u2032 single reverse\u20035\u2032 GTTAAGGTTGGGGGTTTTTTIL-2R forward\u20035\u2032 AAAACTCTACCTAACAACCAAACACIL-2R reverse\u2003PCR primers used in RT-PCR gene expression analysis were5\u2032CAGTGCACGTTTGGCATTTGAAGGgVEZF805anti\u20035\u2032GAAAAGGCTTCTCGAGGCCTGATCGgVEZF716sense\u20035\u2032-6FAM-TCCAGGAGCGTGACCCCAGCA-TAMRAGgGAPDH-247T\u20035\u2032 ATGGGCACGCCATCACTATCGgGAPDH-226F\u20035\u2032 AACATACTCAGCACCTGCATCTGGgGAPDH-302R\u20035\u2032 TGCTGCGCTCGTTGTTGAGgB-ACTIN_F\u20035\u2032 CATCGTCCCCGGCGAGgB-ACTIN_R\u20035\u2032-6FAM-TGGCTCCGGTATGTGCAAGGCC-TAMRAGgB-ACTIN_T\u2003APRT non-island element were:The primers used for methylation specific PCR analysis of the 5\u2032\u2003TCTAGATTGCTAGGAGTAGCAPRTNI_3\u2032\u2003TCTAGAACCACCCCTAGCAPRTNI_Figure S1SacI - HindIII fragment originally defined as the \u201c250 bp core\u201d HS4 element in vitro DNaseI footprints AscI cloning sites present in all functional assays of the HS4 core are shown in italics.Core HS4 sequences. (A) The 244 bp (0.87 MB TIF)Click here for additional data file.Figure S2Identification of FI- and FIII-binding proteins. (A) Peptide sequences obtained from tandem MS sequencing of proteins isolated by FI- and FIII-DNA affinity. (B) Alignment of the amino acid sequences of chicken VEZF1/BGP1, human VEZF1/DB1, and mouse Vezf1 . Peptides obtained from tandem MS sequencing that match VEZF1 are indicated by lines above the alignment. Six C2H2 zinc fingers motifs are boxed. (C) Schematic representation of the domain structure of VEZF1. Only the zinc finger motifs share homology with factors other than VEZF1 orthologs, the nearest relative being the MAZ transcription factor.(4.96 MB TIF)Click here for additional data file.Figure S3A-globin\u03b2 promoter. Gel mobility supershift assays using 32P-labelled FI (A), FIII (B), FV (C), and glo wt (D) oligonucleotide duplexes. Adult chicken erythrocyte nuclear extract (ery) and recombinant chicken VEZF1, SP1, or SP3 used in the reactions are indicated by brackets above the lanes. Proteins were pre-incubated with antibodies (indicated above each lane) prior to incubation with DNA. Supershifts are evidenced by abrogation of specific complexes (asterisks) and/or formation of low mobility ternary complexes (SS). Antibodies alone do not give rise to complexes with any of the duplexes used (not shown).Supershift analysis of VEZF1, SP1, SP3, and ZF5 interactions with HS4 footprints and the (3.49 MB TIF)Click here for additional data file.Figure S432P-labelled FI and FIII oligonucleotide duplexes. Unlabelled competitor duplexes (indicated above each lane) were added at 50 fold molar excess. Recombinant chicken SP1, SP3, and ZF5 used in the reactions are indicated by brackets above the lanes. The competition profile of these proteins does not match that of red blood cell nuclear extract Click here for additional data file.Figure S5in vivo. (A) ChIP analysis of transcription factor interactions at the \u03b2-globin locus in 6C2 erythroid progenitor cells. DNA enrichments at the {lower case beta}A promoter or the HS4 and 3\u2032HS insulators were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the \u03b2-globin locus. (B) ChIP analysis of transcription factor interactions with a stably integrated HS4 element in transgenic human 293 cells. Interactions with the DHFR CpG island promoter are shown as a positive control for SP1 and SP3 binding. DNA enrichments are normalized to an AT-rich negative control locus.SP1, SP3, and ZF5 do not interact with HS4 (0.49 MB TIF)Click here for additional data file.Figure S6IL-2R transgene showing location of QPCR primer sets used to analyze the interaction of VEZF1 with the transgenic HS4 and promoter elements. (B\u2013D) ChIP analysis of (B) VEZF1, (C) CTCF, or (D) USF1 interactions at the endogenous HS4 insulator (eHS4) and the transgenic HS4 (tHS4) and promoter (\u03b2-IL2Rpro) elements in the same 6C2 cell lines used for DNA methylation  Schematic representation of the hylation  and hist(1.09 MB TIF)Click here for additional data file.Figure S7\u03b2-globin locus.VEZF1 binding to HS4 is not significantly affected following VEZF1 RNAi. (A) Quantitative RT-PCR analysis following 48 hours of doxycycline-induction of two chicken 6C2 cell lines harboring lentiviral vectors that express VEZF1-specific miRNA. Expression levels are normalized to those of \u03b2-actin (ACTB) and untreated cells. VEZF1 mRNA levels are reproducibly knocked down by 70%. (B) Western blot analysis of chicken VEZF1 protein levels in one of the above lines with and without 14 days of doxycycline induction of VEZF1-specific miRNA. TBP levels were monitored as a loading control. VEZF1 and TBP band intensities visualized on a FUJI LAS3000 imager were quantified using AIDA software (shown below). Following normalization to TBP levels, VEZF1 protein levels were quantified to be knocked down by \u223c97%. VEZF1-specific miRNA may cause translational inhibition in addition to the mRNA degradation observed by RT-PCR (C) ChIP analysis of VEZF1 interaction with the endogenous HS4 element following 14 days of induced VEZF1 knockdown. DNA enrichments were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the (1.64 MB TIF)Click here for additional data file.Figure S8APRT CpG island elements. VEZF1 and SP1 interact with the wild type APRT CpG island, but the FIII mutant APRT element is only bound by VEZF1. Gel mobility supershift assays using 32P-labelled oligonucleotide duplexes containing either the wild type APRT SP1 sites 1&2 (lanes 1\u20134) and site 3 (lanes 5\u20138) or the mutant sites 1&2 (lanes 9\u201312) and site 3 (lanes 13\u201316). The core sequences of the duplexes are as shown in Transcription factor interactions with (1.78 MB TIF)Click here for additional data file.Table S1VEZF1 sites in the HS4 barrier protect a promoter from DNA methylation. CpG methylation of transgene promoters flanked by wild type or mutant HS4 insulators after 30 or 90 days of culture. The scoring of individual CpG bases from each clone subject to bisulfite sequencing is shown. Methylated bases are marked as \u20181\u2019 and shaded blue. Average CpG methylation values are shown in (0.02 MB PDF)Click here for additional data file.Table S2de novo methylation of HS4. CpG methylation of wild type (WT) or mutant (\u0394I - \u0394V) HS4 insulators after 30 or 90 days of culture. Both copies of HS4 were sequenced, except for \u0394II and \u0394V, where only the outermost copy was sequenced (see Mutations of insulator protein binding sites result in the nced see . The sco(0.03 MB PDF)Click here for additional data file.Text S1DNA binding activities of candidate HS4-binding proteins; RNA interference methods; western blotting methods.(0.04 MB DOC)Click here for additional data file."}
+{"text": "The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors  were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs ( OCT4, SOX2, KLF4, cMYC, LIN28 and NANOGHuman embryonic stem cells (hESCs) have significant therapeutic potential for various diseases, but the generation of these cells from individual patients raises ethical concerns. Recently, a technological breakthrough where somatic cells from mouse and human can be reprogrammed into hESC-like pluripotent cells, termed induced pluripotent stem cells (iPSCs), was made possible through ectopic expression of combinations of reprogramming factors including MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression post-transcriptionally . The miR-302 gene encodes a cluster of eight miRNAs on chromosome 4 (miR-302b*-b-c*-c-a*-a-d-367) that are preferentially expressed in embryonal carcinoma cells, hESCs and hiPSCs miR-223, miR-155, and miR-142-3p are enriched in differentiated cells miR-223 and miR-142-3p are found in cells of primarily hematopoietic origin miR-155 is found in hematopoietic cells as well as in many types of lymphoma and solid cancers miR-155 is also expressed 20\u201350 fold higher in fibroblasts than in hESCs and hiPSCs We constructed a bidirectional vector whereby the reporter gene mCherry is conjugated with perfectly complementary miRNA target sites for miR-302a, miR-302b, miR-302c, and miR-302d or miR-155 results in ablation of EGFP or mCherry expression, respectively, demonstrating sensitivity of the vector to specific miRNAs . In hematopoietic lineage cells  mCherry expression is entirely ablated whereas EGFP is maintained . After a prolonged period of culture on feeder cells, a fraction (<0.01%) of the cells reprograms and appears as hiPSC colonies The reprogramming of HFFs to hiPSCs can be achieved by the introduction of four transcription factors . Expression levels of EGFP and mCherry were monitored over the four week course of reprogramming to assess the activity of the reporter vector during generation of hiPSC  and one hiPSC clone that was not transduced with this reporter vector (hiPSC #19) for characteristics of pluripotent stem cells. These hiPSC clones transduced with the reporter vector stably maintained mCherry expression and lack of EGFP expression for over 20 generations . Althougin vitro into EBs comprising the three embryonic germ layers miR-302a/d expression as the cells differentiated. Concomitantly, mCherry expression was slightly reduced in the cells, reflecting expression of one or more of the differentiation specific miRNAs miR-223, miR-155, or miR-142-3p. However, since the EBs represent multiple lineages of differentiated cells, we were unable to conclude which cells and to what extent these miRNAs are being expressed.hESC as well as hiPSC can be differentiated SOX1, SOX3 and PAX6, markers of neuroectodermal differentiation DNMT3B, REX1 and endogenous OCT4 which are assumed to be expressed in fully-reprogrammed hiPSCs DNMT3B, REX1 and endogenous OCT4 were downregulated, whereas SOX1, SOX3 and PAX6 were upregulated in the differentiated population compared to the undifferentiated population  had reduced mCherry expression compared to two other hiPSC clones transduced with the reporter vector . AlthougThe development of this vector adds to the tools available to monitor hiPSC generation and subsequent differentiation of the hiPSC into different lineages. In addition to taking advantage of differential miRNA expression, other investigators have utilized differential promoters/enhancer expression in different lineages miR-223, miR-142-3p, and miR-155 are enriched primarily in cells of hematopoietic origin The work presented here indicates the potential great utility and flexibility of miRNA-regulatable lentiviral vectors to monitor various stages of reprogramming and the subsequent differentiation into lineage specific cells and tissues. For example, CCCGGGCGAGCAAGCTCAGTTTACACCGAATTCGGATCCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAGGCCTACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGATATCGTCGAC) and differentiated cell specific-miRNA targets (CCCGGGTCGAATTCGGTACCAGATCTGGCGCGCCGTACGTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAGGCCTACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAATTTAAATTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGTACAGTTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGATATCTGCATGCTTCGAAGCTAGCGGGCCC) were synthesized by Genescript . The synthesized fragment of differentiated cell specific-miRNA targets was conjugated to mCherry coding sequence amplified by PCR from pmCherry  with primers (sense: ACGCACCGGTGGATCCAAGCTTGCCACCATGGTGAGCAAGGGCGAGGA and reverse: CTGCGAATTCTCACTACTTGTACAGCTCGTCCATGCCGCCGG) and exchanged with EGFP coding sequence in FG12 lentiviral vector AGTCAGCTAGCGCCACCATGGTGAGCAAGGGCGAG and reverse: CATCGACCCGGGAATTCTCATTACTTGTACAGCTCGTCCATG), and cloned into a pAAV-MCS vector  (pAAV-EGFP). The synthesized fragment of hESC specific-miRNA targets was inserted into the pAAV-EGFP between stop codon of EGFP and hGH poly A signal. The fragment containing CMV minimal promoter, \u03b2-globin intron, EGFP, hESC specific-miRNA targets, and hGH poly A signal was amplified by PCR with primers (sense: GTACTCTCGAGCCCCATTGACGCAAATGGGCGGTAGG and reverse: CTCGTCTAGAAGGACAGGGAAGGGAGCAGTGGT) and cloned into the FG12 lentiviral vector deleted EGFP (EGFP-T/FG12) or into the mCherry-T/FG12 (EGFP-T/mCherry-T/FG12).For construction of the reporter vector, whole sequences of hESC specific-miRNA targets  were substituted with EGFP coding sequence in the FRh11. The infectious titer was determined in 293T cells by infecting with the FRh11 encoding EGFP in the presence of 8 \u00b5g/ml polybrene. Reporter gene expression was monitored by flow cytometry.For reprogramming of HFFs, we substituted the ubiqutin C promoter of FG12 lentiviral vector with the RhMLV promoter (FRh11). The RhMLV promoter is derived from the long terminal repeat (LTR) region of Moloney murine leukemia virus (MLV) in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation miR-302a, miR-302b, miR-302c, and miR302-d (PMIRH302abcdPA-2) and miR-155 (PMIRH155PA-1) from System biosciences . CopGFP sequence was eliminated from the vector.For ectopic expression of miRNAs, we purchased miRNA expressing lentiviral vectors for 2.293T hESCs (H1 clone) HFFs were isolated from the skin of 16 week-old fetus with DMEM supplemented10% FBS and 2 mM Glutamax (fibroblast medium) as reported previously CD34+ HPSCs were prepared from the liver of 16 weeks-old fetus as previously described Lentiviral vector stocks were generated using a vector plasmid, a packaging plasmid pCMV R8.2 \u0394Vpr, and a VSV-G envelope protein-coding plasmid by calcium phosphate-mediated transient transfection as previously described 4 cells per well of 6-well plates and infected with vectors encoding each reprogramming factor  with or without a lentiviral vector encoding EGFP-T/mCherry-T at 300 ng (around multiplicity of infection of 3\u20135) of p24 per each virus. The cells were cultured for 3 days in fibroblast medium and replated at 5\u00d7104 cells per 60 mm dish on irradiated mouse embryonic fibroblast (iMEF) feeder cells. On the next day, the medium was replaced with KO-DMEM (Invitrogen) supplemented with 20% Knockout Serum Replacer , 2 mM Glutamax (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 0.1 mM \u03b2-mercaptoethanol , and 50 ng/ml of recombinant human basic fibroblast growth factor (Invitrogen) (hiPSC medium). The medium was changed on a daily basis. To increase a reprogramming efficiency, the cells were treated with 0.5 mM valproic acid  and 10 \u00b5M Y27632  for first 14 days The day before lentiviral vector transduction, HFFs (passage 1\u20133) were seeded at 5\u00d710Size-controlled EBs (3000 hESCs/EB) were formed using AggreWell\u2122 400 plates  following the manufacturer's protocol. Briefly, hESCs and hiPSCs were incubated with 10 \u00b5M Y-27632 for 24 hrs before EB formation. Cells were harvested with Accutase  as a single-cell suspension and used for EB formation. EBs were harvested into ultra low attachment plates  and maintained in Iscove's Modified Dulbecco's Medium  containing 10% FBS, 2 mM Glutamax, and 0.1 mM \u03b2-mercaptoethanol for differentiation into EBs. The medium was changed every 3 days.Induction of differentiation into neural lineages was performed as previously described with some modifications For detection of EGFP and mCherry expression, single-cell suspensions from 293T, HFF, hESC, and hiPSC were prepared using 0.25% trypsin-EDTA and collected in FACS buffer (2% FBS and 0.01% sodium azide in PBS). U937, Ramos and CEM cells were also collected in FACS buffer. For detection of hESC-specific markers, hESCs and hiPSCs were dissociated with 0.25% trypsin-EDTA into a single cell suspension. Cells were adjusted to 100,000 per sample in 100 \u00b5l of FACS buffer and then labeled with monoclonal antibodies conjugated with a fluorescent dye . For detection of iMEFs, cells were stained with an antibody specific with mouse CD29 conjugated with PE-Cy7 (eBioscience). Data were collected on a Cytomics FC500  and analyzed using FCS express .hESC and hiPSC colonies were grown on poly-L-lysine and Matrigel coated glass coverslips. Cells were fixed with 1.0% formaldehyde/PBS and permeabilized with 0.2% Triton-X 100 for 5 min on ice. Cells were then incubated with anti-human Nanog antibody  and subsequently with DyLight488 conjugated donkey anti-rabbit IgG (BioLegend) and 7-amino-actinomycin D (7-AAD) (Invitrogen) for nuclear staining. After washing, cells were visualized with a LEICA DM IRB  equipped with a SPOT camera and software .RNA extraction from hESC and hiPSC was performed using QIAGEN's RNeasy Mini kit following the manufacture's protocol . Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript RT-kit with a 0.5 ng/ml oligo dT primer (Invitrogen) in 20 \u00b5l reaction. PCR was performed with the HotMaster Taq DNA polymerase , using 0.5 \u00b5l of cDNA template and primers at a concentration of 3 pmol/\u00b5l. Five \u00b5l of PCR products was loaded in a 2% agarose gel containing ethidium bromide. All primer sequences were listed in Table 1."}
+{"text": "Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of  Drosophila melanogaster centromeres, which have long remained elusive despite the high quality of this species\u2019 genome. assembly.Long-read sequencing, CENP-A ChIP, and chromatin fiber imaging reveal the composition and organization of  Drosophila and centromeric histone H3 [CenH3] in plants), which is necessary and sufficient for kinetochore activity  \u00d7 (1 pmol/330 pg) \u00d7 (1/Oligopaint length in nt) = Oligopaint molarity [\u03bcM]G2/Jockey-3 oligo against the consensus of the 3\u2032 region of G2/Jockey-3 elements found within most centromere contigs. A 5\u2032 addition containing 5\u2032-CAGT-3\u2032 followed by universal forward primer binding sites separated by an XhoI cut site (5 \u2032-cacactccggtacgcacctgctcgagcagtgctcgttggcccacac-3\u2032). A 3\u2032 addition containing 5\u2032-ACTG-3\u2032 followed by universal reverse primer binding sites separated by a SpeI cut site (5\u2032-agggtagtcgttgtagctcgactagtggtacgcccagaagcatccc-3\u2032). The G2/Jockey-3 sequence was ordered as a \u201ccustom gene\u201d (IDTDNA.com) and synthesized in the pUCIDT (AMP) vector (pUCIDT-G2). The sequence of the insert is as follows .We designed a 1,643-bp cacactccggtacgcacctgctcgagcagtgctcgttggcccacacCGGACGGCTCTTGGTGCCGCTCTGAAGCCGAAAGAGCTGAAGCGTTTGCAGATCACCTCCAGAATGCATTCACACCATTTGACAGATGCACTGGCGAAGAGCGTGCTGCAACCACCAGGTTCCTAGAGAGTCCATGTCCTCCTAGCCTGCCCATAGAGCCCGTCACCCCAGAAGAGGTTGCGCAAGAGTCGCCTCACTAAAGGCTAGCAAATCCCCAGGACTGGATCGCATCGACGCCACATCCCTTAAAATGCTGCCACCTCCCTGTTCCCAGTTGCTGGCCAACATATACAACAGATGCTTCTCACTAGGGTACTTCCCGAGATCATGGAAACGTGCAGAAGTCATTCTCATCCTCAAACCTGGAAAACCTGAAGCCAATCTTGCCTCATATAGACCGATTAGTCTGCTGGCAATCCTCTCCAAAATACTCGAAAGAGTATTTCTGCGCAGAGTGTTGCCAGTACTGGACGAGGCTGGACTGATCCCTGATCACCAGTTTGGCTTCAGGCGATCCCACGGAACACCCGAGCAATGCCACCGGCTCGTAGCACGCATCCTAGATGCATTCGAGAACAAACGATACTGTTCGGCCGTATTCCTGGATGTCAAGCAGGCGTTCGACAGAGTGTGGCATCCTGGACTCCTCTACAAACTCAAGTCCCACCTTCCCAGTTCCCACTATGCCCTACTCAAATCGTATACTGAAGGAAGAGAGTTCCAAGTGCGATGCGGTTCCTCAACCAGCACGACAAGGCCTATACGAGCCGGAGTACCTCAAGGCAGCGTCCTTGGTCCCATCCTCTACACCCTGTTTACAGCAGACCTCCCTATCATACCCTCCCGTTACCTCACAGCAGCCACCTATGCAGATGACACGGCGTTCCTTGCCACCGCAACAAACCCTCAACTAGCATCAGCCATCATCCAGAGGCAACTGGATGCATTGGATCCATGGCTGAAACGCTGGAACATCGTGATCAACGCTGATAAATCCTCCCACACCACCTTCTCTCTGCGCAGAGGAGAATGCCCCCCGGTCTCACTCGACGGCGACACAATCCCTACCTCCAGCACCCCCAAATATTTAGGGCTGACCCTGGACAGAAGGCTGACTTGGGGCCCCCACATCAACAGAAAGCGTATCCAGGCCAACATACGCCTAAAGCAACTCCACTGGCTCATCGGTAAAAAGTCCAAGCTGCGAGAGAAACTAAAGATTCTCGTCTACAAGACTATTCTCAAGCCAATCTGGACGTACGGAATTCAGCTGTGGGGCACTGCAAGCACATCACATAGAAGGAAGATCCAGCGATTTCAAAACAGATGTTTGAGAATAGTCTCCAACGCCCATCCCTACCACGAAAATTCCGCCATCCACGAGGAGCTCGGGATTCCATGGGTAGACGACGAAATCTACAGACACAGTGTGAGATATGCTAGCAGACTGGAGAACCACCACAACCACCTGGCCGTCAACCTTCTAGACCATAGCCAATCCCTAAGACGCCTGCAGAGAACGCACCCGCTTGACCTTACTCAACATACTTAATCATACTTAACCCCTACCCAAGTACACTCGATGTACTCCCCTTAAGTTAATGTTTCCCTCCAAAAAATTTAATTATTGTCCACTAGGACAGgggatgcttctgggcgtaccactagtcgagctacaacgactaccctcagt-3\u20325\u2032-cagtG2 (500 ng) was digested using SpeI and XhoI restriction enzymes in 1x Cutsmart Buffer for 1 h at 37\u00b0C. The digest was run on a 1.0% SeaPlaque GTG agarose gel (Lonza), and a 1,689-bp band containing the G2 sequence was gel extracted and purified using the PureLink Quick Gel Extraction Kit (Invitrogen). DIG-labeled G2 probes were generated via PCR in 50-\u03bcl reactions consisting 0.09 ng of gel extracted G2 DNA, 0.5 \u03bcM of forward and reverse primers from the Universal_2 primer set  to provide additional support for our candidate centromeric contigs . We mappnogaster  to our anogaster  and the We calculated the significance between different categories using a Kruskal-Wallis test by ranks with Dunn\u2019s test for post hoc analysis and the pairwise Wilcoxon rank sum test with false discovery rate (FDR) correction  of type G2/Jockey-3 sequences based on RepeatMasker annotations and custom scripts. We aligned and manually inspected G2/Jockey-3 and IGS alignments using Geneious v8.1.6 [https://doi.org/10.5061/dryad.rb1bt3j [G2/Jockey-3 and IGS using RAxML v.8.2.11 with settings \u201c-m GTRGAMMA -T24 -d -p 12345 -# autoMRE -k -x 12345 -f a\u201d [We extracted all IGS elements from the genome using BLAST v2.7.1  with sets v8.1.6  (see Dry.rb1bt3j ). We con45 -f a\u201d . We used45 -f a\u201d  to plot G2/Jockey-3 non-LTR retroelements have evidence for recent activity based on insertion polymorphism and expression. We examined RNA-seq reads from testes for evidence of G2/Jockey-3 because of the enrichment of these elements on the Y chromosome. We mapped poly-A [We investigated whether d poly-A  and totad poly-A  (S6 Tabld poly-A  and estid poly-A ).S1 FigPlot of normalized CENP-A/input for simple tandem repeats for each ChIP-seq replicate, sorted by median (red lines). Shown are only the simple tandem repeats with median CENP-A/input > 1 in all four CENP-A ChIP replicates (see details in (TIF)Click here for additional data file.S2 FigG2 and Jockey-3 sequences in D. melanogaster genome and closely related species in the simulans clade (D. simulans and D. sechellia) and D. yakuba. In D. melanogaster, G2 and Jockey-3 are interleaved across the phylogeny and thus likely correspond to the same repeat type. We therefore refer to these elements collectively as G2/Jockey-3 throughout the manuscript. (See Dryad repository files 13 and 15: https://doi.org/10.5061/dryad.rb1bt3j [A maximum-likelihood phylogenetic tree showing the relationship between .rb1bt3j ). LTR, l(TIF)Click here for additional data file.S3 FigLocations of the top 100 strongest peaks for each ChIP experiment. (A) Plot of the location of top 100 strongest peaks for each ChIP experiment on the diagonal (see details in (TIF)Click here for additional data file.S4 FigJockey elements are shown in one color even though they are distinct elements). The normalized CENP-A enrichment over input  is shown for three replicates (replicate 2 is in Organization of each CENP-A-enriched island corresponding to centromere candidates: (A) X centromere, (B) centromere 4; (C) Y centromere; (D) centromere 3; (E) centromere 2. Different repeat families are color coded Click here for additional data file.S5 FigRpL32 promoter region as a noncentromeric control. (C) Graph showing our ChIP-qPCR results using primers targeting other regions that showed CENP-A enrichment but that were not in our contigs. Again, the enrichment is calculated relative to the input and is normalized by RpL32 promoter as a noncentromeric control. We did not observe a robust CENP-A enrichment at these sites. The underlying data can be found in (A) Diagram showing putative centromere contigs showing the locations of CENP-A ChIPtigs in black and CENP-A MACS peaks in orange as in (TIF)Click here for additional data file.S6 FigPacBio reads were mapped to the genome using Minimap (v 2.11) and the setting \u201c-ax map-pb.\u201d Shown are (A) X centromere, (B) centromere 4, (C) Y centromere, (D) centromere 3, and (E) centromere 2. The depth of only the high-quality mapped reads (mapped Q \u2265 30) was estimated for each position and normalized by the median depth of other genomic regions (98.32\u00d7 for autosomes and 49.16\u00d7 for sex chromosomes) to get relative depth. The relative depths of the TE-rich islands are close to 1, whereas the depth of the flanking simple satellites is uneven, with some regions > 1 and some < 1. We therefore exclude simple repeats from any assembly-based analyses and color these regions gray in (TIF)Click here for additional data file.S7 FigProdsat (magenta) and AAGAG (blue); (C) AATAG (magenta) and AAGAG (blue) with AATAG blocks identified by white  and yellow (large block) arrows; (D) Prodsat (magenta) and dodeca (blue); (E) AATAT (magenta) and SATIII (blue); (F) AATAT (magenta) and AAGAG (blue); (G) AATAG (magenta) and Prodsat (blue) with AATAG blocks identified by white  and yellow (large block) arrows. DAPI is shown in gray. The underlying data can be found in Prodsat, Prod satellite.IF-FISH using an anti-CENP-C antibody (green) and satellite FISH probes in the following combinations: (A) AAGAT (magenta) and AAGAG (blue) with a high-contrast inset of AAGAT on the X chromosome; (B) (TIF)Click here for additional data file.S8 FigSATIII (blue); (B) dodeca (magenta) and Prodsat (blue); (C) AATAG (magenta) and Prodsat (blue) with a high-contrast inset of AATAG and Prodsat on cf(2R); (D) AAGAG (magenta) and Prodsat (blue); (E) AAGAG (magenta) and AATAG (blue) with a high-contrast inset of AATAG on chromosome 3; (F) AAGAT (magenta) and AAGAG (blue). DAPI is shown in gray. See also Prodsat, Prod satellite; S2, Schneider 2.IF-FISH using an anti-CENP-A antibody (green) and satellite FISH probes in the following combinations: (A) AATAT (magenta) and (TIF)Click here for additional data file.S9 FigG2/Jockey-3 consensus element obtained from mapping total and poly-A RNA-seq data from testes [G2/Jockey-3 copies surveyed on centromere (\u201cCen\u201d) X, 4, and 3 but not Y and 2 show low levels of transcription compared to the housekeeping gene Actin. Although the primers (G2/Jockey-3 copies not included in our assembly. Error bars = SD. The underlying data for this figure can be found in (A) Shown is the plot of the normalized reads depth from uniquely mapped reads  across the m testes , 122 to  primers  are spec(TIF)Click here for additional data file.S10 FigD. melanogaster genome with related outgroups  are similar to the IGS at 3Giglio but are on small contigs, tig00022795 and id = 102159_0. Contig tig00022795 is also moderately enriched in CENP-A. CENP-A, centromere protein A; IGS, intergenic spacer of the ribosomal genes.Maximum-likelihood phylogenetic tree of all individual IGS sequences found in the .rb1bt3j ). Node s.rb1bt3j ). All ce(TIF)Click here for additional data file.S11 FigPROTOP). PROTOP are DNA transposons that have not been recently active, and their distribution is primarily in heterochromatin. TART elements are non-LTR retroelements highly enriched at telomeres and are also moderately CENP-A enriched. DM1731 is a retroelement moderately enriched for CENP-A but not enriched in the centromere islands. Doc2, G, Jockey-1, and G2/Jockey-3 are CENP-A enriched non-LTR retroelements abundant in the centromere islands  along the following chromosomes: (A) chromosome 2, (B) chromosome 3, (C) chromosome 4, (D) chromosome X, and (E) chromosome Y. Contigs from each chromosome were concatenated in order with an arbitrary insertion of 100 kb of \u201cN.\u201d Distances along the x-axis are approximate. The order and orientation of the Y chromosome contigs are based on gene order (see ). Each tands see  Tables. (TIF)Click here for additional data file.S12 FigMaupiti  and AAGAG (blue); (B) Lampedusa  and AAGAT (blue); (C) Lipari  and AATAT (blue); (D) Giglio  and dodeca (blue). White boxes show the separate signals at the targeted centromeres. Yellow boxes show centromeric hybridizations at other centromeres. DAPI is shown in gray. Bar 5 \u03bcm. The underlying data for this figure can be found in IF-FISH using an antibody for CENP-C (green), centromere Oligopaint FISH probes (magenta), and FISH probes for centromeric satellites (blue) in the following combinations: (A) (TIF)Click here for additional data file.S13 FigMaupiti (X), (B) Lampedusa (4), (C) Lipari (Y), (D) Giglio (3). The \u201cSignal Adjusted\u201d panels in (A) and (B) show high-contrast Oligopaint hybridization for visualization of weak foci. Bar 5 \u03bcm. See also IF-FISH using an antibody for CENP-A (green) and centromere Oligopaint FISH probes designed to target centromere contigs (magenta). (A) (TIF)Click here for additional data file.S14 FigP < 0.0001; *adjusted P < 0.02, pairwise Wilcoxon rank sum test with FDR correction; Kruskal-Wallis test by ranks with Dunn\u2019s test for post hoc analysis. The underlying data for this figure can be found in Plots showing intra- and interchromosomal interactions between regions in Hi-C data from: (A) stage 16 embryos (end of embryogenesis) and (B) embryonic cycles 1\u20138 . The di(TIF)Click here for additional data file.S15 FigRsp locus ; (B) 100-kb Oligopaint for a heterochromatic region on chromosome 3L ; (C) 100-kb Oligopaint for a euchromatic region approximately 600 kb from the telomere of chromosome 3L . Arrows show the region of the fiber that was measured. Bar 5 \u03bcm. (D) Scatterplot showing the quantification of fiber lengths. Mean lengths were used to estimate the size in kb (approximately 10 kb/1 \u03bcm). Error bars show the standard deviation. P = 0.085 (n.s.) for each pair of measurements compared (two-tailed t test). The underlying data can be found in Rsp, Responder.Stretched chromatin fibers from female third instar larval brain cells using the following probes: (A) (TIF)Click here for additional data file.S16 FigMaupiti (magenta), and AAGAG probe (cyan) on female third instar larval brain cells. DAPI is shown in gray. CENP-A occupies Maupiti and the AAGAG satellite. We observed some variation in FISH signals and Maupiti and CENP-A domain lengths, likely because of the efficiency of Oligopaint binding and variable stretching in this region. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Maupiti FISH and CENP-A IF signals. Error bars show the standard deviation. N = 24 fibers. Bar 5 \u03bcm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized with IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S17 FigLampedusa (magenta), and AAGAT probe (cyan). DAPI is shown in gray. CENP-A occupies predominantly the island Lampedusa. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Lampedusa FISH and CENP-A IF signals. Error bars show the standard deviation. N = 25 fibers. Bar 5 \u03bcm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH Oligopaint FISH for (TIF)Click here for additional data file.S18 FigLipari (magenta). DAPI is shown in gray. We did not include satellite FISH because no centromeric satellites are known for the Y. Note that the Oligopaints only target part of Lipari  Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for pari see . CENP-A (TIF)Click here for additional data file.S19 FigGiglio (magenta), and a probe for the centromere 3\u2013specific dodeca satellite (cyan). DAPI is shown in gray. CENP-A occupies primarily Giglio and a small stretch of dodeca satellite. Note that the binding of the dodeca (an LNA probe) is quite variable between fibers and results in several gaps that could be a result of the higher stringency conditions needed for Giglio Oligopaint FISH. Arrows show the region of the fiber that was measured. (F) Scatterplot showing the quantification of the length of Giglio FISH and CENP-A IF signals. Error bars show the standard deviation. N = 30 fibers. Bar 5 \u03bcm. The underlying data can be found in (A-E) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S20 Figdodeca from the experiment in Giglio (magenta), and FISH with dodeca probe (cyan). DAPI is shown in gray. Note the presence of Giglio signal on the dodeca CENP-A region. Multiple, overlapping panels were often acquired to follow an individual fiber. Panels were then cropped and juxtaposed in the figure, with white lines showing the separate images. White boxes show the CENP-A domain on Giglio, and yellow boxes show the smaller domain on dodeca. N = 5 (these are rare fibers to find in our preparations because of their length). Bar 5 \u03bcm. CENP-A, centromere protein A; FISH, fluorescence in situ hybridization; IF, immunofluorescence.(A-D) Examples of longer fibers tracked along (TIF)Click here for additional data file.S21 FigProdsat (magenta) and AATAG (cyan). (C-D) Examples of fibers with AAGAG (cyan) and Prodsat (magenta). (E) Example of fiber with AAGAG (magenta) and AATAG (cyan). We propose that Capri is located between flanking blocks of AAGAG and AATAG satellites that reside very close to where the Prodsat begins. Arrows show the region that was measured for each fiber. (F) Scatterplot of CENP-A IF signal lengths. (G) Model for the organization of centromere 2 showing a possible location of Capri. Error bars show the standard deviation. N = 18 fibers. Bar 5 \u03bcm. The underlying data can be found in Prodsat, Prod satellite.(A-D) Examples of fibers visualized with IF with anti-CENP-A antibody (green) and FISH with satellites. DAPI is shown in gray. (A-B) Examples of fibers showing colocalization of CENP-A (green) with (TIF)Click here for additional data file.S1 TableWe used kseek  to estim(XLSX)Click here for additional data file.S2 TableRows correspond to complex repeat families (TEs and complex satellites), with the counts per family in the ChIP and input reads from every dataset. We calculated enrichment for each repeat type by normalizing by total mapped reads for each dataset and taking the ratio of normalized values for each ChIP and its corresponding input. ChIP, chromatin immunoprecipitation; TE, transposable element.(XLSX)Click here for additional data file.S3 TableWe mapped all ChIP-seq data to the de novo assembled ChIPtigs and called peaks using MACS with high-quality reads . We also mapped ChIPtigs to the genome to determine its genomic location and assigned repeat IDs based on BLAST results.(XLSX)Click here for additional data file.S4 TableWe mapped the ChIP and input reads to our genome assembly and used the high-quality reads  to call ChIP peaks with MACS. We show the peak locations for each dataset. ChIP, chromatin immunoprecipitation.(XLSX)Click here for additional data file.S5 TableWe used IDR to compare MACS peaks from different ChIP-seq replicates. We show the statistics for shared peaks from each comparison. ChIP-seq, chromatin immunoprecipitation sequencing; IDR, irreproducible discovery rate; OreR, Oregon-R.(XLSX)Click here for additional data file.S6 TableWe list reads and mapping summaries of all Illumina and long-read datasets generated in this paper or downloaded from NCBI\u2019s SRA.(XLSX)Click here for additional data file.S7 TableList of primers used for qPCR in this study. The centromere contig that each target is associated with  is designated in the \u201cCentromere\u201d column. Note that in silico PCR for the 3_G2 primers predicted three specific products from centromere 3 as well as two products on contig tig00022795 and additional nonspecific products from the X chromosome when three or more mismatches are allowed all of the same 145-bp size. qPCR, quantitative PCR.(XLSX)Click here for additional data file.S8 TableG2/Jockey-3 outside of the centromeric islands. ChIP, chromatin immunoprecipitation; IDR, irreproducible discovery rate; OreR, Oregon-R.We listed peaks outside canonical centromeres with any agreement between replicate ChIP experiments (IDR \u2264 0.05). We also report any genes or repeat annotations that overlap the MACS peaks. Note that there is no general enrichment in (XLSX)Click here for additional data file.S9 TableP values. G2/Jockey-3, G, Doc2, and Jockey-1 are significantly enriched in centromeres relative to other heterochromatic regions and to the whole genome. Asterisk signs show that TART and ProtoP are significantly underrepresented in centromeres relative to other heterochromatic regions. FDR, false discovery rate; FISH, fluorescence in situ hybridization; TART, Telomere-associated retrotransposon; TE, transposable element.We show the copy numbers of TEs in different genomic regions. The sums of base pairs in the assembly size in centromeres , pericentromeric heterochromatin , and other regions  were used to compute the distribution statistics of TEs. We created a 2-by-2 contingency table for each TE comparing observed to expected (based on the sum of bp) for each comparison: centromere to heterochromatin (\u201ccen-het\u201d) regions or centromeres to whole genome (\u201ccen-genome\u201d). We computed a Fisher\u2019s exact test with FDR correction to get adjusted (XLSX)Click here for additional data file.S10 TableHybridization conditions used for FISH with specific Oligopaints. FISH, fluorescence in situ hybridization.(XLSX)Click here for additional data file.S11 TableInformation on the fluors used and sequences of satellite FISH probes used in this report. * = \u201c+N\u201d designates the incorporation of an LNA. FISH, fluorescence in situ hybridization; LNA, locked nucleic acid.(XLSX)Click here for additional data file.S12 TableInformation on the 5\u2032 secondary oligo adapter site and sequence of satellite probes used in this report.(XLSX)Click here for additional data file.S13 TableSequence and fluors of secondary oligo probes used for fluorescence detection of Oligopaints and unlabeled satellite probes.(XLSX)Click here for additional data file.S14 TableList of primer sets used for library amplification and G2 probe synthesis.(XLSX)Click here for additional data file.S15 TableList of primer sets used for sublibrary amplification and Oligopaint synthesis.(XLSX)Click here for additional data file.S16 TableWe assigned contigs from the assembly to a chromosome and a chromatin status Click here for additional data file.S17 TableG2/Jockey-3. CENP-A, centromere protein A; IDR, irreproducible discovery rate; S2, Schneider 2.We compared the MACS peaks shared between \u201cnormal\u201d S2 (this study) and S2 with CENP-A overexpression using the IDR test. Some noncentromeric regions should have more CENP-A enrichment after CENP-A overexpression; however, only four peaks have IDR \u2264 0.05. None of these peaks have (XLSX)Click here for additional data file.S18 TablePercentage of probe signals that overlap with different cytological locations  in S2 cells. The underlying data can be found in (XLSX)Click here for additional data file.S19 Tables, using an anti-CENP-A antibody to mark the centromere. Locations were designated as centromeric (\u201cCen\u201d), pericentric (\u201cPeri\u201d), or heterochromatic (\u201cHet\u201d). See also s, small chromosome 4; CENP-A, centromere protein A; cf(2L), centric fragment of chromosome 2L; cf(2R), centric fragment of chromosome 2R; S2, Schneider 2; X;4, Robertsonian translocation between chromosomes X and 4.Summary of the locations of satellite repeats determined by IF-FISH on S2 cell chromosomes X, X;4, 2, cf(2R), cf(2L), 3, 4, and 4(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 DataThe columns indicate the centromere contig ID, start and end coordinates of sequence, followed by the oligo sequence, and the melting temperature . Included are also the same Oligopaint sequences with 5' and 3' extensions containing the universal primer followed by library-specific barcodes (oligos.with.adaptors).(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file."}
+{"text": "Concerns were raised that some of the sequences reported in this article  may not In Table 1, the sequences are written as 5\u2019 to 3\u2019, and for shRNA sequences, both rows in the first white band comprise a single sequence per column. The shRNA sequences were designed according to the polyclonal loci of pGenesil-1 vector, with the shRNA structure: BamHI+ Sense + Loop (TTCAAGACG) + Antisense+ Termination signal+ Sal I+ Hind III.As noted in Table 1, the COMMD7 shRNA Sense sequence is as follows:CTCTGGGTCTTAGTGAGGATTCAAGACGTCCTCACTAAGACCCAGAGTTTTTTGTCGACA-3\u20195\u2019-GATCCGFor clarity, the targeting  sequences are in bold. The targeting sequence aligns to nucleotides 910\u2013928 of COMMD7 transcript variant 1 mRNA sequence (NM_053041.2).There is an error in Table 1 in that the COMMD7 shRNA Antisense sequence is reported in 3\u2019 to 5\u2019 orientation. The COMMD7 shRNA antisense sequence used in the study with targeting sequences in bold is as follows:GAGACCCAGAATCACTCCTAAGTTCTGCAGGAGTGATTCTGGGTCTCAAAAAACAGCTGTTCGA-5\u20193\u2019-GCIn Table 1, the Sense and Antisense sequences for COMMD7 (for PCR) are switched. The 5\u2019 to 3\u2019 sequence listed in Table 1 for COMMD7 Antisense (for PCR) is actually the Sense sequence, corresponding to nucleotides 758\u2013779 of COMMD7 transcript sequence NM_053041.2. The sequence listed for COMMD7 Sense is actually the Antisense sequence, corresponding to nucleotides 938\u2013915 of COMMD7 transcript sequence NM_053041.2.A 2009 report , as a ce3 are reported. As noted in the Methods, tumor sizes were estimated using the equation, V = L x W2 . This resulted in reported tumor volume estimates twice as large as would be calculated using the standard technique for this type of experiment in which tumor volumes are instead estimated as V = (L x W2)/2. [In Fig 5, tumor sizes up to 5000 mmx W2)/2. .The authors also provide here additional information about the controls used for the reported experiments:\u201cControl\u201d indicates HepG2 cells without plasmid transfection.\u201cScramble\u201d group was transfected with an shRNA construct generated using the following oligonucleotides:GACTTCATAAGGCGCATGCTTCAAGACGGCATGCGCCTTATGAAGTCTTTTTTGTCGACA-3\u2019(Sense) 5\u2019-GATCCCTGAAGTATTCCGCGTACGAAGTTCTGCCGTACGCGGAATACTTCAGAAAAAACAGCTGTTCGA-5\u2019(Antisense) 3\u2019-GThe targeting sequence (bold) for the scramble construct includes a 14 nucleotide stretch that is 100% identical to the human ODF2 transcript (variant X34).For electrophoretic mobility shift assays, the core \u03baB binding sequence  in the rThe data underlying results in this article are no longer available."}
+{"text": "This article presents data on initial experiments that were carried out to investigate new thermostable transketolase (TK) activities with l-arabinose. Transaminase (TAm) sequences from an in-house library of thermophilic strains were analyzed to compare homologies to characterized TAms with desired activity. DNA and amino acid sequences are presented for all the enzymes investigated. Calibration curves for products of the TK and TAm reactions are also presented along with chromatographic analysis of the various one-pot reactions.The dataset presented in this article is related to the research article entitled \u201cOne-pot, two-step transaminase and transketolase synthesis of  Specifications TableValue of the data\u2022The data presented in this article gives new insight into the activities of thermostable enzymes not published before.\u2022The data represents a rationale behind why TKs and TAms were selected for the one-pot reaction.\u2022l-gluco-heptulose, is a pharmaceutically-relevant compound.Product of one-pot reaction, 1l-Arabinose is a major monosaccharide of sugar beet pulp (SBP), a by-product of sucrose extraction which is currently produced and sold as a low value animal feed l-gluco-heptulose, a high value, pharmaceutically relevant compound from l-arabinose using a two-step thermostable enzyme cascade. A thermostable TK catalyzed the synthesis of l-gluco-heptulose from l-arabinose and \u03b2-hydroxypyruvate (HPA) in which the latter was produced in situ from l-serine and \u03b1-ketoglutaric acid using a thermostable TAm.l-arabinose via the Seliwanoff assay l-gluco-heptulose.22.1E.coli BL21 DE3. Cell lysates were used to determine activity towards l-arabinose using the colorimetric assay, Seliwanoff assay. The Seliwanoff assay distinguishes between ketoses and aldoses using 6\u2009M HCl and resorcinol (Seliwanoff\u05f3s reagent) l-arabinose, Seliwanoff reagent was added to the reaction and heated at 100\u2009\u00b0C. Colour formation due to the presence of the ketose, l-gluco-heptulose, was observed within 15\u2009min  equipped with a Dionex AminopacTM PA1 anion exchange column 4\u00d7250\u2009mm2 fitted with a Dionex AminopacTM PA1 guard column 4\u00d750\u2009mm2, an electrochemical detector system, and an eluent generator with a KOH 500 cartridge. The elution times of each compound can be observed in Dgeo. Standard calibration curves of l-gluco-heptulose and HPA were used for quantification purposes (Quantitative analysis of purposes , Fig. 5.2.4>TKDgeoDNA sequences were retrieved from the NCBI database >TKDgeoATGAGTCCCGAACAGCAGGCCGTGCGTCAGGATGTCGATCAGCTGAGCATCAACACCATCAGGACGCTTGCCATCGATGCGGTGCAGCGGGCCAACAGTGGCCACCCCGGCGCGCCGCTCGGCATGGCCCCGATGGGCTACGTGCTGTGGCAGCGCTTCCTGCGCCACAATCCGAAACATCCCGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCAGGGCACGCCAGCATGCTGATCTACTCGCTGCTGCACCTCACCGGCTACGACCTGCCGCTGGAGGACATCAAGAACTTCCGCCAGTGGGGCAGCAAGACGCCTGGGCATCCCGAGTTCTTCCACACCCCAGGCCTAGACGCCACCACCGGCCCGCTCGGTCAGGGTGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCGCACCTCGCCGCACGCTACAACCGCCCCGGCTTCAAGGTCTTTGACAACTACACCTACGCGATCTTGGGGGACGGCGACCTGCAAGAAGGCGTCAACCACGAGGCCGCGTCGCTGGCAGGGCACCTCAAGCTGGGCAAGCTGATCTGGCTGCACGACGACAACCAGGTGCAGCTGGACACCGCCACGTTCAAGGCGGCCAACGAGGATACTGCGGAGCGTTACCGCGCCTACGGCTGGGAAGTTCTGCGTGTGCAAGACGGCAACAATCTCACGGAGATCGAGAACGCGATCCGCCAGGCACGGATGAACACCGAGCAGCCCACCCTGATCCAGGTTCGCACGGTGATCGGCTTCGGCAGTCCCCGTGCGGGCACCAGCAAGGCGCACGGCGAGCCGCTGGGCGAGGAAGGCGTGCAGGAGACCAAGGCGGCCCTGGGCTGGGACTACCCGCCCTTCACGGTGCCCGACGAGGTCAAGGCGCATATGGACGCGACTGAGCGTGGCGCGGAGTGGGAGGCCGACTGGAACGCGCTGATGGAGCGCTACCGTGCCGAGTACCCCGATCTCGCGGCGGAGGTTGACGCGCTGCTGGCGCGCGAACTGCCCGCCAATCTCGCCGAAGTGCTCCCCTCCTACGAAGTGGGCAGCAAGGCCATCGCCACCCGCAACGCGAGCGGTGAAGTCATCAATGCGCTGGCGCAGGTGGTGCCGGGCCTGATGGGGGGCAGTGCGGATCTCTCCGGCAGCACCAAAACCACCATCAAGGACGGCGGCGAGTTTCTGCCAGGAAACTACGGGGGCCGCAACGTCTACTTTGGCGTCCGCGAGTTTGGGATGGCCGCAGCGGGCAATGGCCTTTCGCTCTACGGAGGTGTTCGGCCCCTGGTGGGGACCTTCCTGGTGTTTGCGGACTACCTCAAGCCCGCCTTCCGCCTCTCCGCCCTTCAGTTCCAGCCGGTTACCTATGTCCTGACCCATGACTCCATTGGCCTGGGCGAAGACGGCCCAACCCACCAGCCTATTGAGCAGCTCGCCATGCTGCGCGCCGTGCCGGGTGCCCACGTGATTCGCCCCGCCGACGCCAACGAGACGGCGGCGGCCTGGCAGATGGCGCTGGAGTACGACAAGGGACCAACCGCTCTGGCCCTCTCCCGCCAGGATCTCCCAGTGCTGCCCCGCAACCACGCGGGCGTGAAGAAGGGCGCCTACGTGGTTCGCGACGCCGAAGGGGGGCCGGCACAGATCATCTTGATCGCCACCGGCTCGGAGGTGAGCCTGGCGCTGGATGCTGCCCAAGCGCTGGCGGAGGAAGGCATCCAGGCTCGGGTCGTCTCAATGCCCTGCATGGAAGTCTTCCGCCAGCAGGACGCCAGTTATCGGGACAGCGTGCTCACCCCCGGCGTGAAACGCGTGGCCATCGAGGCTGCCAGCCCGCTCCCCTGGTATGAGTGGGTGGGCTTTGACGGCGCGGTGATCGGAATGACCACCTTTGGCGCCTCGGCCCCAGCCAAAGTCCTCTTTGAGAAATTCGGCTTCAACGTGCCGAACGTCGTGCAGGTCGTCAAGGGCGTTTTGCAGAGGTGA>TKDradMSPEQQAVRQDVDQLSINTIRTLAIDAVQRANSGHPGAPLGMAPMGYVLWQRFLRHNPKHPEWPGRDRFVLSAGHASMLIYSLLHLTGYDLPLEDIKNFRQWGSKTPGHPEFFHTPGLDATTGPLGQGAAMTVGMAMAEAHLAARYNRPGFKVFDNYTYAILGDGDLQEGVNHEAASLAGHLKLGKLIWLHDDNQVQLDTATFKAANEDTAERYRAYGWEVLRVQDGNNLTEIENAIRQARMNTEQPTLIQVRTVIGFGSPRAGTSKAHGEPLGEEGVQETKAALGWDYPPFTVPDEVKAHMDATERGAEWEADWNALMERYRAEYPDLAAEVDALLARELPANLAEVLPSYEVGSKAIATRNASGEVINALAQVVPGLMGGSADLSGSTKTTIKDGGEFLPGNYGGRNVYFGVREFGMAAAGNGLSLYGGVRPLVGTFLVFADYLKPAFRLSALQFQPVTYVLTHDSIGLGEDGPTHQPIEQLAMLRAVPGAHVIRPADANETAAAWQMALEYDKGPTALALSRQDLPVLPRNHAGVKKGAYVVRDAEGGPAQIILIATGSEVSLALDAAQALAEEGIQARVVSMPCMEVFRQQDASYRDSVLTPGVKRVAIEAASPLPWYEWVGFDGAVIGMTTFGASAPAKVLFEKFGFNVPNVVQVVKGVLQRATGACAGACCAGAGCGTTTCCCAAAACGTGGCGCGGCTGAGTGTGAACACCATTCGCACGCTCGCCATTGACGCGGTGCAGGCCGCCAACTCGGGCCACCCCGGTGCGCCGCTGGGCATGGCCCCGATGGGCTACGTGCTGTGGCACAAGTTCCTGCGCCACAACCCCGCGCACCCTGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCGGGGCACGCCTCCATGCTGATCTACAGCCTGCTGCACCTGACCGGCTACCAGGAAATGACGCTCGACGACCTGCGCCACTTCCGGCAGTGGGGCTACCACACCCCCGGCCACCCCGAGTTTTTCCACACCAAGGGTCTGGACGCGACCACCGGCCCGCTTGGGCAGGGCGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCACACCTCGCCGCCCGCTACAACCGCGAAGGCTTTCCGATTTTCGACAACCGCACCTACGCCATCATGGGCGACGGCGATCTGCAAGAAGGCATCAACCACGAAGCCGCCGCGCTCGCCGGGCACCTGAAACTCGGCAAGCTGATCTGGCTGCACGACGACAACCACATCCAGCTCGACACGCCCACGAACAAGGCCGAGTCCGAGGACACCGCCGCCCGCTTCCGCGCCTATGGCTGGAACGTGCTGAAGGTGGAAGACGGCGACAATCTGGACGAAATTGAAAAGGCGATTGCCGAGGCCCGCAGCCAAAGCGAGCGGCCCACGCTGATTCAGGTGCGCACCATCATCGGCTTCGGCAGCCCGCGCGCCGGCACGAGCAAGGCGCACGGCGAGCCGCTCGGCGAAGAGGGCGTGGCCGAGACGAAGAAAGCGTTGGGCTGGGAGTACCCCGCTTTTACCGTGCCCGACGAAGTGGCTGCGCACATGAACGCTCGCGCTAAGGGTGCTCAACTCGAAGCCGACTGGGAAAAACTGATGGCCGACTACCGCACCGCGCACCCCGACCTCGGCAAGGAAGTGGACGCGCTGCTCGCCCGTGAACTGCCCGCCAACCTCGCCGACCTGCTGCCCAAGTACGAAGTCGGCGGCAAGGCGGCGGCCACCCGCAACGCGAGCGGCGAAGTCATCAACGCGCTGGCGAAGGTGCTTCCCGGTTTGATGGGCGGCAGCGCGGACCTCTCGGGCTCGACCAAGACCACCATCAAGGACGGCGGCGAGATGGAAGCGGGCACCATGGGCGGGCGCAACGTGCTGTTCGGCGTGCGCGAGTTCGGCATGAGCGCCGCGGGCAATGGCCTGAGCCTCTACGGCGGCCTGCACCCGATGGTAGGCACCTTCCTGGTATTCGCCGACTACCTCAAGCCGGCTTTCCGCCTCTCGGCGCTGCAAATGCAGCCGGTGACTTACGTGCTGACCCACGACTCCATCGGTCTGGGCGAAGACGGGCCGACCCACCAGCCGGTGGACCAGCTCGCCATGCTGCGAGCGGTGCCGGGCGCCCACGTCATTCGCCCCGCCGACGCCAACGAAACCGCCGCCGCGTGGCTGATGGCGCTGGAATACGACAAGGGCCCCACCGCGCTCGCCCTCTCGCGCCAGGATCTGCCGATTCTGCCCGCCAACATCGAAGGCGTGAAGAAGGGCGCGTATGTCCTCCGAGATGTGGACGGTGCCGATGGTCAGGGGGCTCAAGTCATCCTGATC>TKDradGCCAGCGGCTCGGAAGTCGCCCTGGCCCTGAGCAGCGCCGAGCGGCTGGCCGAAGAGGGCGTGCAGGCCCGCGTGGTGTCCATGCCGTGCATGGAGGTCTTTCGCCAGCAGGAGCAGAGCTACCGCGACAGCGTGCTGACCCCCGGCGTGAAGCGCGTCGCCATCGAGGCCGCCAGCCCGCAGCCCTGGTACGAGTGGACGCTCGGCGGCCCAGTCATCGGCATGACGACCTTCGGTGCGTCGGCCCCGGCCAAGGTGCTGTTTGAGAAGTTCGGCTTCAGCGTGGAAAACGTGGTGAAGGTGGTCCACTCCGTGCTGTAA>pQR1743MTDQSVSQNVARLSVNTIRTLAIDAVQAANSGHPGAPLGMAPMGYVLWHKFLRHNPAHPEWPGRDRFVLSAGHASMLIYSLLHLTGYQEMTLDDLRHFRQWGYHTPGHPEFFHTKGLDATTGPLGQGAAMTVGMAMAEAHLAARYNREGFPIFDNRTYAIMGDGDLQEGINHEAAALAGHLKLGKLIWLHDDNHIQLDTPTNKAESEDTAARFRAYGWNVLKVEDGDNLDEIEKAIAEARSQSERPTLIQVRTIIGFGSPRAGTSKAHGEPLGEEGVAETKKALGWEYPAFTVPDEVAAHMNARAKGAQLEADWEKLMADYRTAHPDLGKEVDALLARELPANLADLLPKYEVGGKAAATRNASGEVINALAKVLPGLMGGSADLSGSTKTTIKDGGEMEAGTMGGRNVLFGVREFGMSAAGNGLSLYGGLHPMVGTFLVFADYLKPAFRLSALQMQPVTYVLTHDSIGLGEDGPTHQPVDQLAMLRAVPGAHVIRPADANETAAAWLMALEYDKGPTALALSRQDLPILPANIEGVKKGAYVLRDVDGADGQGAQVILIASGSEVALALSSAERLAEEGVQARVVSMPCMEVFRQQEQSYRDSVLTPGVKRVAIEAASPQPWYEWTLGGPVIGMTTFGASAPAKVLFEKFGFSVENVVKVVHSVL> pQR1743ATGGCGCATTCGATCGAAGAATTAGCGATTACCACCATTCGAACGCTGTCGATTGACGCGATCGAAAAAGCGAAATCCGGGCACCCGGGCATGCCGATGGGCGCGGCCCCGATGGCGTATACGCTCTGGACGAAATTTATGAACCATAATCCAGCGAATCCCAACTGGTTTAACCGCGACCGGTTTGTTTTGTCCGCTGGGCACGGGTCGATGCTGCTTTACAGCCTGCTTCATCTAAGCGGCTACGATGTCACGATGGACGACTTGAAACAGTTCCGCCAATGGGGAAGCAAAACGCCGGGCCATCCGGAATACGGCCATACGCCAGGGGTGGAGGCAACGACCGGCCCGCTCGGCCAAGGGATTGCGATGGCGGTCGGCATGGCGATGGCGGAACGGCATTTGGCGGCTGCATACAATCGCGATGGATTTGACATTATCAACCACTACACGTATGCGATTTGCGGCGACGGCGATTTGATGGAAGGAGTGGCGAGCGAAGCGGCGTCACTCGCCGGCCACTTGAAGCTCGGCCGTCTGATCGTCCTGTATGACTCGAACGACATTTCGCTGGACGGCGAGCTCAACTTGTCGTTTTCGGAAAACGTCGCCCAACGTTTCCAAGCGTACGGCTGGCAATATTTGCGCGTTGAGGACGGCAACAATATTGAAGAAATCGCCAAAGCGCTCGAGGAGGCGCGGACGGACCTCAGCCGGCCGACGCTCATTGAAGTAAAAACGACGATTGGCTACGGCGCGCCAAATAAAGCGGGCACGTCCGGCGTCCACGGCGCTCCGCTCGGCGCCCAAGAGGCGAAGTTGACGAAAGAAGCGTACCGCTGGACGTTTTCCGAAGATTTCTACGTGCCGGATGAAGTGTACGCTCATTTCCGGGAAACGGTGCAAGAAGCCGGAGCGAGAAAAGAAGCGGAGTGGAATGAGCGCTTCGTTGCTTACGAGCGGGCGCATCCGGAATTGGCCGCCGAGCTGAAGCAGGCGATTGAAGGGAAGCTTCCGGATGGCTGGGAAACATCGCTGCCGGTGTATGAAGCGGGCAAAAGCTTGGCGACCCGCTCATCGTCCGGGGAAGTGATCAACGCCATCGCCAAAGCGGTGCCGCATTGTTTGGCGGTTCGGCGGACTTGGCAAGCTCGAATAAAACGCTTATCAAAGGCGGCGGCAACTTCTTGCCGGACAGCTACGAAGGGCGCAACATTTGGTTTGGCGTGCGCGAGTTTGCCATGGGCGCGGCGTTAAACGGCATGGCGCTTCACGGCGGGTTGAAAGTGTTCGGCGGCACGTTCTTCGTGTTCTCCGACTACTTGCGCCCGGCGATTCGGCTGGCGGCGCTCATGGGCTTGCCGGTGACGTACGTGCTGACGCACGACAGCATCGCCGTCGGGGAAGACGGCCCGACGCATGAGCCGGTCGAGCATCTCGCTTCACTTCGGGCGATGCCGAACTTGTCAGTCATCCGGCCGGCTGACGCAAACGAAACGGCGGCCGCCTGGCGGCTGGCGCTCGAGTCGACGAACAAGCCGACTGCGCTCGTCTTGACGCGTCAAGATGTGCCGACATTGCCGACAACCGCTCAGTTGGCGTATGAAGGCGTGAAAAAAGGCGCGTACGTCGTTTCACCGGCGAAAAACGGCGCTCCGGAGGCGCTGTTGTTGGCGACTGGCTCGGAAGTCGGTCTGGCCGTCAAAGCGCAAGAAGCGCTCGCCGCTGAGGGCATCCATGTCTCCGTCATCAGCATGCCATCGTGGGACCGCTTCGAAGCGCAGCCAAAATCGTACCGCGATGAAGTGCTGCCGCCGGCCGTGACGAAGCGGCTCGCCATTGAAATGGGCGCCTCGCTCGGTTGGGAGCGCTACGTCGGCGCCGAGGGCGACATTTTGGCCATCGACCGATTCGGTGCTTCCGCTCCGGGAGAGAAAATCATGGCCGAGTATGGCTTTACGGTTGACAACGTCGTCCGCCGCACAAAAGCGCTGCTCGGCAAGTAA>pQR1744MAHSIEELAITTIRTLSIDAIEKAKSGHPGMPMGAAPMAYTLWTKFMNHNPANPNWFNRDRFVLSAGHGSMLLYSLLHLSGYDVTMDDLKQFRQWGSKTPGHPEYGHTPGVEATTGPLGQGIAMAVGMAMAERHLAAAYNRDGFDIINHYTYAICGDGDLMEGVASEAASLAGHLKLGRLIVLYDSNDISLDGELNLSFSENVAQRFQAYGWQYLRVEDGNNIEEIAKALEEARTDLSRPTLIEVKTTIGYGAPNKAGTSGVHGAPLGAQEAKLTKEAYRWTFSEDFYVPDEVYAHFRETVQEAGARKEAEWNERFVAYERAHPELAAELKQAIEGKLPDGWETSLPVYEAGKSLATRSSSGEVINAIAKAVPQLFGGSADLASSNKTLIKGGGNFLPDSYEGRNIWFGVREFAMGAALNGMALHGGLKVFGGTFFVFSDYLRPAIRLAALMGLPVTYVLTHDSIAVGEDGPTHEPVEHLASLRAMPNLSVIRPADANETAAAWRLALESTNKPTALVLTRQDVPTLPTTAQLAYEGVKKGAYVVSPAKNGAPEALLLATGSEVGLAVKAQEALAAEGIHVSVISMPSWDRFEAQPKSYRDEVLPPAVTKRLAIEMGASLGWERYVGAEGDILAIDRFGASAPGEKIMAEYGFTVDNVVRRTKALLGK>pQR1744ATGAACACCGGCACCCCAAAGACCCTGGACTGGTCTGATCTCGATAGACGTACCGTAGACGTGGTTCGTGCCCTGGCGATGGACGCGGTCGAAGAAGCGGGATCCGGGCACCCTGGAACCGCGATGAGTCTGGCGCCTGTGGCCTACCTGCTCTTCCAGAAGGTGATGCGGCACGATCCGACAGATCCGAAGTGGATCGGCCGCGACCGCTTCGTCCTGTCCTGCGGGCACTCCAGCCTCACGCTCTACATCCAGCTCTACCTGGCTGGCTACGGGCTGAGCCTGAACGACATCAAGCGGCTGCGCCAGTGGGGCAGCCTCACCCCGGGCCACCCCGAATACGGGCACACCGCCGGGGTGGAAACCACCACCGGCCCCTTGGGGCAGGGCATCGGCAACGCGGTCGGCATGGCCATGGCCGCCCGCCGGGAGCGGGGCCTGTTCGACCCGGACACCCCGATCGGGGAAAGCCCGTTCGACCACTACATCTACGTCCTGTGCTCTGACGGCGACGTCCAGGAGGGCATCAGCCACGAAGTAAGTGCCCTCGCCGGCACGCAGAAGCTCGGCAACCTCATCGTCATCTGGGACGACAACCGCATCTCCATCGAAGACGACACCCAGATCGCATTCACCGAAGACGTCGTCGCCCGCTACGCCGCCTACGGCTGGCACGTCCAAGAGGTCGAGTGGGTCGGCGAGGACGGCTCCTACCACGAAGACGTGGCGGCGCTGTACGACGCGATCCGGGCCGCCCAGGCGGAGACGGAACGTCCCTCTTTCATCCGGCTGCGCACCATCATCGGCTGGCCGTCCCCGAACAAGCAGAACACGGGGGCGATCCACGGCGCCGCGCTGGGGGCTGAAGAGGTCGCCGCCACCAAGCGGGTGCTGGGCCTCAACCCTGAGGCGCAGTTCGACGTGCCCAACGAGCTGCTGGAGCACGCCCGGGGCGTGGTGGCGCGGGGCCGCGCCGCCCGCCAGGAATGGGAGGCCTTGTTCGCCAAGTGGCGGGCCAACGCGGGCGAGCGTGCCGAACTGTTCGACCGGCTGATGGCAGGCTCGCTCCCGGACGGTTGGGAGAAGGCGATCCCGACCTTCGAGCCCAGCGCTAAGGGCATGGCCACCCGGAAAGCGTCCGGTGAGGTGCTGAGCGCGATCGCCCCGGTGCTGCCGGAGCTGTGGGGCGGCTCGGCGGACTTGGCCGGATCCAACAACACCACGCCTAAGGGCGAGCCGTCGTTCATCCCCGAGGAGCGGTCCACGAAGGCGTTCTCCGGCCACCGCTACGGCCGGGTGCTGCACTTCGGGATCCGTGAACACGGCATGGGGGCGATCCTCAACGGGATCGCGCTGCACGGCCCCACCCGCCCCTACGGTGGCACCTTCCTCGTGTTCAGCGACTACATGCGGCCGTCGGTGCGGCTGGCTGCCCTGATGAAGCTGCCGGTCACGTACGTGTGGACCCACGACTCGATCGGTCTGGGCGAAGACGGACCCACCCACCAGCCGGTGGAGCACCTGTGGTCGCTGCGCGCCATCCCCGGCCTGGCGGTGGTGCGTCCCGCCGACGCCAACGAGACGGCAGTGGCCTGGCGCACCATCCTGGAACGCAATGACGGCCCGGTGGCGCTCGCGCTGACCCGGCAGTCGGTTCCGGTTCTGGACCGCTCCGAGCTCGCCTCTGCGGAGCTGGTCTCCCGCGGCGGGTACATCCTGGCCGAAGCCAGCAACGGCCGTCCGGAGGCGATCATCATCGCCACCGGAAGTGAGGTGCAGATCGCGTTGGAGGCGCGTTCCCGCCTGGAGGAGTCGGGTACTCCTACCCGTGTGGTGTCGATGCCGTGCCTGGAGTGGTTCAACGAGCAGGACGACGCCTACCGCCAGCAGGTGCTTCCACCGTCGGTCCGGGTCCGGGTCTCCGTGGAAGCCGGGGTCGCCTTGGGCTGGCGCGAGCTGGTGGGCGAGTATGGCGAGTCGGTGAGTCTGGAACACTTCGGCGCTTCGGCTCCGTACGCGACTCTCTACGAGCAGTTCGGGCTCACCGCCGACCGGGTAGTGGCAGCCGTACACTCCAGCGCTGCCAAGCTCGGCGGTGACCGTGGATCAACGACCGGCAACTGA>pQR1745MNTGTPKTLDWSDLDRRTVDVVRALAMDAVEEAGSGHPGTAMSLAPVAYLLFQKVMRHDPTDPKWIGRDRFVLSCGHSSLTLYIQLYLAGYGLSLNDIKRLRQWGSLTPGHPEYGHTAGVETTTGPLGQGIGNAVGMAMAARRERGLFDPDTPIGESPFDHYIYVLCSDGDVQEGISHEVSALAGTQKLGNLIVIWDDNRISIEDDTQIAFTEDVVARYAAYGWHVQEVEWVGEDGSYHEDVAALYDAIRAAQAETERPSFIRLRTIIGWPSPNKQNTGAIHGAALGAEEVAATKRVLGLNPEAQFDVPNELLEHARGVVARGRAARQEWEALFAKWRANAGERAELFDRLMAGSLPDGWEKAIPTFEPSAKGMATRKASGEVLSAIAPVLPELWGGSADLAGSNNTTPKGEPSFIPEERSTKAFSGHRYGRVLHFGIREHGMGAILNGIALHGPTRPYGGTFLVFSDYMRPSVRLAALMKLPVTYVWTHDSIGLGEDGPTHQPVEHLWSLRAIPGLAVVRPADANETAVAWRTILERNDGPVALALTRQSVPVLDRSELASAELVSRGGYILAEASNGRPEAIIIATGSEVQIALEARSRLEESGTPTRVVSMPCLEWFNEQDDAYRQQVLPPSVRVRVSVEAGVALGWRELVGEYGESVSLEHFGASAPYATLYEQFGLTADRVVAAVHSSAAKLGGDRGSTTGNATGGAAAGGTTTCCCTATGAAAAACTTCCAGAAAGCGAACTCAAAGAGTTGAAAGAACTCGGAAGGCTCTGCCGTGGCGACATACTGAAAATGACCTACATAGCTAACTCAGGCCATCCTGGAGGATCCATGTCTTCGATCGATCTTTATCTTACCGTCTTCAAGTACGCAAAACTCAGACCCGTCGATGATCCTGCAAGAGACAGAATCGTGATCAGCCATGGACACACTTCTCCGGGTGTCTACGCAGCTATGGCTCGTTTGGGGTTTGTCGATCTCGATGAAGTCCTCGCAGGATTCAGACACCCCGCTTCCGTTTTTGAAGGACACGTGACCCGAGGTGTTGGGATCATCGACTGGACAACCGGAAACCTCGGTCAGGGTCTTTCAGCCGGACTCGGTTTTGCCCTCGCATCCAGGTTCACAGGAAAAGATTACCACGTCTTTGTTCTCATGAGTGACGCAGAACAGGCAAAAGGACAGGTGGCGGAGGCAAGAAGAGTGGCGAAAAAGTACGGTGTCACGAATCTCACAGTGATCATCGACTACAACGACGCCCAGATCAGTGGCCGTGCCAGAGACGTCATGCCCGTGAACATAAAGGAAAACTACTTAGCGGACGGCTGGAGGGTCATCGAGATCGATGGGCACGACTACGAACAGATCTATCTCGCACTGAAAGAAGCGGTAGAAGACGAAC> pQR1745TGAATCCCGTTGCCATAATCGCCAAAACGGTCATGGGAAAAGGCGTATCTTTCATGGAAAACGAGGTGAAATACCACGGAAAGCCTTTGAACAGAGAAGAACTCGAAAAAGCCCTCGCGGAACTCGGAATTGAAAACGATGTTGATGTGTACATCGAAAAAAGAAAACAACTTCCAGTGGAAAAACACAAGAAAGTCTACAAAACTTACCCGATCAAGATCGACACGGGAGAGCCCATCACCTACACCTCACCCACTGACAACAGAAGCGCATTCGGAAAAGCTATTCTGGATCTGGTGAAGAAGAACGTAAACAATCCAGAAACCACACCCATCGTCGCTGTGGACTGCGACCTGAAGGGATCGGTCAAACTCGACCTGCTCGACAAAGAGTTCCCTGAGAGACTCCTGGAAGTGGGCGTTCAGGAACACAACGCTGCCGCTATGGCGGGGGCACTCTCCGCAGAGGGTGTGATCACGTTCTTCGCTGATTTTGGTGTTTTTGGAATTTCTGAAACCTACAACCAGCACAGGCTGAACGCCATCAATGGAACGAACCTCAAAGTCGTTGTCACACACTGCGGACTCAACGTGGGAGAGGACGGAAAAACTCATCACGGACTCGACTACGTTTCCGGGCCGATGAACTGGTACGGTTTCAAAGTGATCGTCCCTGGTGATCCCAACCAGACGGATAGAGTTGTCAGATACGCCGCGAAGGAATACGGGAACTTCGTAATCGCCATGGGAAGATCTAAGCTTCCCATCATCCTCGATGAAAACGGGAAACCTTTCTTCGGAGAGGGTTACACCTTCGAATATGGGAAGATCGATGTCGTTAGAAAAGGTGACGACGCGGTGATCATAACTTACGGTTCTACACTCTGTGAAGCCGTAAATGCCGCAGACGAACTCAAGAAAGAAGGAGTAAACGTAGCCGTTCTGAATGTCTCCTGTCCGGTGGATCTCGATATAGAGACCTTGAAGATGGTCGATGGAAAACCCGTTCTCGTTGTGGAGGATCACAACGTTTTCACAGGACTTGGAAGCTTCCTTGGAACCACCCTTCTTGAAAACGGCATCATCCCGAAGAAATACGTGAGAGTAGGTGTTCCAGAATTCGCCGTGTCCGGCAGTTACACGATGCTCTACAAACTCTACGGCCTGGATAAAGATGGAATAATTTCCAGACTCAGAGAGATGCTCTAAMERFPYEKLPESELKELKELGRLCRGDILKMTYIANSGHPGGSMSSIDLYLTVFKYAKLRPVDDPARDRIVISHGHTSPGVYAAMARLGFVDLDEVLAGFRHPASVFEGHVTRGVGIIDWTTGNLGQGLSAGLGFALASRFTGKDYHVFVLMSDAEQAKGQVAEARRVAKKYGVTNLTVIIDYNDAQISGRARDVMPVNIKENYLADGWRVIEIDGHDYEQIYLALKEAVEDELNPVAIIAKTVMGKGVSFMENEVKYHGKPLNREELEKALAELGIENDVDVYIEKRKQLPVEKHKKVYKTYPIKIDTGEPITYTSPTDNRSAFGKAILDLVKKNVNNPETTPIVAVDCDLKGSVKLDLDKEFPERLLEVGVQEHNAAAMAGALSAEGVITFFADFGVFGISETYNQHRLNAINGTNLKVVVTHCGLNVGEDGKTHHGLDYVSGPMNWYGFKVIVPGDPNQTDRVVRYAAKEYGNFVIAMGRSKLPIILDENGKPFFGEGYTFEYGKIDVVRKGDDAVIITYGSTLCEAVNAADELKKEGVNVAVLNVSCPVDLDIETLKMVDGKPVLVVEDHNVFTGLGSFLGTTLLENGIIPKKYVRVGVPEFAVSGSYTMLYKLYGLDKDGIISRLREML2.5>TAmGsteDNA sequences were retrieved from the NCBI database >TAmGsteATGAAATTGGCAAAACGGGTGGCGTCGCTGACGCCATCGGCGACTTTGGCCATTACGGAGAAAGCAAAAGAACTAAAAGCGGCCGGGCATGACGTGATTGGTCTCGGAGCTGGCGAACCGGATTTCAACACGCCACAGCACATTCTTGATGCCGCCATCAAGGCAATGAACGAAGGACATACGAAATATACACCATCGGGCGGTTTGCCGGCGTTAAAGGAGGAAATTATAAAAAAATTCGCCCGCGACCAAGGCTTGGATTATGAGCCGGCTGAAGTGATTGTATGCGTCGGAGCGAAGCACGCCCTTTACACGCTGTTCCAAGTATTGCTCGATGAAGGCGACGAAGTGATCATTCCGACGCCATACTGGGTGAGCTATCCGGAACAAGTGAAACTGGCGGGCGGTGTTCCGGTTTACGTCGAAGGGCTTGAACAAAATCATTTTAAAATTACGCCGGAGCAGCTGAAACAGGCAATCACGCCGCGGACGAAAGCGGTTATCATCAACTCGCCGAGCAACCCGACTGGCATGATTTATACAGCCGAAGAGTTGAAGGCGCTTGGTGAGGTGTGCCTAGCGCATGGTGTATTGATCGTGTCAGATGAAATTTACGAAAAATTGACTTACGGCGGGGCGAAGCATGTGTCCATCGCTGAGTTGTCGCCGGAGCTGAAGGCGCAGACAGTCATCATTAACGGCGTGTCAAAGTCGCATTCGATGACGGGCTGGCGCATTGGTTATGCGGCGGGGCCGAAAGATATTATTAAGGCAATGACAGATTTGGCGAGCCACAGCACGTCCAACCCGACGTCAATCGCCCAATACGCGGCCATCGCTGCTTACAGCGGGCCGCAGGAGCCGGTCGAACAAATGCGCCAAGCGTTTGAACAACGGCTCAATATCATTTACGACAAGCTCGTGCAAATTCCAGGATTCACGTGCGTTAAGCCACAAGGGGCGTTTTATTTGTTCCCGAACGCCCGCGAAGCGGCTGCAATGGCCGGCTGCCGCACGGTCGACGAGTTCGTCGCTGCCTTGTTGGAGGAAGCGAAAGTCGCGCTTGTGCCCGGCTCTGGGTTTGGAGCGCCGGATAACGTTCGCTTGTCATACGCGACATCGCTCGATGCACTGGAAACCGCCGTGGAACGCATCCACCGGTTTATGGAAGCGCGCGCTTAA>TAmDGeoMKLAKRVASLTPSATLAITEKAKELKAAGHDVIGLGAGEPDFNTPQHILDAAIKAMNEGHTKYTPSGGLPALKEEIIKKFARDQGLDYEPAEVIVCVGAKHALYTLFQVLLDEGDEVIIPTPYWVSYPEQVKLAGGVPVYVEGLEQNHFKITPEQLKQAITPRTKAVIINSPSNPTGMITAEELKALGEVCLAHGVLIVSDEIYEKLTYGGAKHVSIAELSPELKAQTVIINGVSKSHSMTGWRIGYAAGPKDIIKAMTDLASHSTSNPTSIAQYAAIAAYSGPQEPVEQMRQAFEQRLNIIYDKLVQIPGFTCVKPQGAFYLFPNAREAAAMAGCRTVDEFVAALLEEAKVALVPGSGFGAPDNVRLSYATSLDALETAVERIHRFMEARA>TAmDGeoATGTTCGAGGACACGCCCGCACCCTTTCCACCGCACATTCTGCTGACGCCCGGTCCGACACCGATTCACCCCCGGGCCCAGCGGGCGCTGCTGCGCGAGATGCTGGGGCACATGGACCCTGAGGTGTTCGCCCTGAACCGCGAGATCCAGGCGGACTTGCGGGTGATGTACGGGACGGGGCCCCAGACCTTTACGGCGCTGCTGGCGGGCACCGGGAGCCTGGGCATGGAGGCGGGCTTCGCCAACTTGGTGGAGAGGGGAGACGACGTGCTGATCTGCGTCAATGGTGCCTTTGGTCAGCGCATGGCCGAGATGGCGGCGCGCTACGGTGCGAATGTACGGCGGGTGACCGCGCCGCTGGGCGAGCCGATCGACCCGGCCGACGTGGCTGCGCGGTTGAGCGGCGCGCGGCTGGTGGCGGTGGTGCATGGGGAGACGAGCACGGGTGTGCTCAATCCGCTTCCGGAGATTGCCGAGGCCGTGCGCGGGAGCGGGGCATTGCTGGCCGTGGACGCCGTGACGACCGCCGGGATGGAACCCTTCCATATGGCGGACTGGGGCGTGGACTACGCCTATACCGGCGCGCAGAAGTGCCTCTCGGCACCGCCCGGCCTGGCCCCGGTGGCGATCAGCGACCGTGCTCTCGCTCGCCACGCGGCCCGCCGCACGCCCACGCCGCTGTGGTACTGCGATTTTGAGGGCCTGCGCGACTACTGGGACCGGCACAGCTACCACCACACGGTCCCGGTGAATCTGCACTACGCCTTCCACGCCGCCCTGCGCGCCGCACTCGAAGAAGGCCTCCAAGCCCGGCAGGCCCGCGTGCGCGACCTTGGCCAGGCGGTGCTGGCGGCCCTGACGCCGCTGGGCTTCACGCCGTATGTGGCCGATCCCGCCGCGCGGCTGCCCACCGTCTTGGCCCTGCGTCTTCCTCCCGGCTTCGACGACGCGGGCGTTCGCCAGGCCCTACGGGAACGCGGGATCAGCGTGACCGGCGGCCTGGGACCGACGGCAGGGCTGATCTGGCGTCTGGGCCTGATGGGGGAAGCGGCTCGCCCCGCGCCCTACCGCGCGCTGATGCTCGCCCTGGAAGACCTGCTGGGCGAGCGGGGCTTGGTGGCGCGCTTCGAGGAGGCGCTGGGCGTCGCGGCCTGA>pQR1746MFEDTPAPFPPHILLTPGPTPIHPRAQRALLREMLGHMDPEVFALNREIQADLRVMYGTGPQTFTALLAGTGSLGMEAGFANLVERGDDVLICVNGAFGQRMAEMAARYGANVRRVTAPLGEPIDPADVAARLSGARLVAVVHGETSTGVLNPLPEIAEAVRGSGALLAVDAVTTAGMEPFHMADWGVDYAYTGAQKCLSAPPGLAPVAISDRALARHAARRTPTPLWYCDFEGLRDYWDRHSYHHTVPVNLHYAFHAALRAALEEGLQARQARVRDLGQAVLAALTPLGFTPYVADPAARLPTVLALRLPPGFDDAGVRQALRERGISVTGGLGPTAGLIWRLGLMGEAARPAPYRALMLALEDLLGERGLVARFEEALGVAA>pQR1746ATGACCTCTCCTTTCCGCCTCTCCGCCCGCGCCCAGAGCCTCAAGCCGTCTGCGACAGTGGCGGTCACGTCCCGCGCCCTGGAACTCCAGCGTCAGGGCCTGGACGTGATTTCCATGAGCGTGGGCGAGCCGGATTTCGACACGCCGCCACATGTCAAGGCCGCCGGCATCGCCGCCATCGAGGAAGGCAAGACCAAATACACCCCGGTCAGCGGCATTCCCGAACTGCGCGAGGCCATCAGCGCCAAGTTTCGGCGCGAAAACGGCCTGGACTACGCGCCGAACGCCGTGACGGTAACGAGCGGCGGTAAACAGGCGCTGTTCAACGCCTTTTTCGCGTTGCTGAACCCCGGCGACGAGGTGCTGATTCCCGCGCCCCACTGGGTCAGCTACCCCGAAATGGTCGCGCTGACCGGCGCGGTGCCGGTAACCGTACCCACTACGCCGCAGCAGGGCTTTCAACTCGACCCGGACGCCCTCGCCGCCGCCATCACGCCGCGCACCCGCATGGTGATTCTCAACAGCCCCGGCAACCCGACGGGCGCGGTGTTTCCGCCGGAAACCTTGCGGGCGGTGGCCGACCTCGCCACGCAGCACGGCTTGATGATCGTCACCGACGAAATCTACGAGCACCTCGTCTACGACGCCGAGCAGGTCAGCATCGGCACCTACGCGCCGGAGCACACCCTGACCATCAATGGCGCGAGCAAAGCGTATGCCATGACCGGCTGGCGCATCGGCTACGCGGGCGGGCCGCGCGAGGTGATTGCCGCCATGAACGCGCTGCAATCGCAAAGCACCAGCAACGCCAGCAGCGTCAGCCAGTACGCCGCCCTCGCCGCGCTGGAACAGCACGAGGAAACCATGCGCTTCATCGACAGGGCCCGCACCGCCTACCGCGAACGGCGCGACCGCATCGTGGCGGGCCTCAACGCGCTGGGGTTGCCCACGCCCACGCCGCAAGGGGCCTTTTACGTGATGGCCGACACCCGCGCCATTCACACCGACGAACTCGAAGCCGCCCGCATCATTCTGGATGAGGCGCAGGTCGCCGTCGTGCCCGGCACCGATTTCGCCGCGCCGGGACAGGTGCGCCTGAGCTACGCGACCAGCATGGACAACATCGAGGAAGTGCTGCGGCGGCTGGAAGGGGTCGTGCGGCGCTAA>pQR1748MTSPFRLSARAQSLKPSATVAVTSRALELQRQGLDVISMSVGEPDFDTPPHVKAAGIAAIEEGKTKYTPVSGIPELREAISAKFRRENGLDYAPNAVTVTSGGKQALFNAFFALLNPGDEVLIPAPHWVSYPEMVALTGAVPVTVPTTPQQGFQLDPDALAAAITPRTRMVILNSPGNPTGAVFPPETLRAVADLATQHGLMIVTDEIYEHLVYDAEQVSIGTYAPEHTLTINGASKAYAMTGWRIGYAGGPREVIAAMNALQSQSTSNASSVSQYAALAALEQHEETMRFIDRARTAYRERRDRIVAGLNALGLPTPTPQGAFYVMADTRAIHTDELEAARIILDEAQVAVVPGTDFAAPGQVRLSYATSMDNIEEVLRRLEGVVRR>pQR1748ATGACTGACCGACCTCGTATCTCCGCACGCATCGGCGGTATCTCCGAGTCAGCGACCCTGGCGGTGGACGCCAAGGCCAAGGCCCTGAAGGCCGCTGGGCATCCCGTGATCGGCTTCGGCGCCGGGGAGCCTGACTTCCCCACGCCCGACTACATCGTGGAGGCAGCGGTCGCCGCCTGCCGCGACTCGCGCTTCCACCGCTACACCCCGGCGGGAGGCCTCCCCGAACTCAAGGAAGCCATCGCGGCTAAGACGCTGCGCGACTCCGGCTACCGGGTGGAGCCGAACCAAGTCCTGGTCACCAACGGCGGCAAGCAAGCGATCTACGAGGCGTTCGCCACGCTGCTCGATCCGGGCGACGAGGTCATCGTGATCGCGCCCTACTGGACCACCTACCCTGAATCGATCCGGCTGGCCGGAGGAACCCCGGTCTACGTGGTCACCGACGAGTCCACTGGCTACCTGGCCACGGTCGAGCAGCTGGAGGCGGCCCGCACCGACCGCACCAAGGTGCTGCTGTTCGTCTCCCCCTCGAACCCGACCGGCGCGGTGTACTCGCCCGAGCAGGTCCGCGAGATCGGCCGGTGGGCCCTCGAACACAACCTGTGGGTGCTCACCGACGAGATCTACGAGCACCTCGTCTACGGGGACGCCCGGTTCTCCTCGATGCCGGTGGAAGTTCCGGAACTGGCCGACCGCACCGTGGTGGTCAACGGGGTGGCCAAGACCTACGCCATGACCGGGTGGCGGGTCGGCTGGCTCATCGGCCCCGTGGACGTGGTCAAGGCTGCGACCAACCTGCAGTCGCACGCCACCTCCAATGTGGCCAACGTCTCGCAGGCCGCGGCTCTGGCAGCGGTCTCCGGCGACCTGTCGGCCGTGGAGGAGATGAAGCAGGCCTTCGACCGGCGGCGGCAGACCATTGTGCGGATGCTCAACGAGATCCCCGGTGTGGTGTGCCCCGAGCCCCAGGGCGCGTTCTACGCCTACCCGTCGGTCAAGGAGATCCTCGGCAAGGAGATCCGCGGTCAGCGTCCGCAGACCTCCAGCGAGCTGGCGTCGCTGATCCTGGAGCACGCCAAGGTCGCGGTGGTCCCGGGCGAGGCGTTCGGCACTCCGGGCTACCTGCGGTTGTCCTACGCGTTGAGCGACGCCGATCTGGTCGAAGGGGTCAGCCGGATCGCCAAGCTGCTGAGCGAAGCCCACTGA>pQR1749MTDRPRISARIGGISESATLAVDAKAKALKAAGHPVIGFGAGEPDFPTPDYIVEAAVAACRDSRFHRYTPAGGLPELKEAIAAKTLRDSGYRVEPNQVLVTNGGKQAIYEAFATLLDPGDEVIVIAPYWTTYPESIRLAGGTPVYVVTDESTGYLATVEQLEAARTDRTKVLLFVSPSNPTGAVYSPEQVREIGRWALEHNLWVLTDEIYEHLVYGDARFSSMPVEVPELADRTVVVNGVAKTYAMTGWRVGWLIGPVDVVKAATNLQSHATSNVANVSQAAALAAVSGDLSAVEEMKQAFDRRRQTIVRMLNEIPGVVCPEPQGAFYAYPSVKEILGKEIRGQRPQTSSELASLILEHAKVAVVPGEAFGTPGYLRLSYALSDADLVEGVSRIAKLLSEAH>pQR1749ATGGTATCCAGGAGAATATCAGAGATTCCCATATCGAAAACCATGGAACTCGACGCGAAGGCCAAAGCCCTCATAAAAAAGGGAGAAGACGTGATCAATCTAACGGCTGGTGAGCCGGATTTTCCCACACCGGAACCCGTCGTGGAAGAAGCGGTGAGATTTCTCCAGAAAGGAGAAGTGAAATACACAGATCCTCGTGGTATCTACGAACTCAGAGAGGGTATAGCGAAAAGGATAGGCGAGAGATACAAAAAAGATATCTCACCGGATCAGGTCGTGGTGACGAATGGAGCGAAACAGGCTCTGTTCAATGCTTTCATGGCCCTTCTCGATCCCGGTGACGAAGTGATCGTGTTTTCTCCCGTCTGGGTCAGCTACATTCCTCAGATCATCCTTGCTGGTGGCACGGTGAACGTGGTTGAGACGTTCATGAGTAAAAATTTCCAGCCCAGTCTGGAAGAGGTGGAAGGGCTTCTTGTTGGGAAAACGAAAGCCGTTCTTATCAACTCGCCGAACAATCCCACTGGTGTGGTGTACAGAAGAGAGTTCCTTGAAGGACTTGTGAGACTTGCCAAGAAGAGGAATTTCTACATAATCAGCGACGAAGTCTACGATTCCCTTGTTTACACGGATGAATTCACATCGATACTCGATGTTTCTGAAGGATTCGACCGGATAGTTTACATAAACGGCTTCTCGAAGTCTCACTCCATGACCGGCTGGAGGGTGGGTTACCTGATATCGAGCGAAAAAGTAGCGACCGCTGTGTCGAAGATCCAGTCTCACACCACCTCCTGTATCAACACGGTAGCACAGTACGCCGCCTTGAAGGCTCTGGAAGTGGACAACTCTTACATGGTTCAGACCTTTAAAGAAAGAAAAAATTTCGTGGTGGAAAGATTGAAAAAGATGGGTGTTAAGTTCGTGGAACCAGAAGGTGCGTTCTACCTCTTTTTCAAAGTCCGGGGTGACGATGTGAAATTCTGTGAAAGGCTCCTCGAAGAAAAGAAGGTTGCACTCGTTCCAGGATCCGCTTTTCTGAAGCCTGGATTTGTGAGGCTTTCTTTTGCCACATCTATAGAAAGACTTACGGAGGCGCTGGATAGAATTGAAGACTTCCTCAATTCTCGTTGA>pQR1751MVSRRISEIPISKTMELDAKAKALIKKGEDVINLTAGEPDFPTPEPVVEEAVRFLQKGEVKYTDPRGIYELREGIAKRIGERYKKDISPDQVVVTNGAKQALFNAFMALLDPGDEVIVFSPVWVSYIPQIILAGGTVNVVETFMSKNFQPSLEEVEGLLVGKTKAVLINSPNNPTGVVYRREFLEGLVRLAKKRNFYIISDEVYDSLVYTDEFTSILDVSEGFDRIVYINGFSKSHSMTGWRVGYLISSEKVATAVSKIQSHTTSCINTVAQYAALKALEVDNSYMVQTFKERKNFVVERLKKMGVKFVEPEGAFYLFFKVRGDDVKFCERLLEEKKVALVPGSAFLKPGFVRLSFATSIERLTEALDRIEDFLNSR>pQR1751ATGGCACCTGACCTGCGCCACCTGCACACCTTCGGCGAACTGGATCCGCCGCAACGCCTGTTGATGGGCCCCGGCCCGGTCAATGCGCATCCACGCGTGCTGCGTGCGATGGCGGCCGACCTGCTTGGCCAGTTCGACCCGGAAATGACCACCTACATGAACGAGGTGATGGCGCTGTACCGCCCCTTGTTCGGCACCCAGAACCGCTGGACCTTTCTGGTCGATGGCACGGCGCGCGCCGGCATCGAAGCCGCGCTGGTGTCGCTGGTGCAGCCGGGCGACCGTGTGCTGGTGATCAACTTCGGCCGCTTCGGTTTGTTGCTGACCGAAATCCTTGGCCGGCTCGGCGCCGACGTCCACACCGTGGATGCGCCGTGGGGCGAGGTGGTGCCGCTGGCGGCGATTGCCGAGGCGATCGCAAGCGTGGCACCCAAGCTGGTGGCCACCGTGCACGGCGACACCTCCACCACCATGGCGCAGCCGCTCGATGGCCTAGGCGCGCTATGCCGGGCGGCCGGCGCGCTGAGTTACGTAGACGCCACAGCCACCATCGGCGGCATGGACATCGCCAGCGACCGCTGGGAGGTGGACGTGGTCACCGCGGGGCTGCAGAAATGCCTGGGCGGGCCGTCCGGCTCGGCGCCGATCACTGTCTCTGCCGCGGCAGCGGAGGCGATCTTTGCGCGGCGGCATGTCGAACGCGGCATCGTGCGCGAGGACATCGCCAACGGCAGCGGCCCACGCATCGCCTCGAATTATTTCGACCTGGCGATGATCATGGATTACTGGTCCGACAAGCGCCTCAATCACCACACCGAAGCCACCACCATGCTGTACGGCGCGCGCGAATGCGCACGCGTGGCCTTGCAGGAAGGCCTGGAGGCGCGCTACGCCCGGCATGCGGCTGCCGGCCGCGCGGTCAGCGCCGGCGTGCGCGCACTGGGGCTGGAGGTGTTCGGCGACGATGCGCACCGCATGAGCAATGTCACCGGCGTGGTGATCCCGCACGGCGTCGACAGTGAAGCAGTGCGGCGGCGCATGCGCGAGGATTTCGAAATCGAGATCGGCACCGCGTTCGGCCCGCTGCAAGGCAGGATCTGGCGCATCGGTGCGATGGGCTACAACGCGATGAAGCACAAGGTGCTGCTCACCCTGGCCGCACTGGAAGCGGTGCTGCGCGCCGAGGGCTACGCGTGCACCCAAGGCCTGGCGGTCGAAGCCGCACGCGCCGCCTGGCATGCGGAGCCGGCTGCATGA>pQR1752MAPDLRHLHTFGELDPPQRLLMGPGPVNAHPRVLRAMAADLLGQFDPEMTTYMNEVMALYRPLFGTQNRWTFLVDGTARAGIEAALVSLVQPGDRVLVINFGRFGLLLTEILGRLGADVHTVDAPWGEVVPLAAIAEAIASVAPKLVATVHGDTSTTMAQPLDGLGALCRAAGALSYVDATATIGGMDIASDRWEVDVVTAGLQKCLGGPSGSAPITVSAAAAEAIFARRHVERGIVREDIANGSGPRIASNYFDLAMIMDYWSDKRLNHHTEATTMLYGARECARVALQEGLEARYARHAAAGRAVSAGVRALGLEVFGDDAHRMSNVTGVVIPHGVDSEAVRRRMREDFEIEIGTAFGPLQGRIWRIGAMGYNAMKHKVLLTLAALEAVLRAEGYACTQGLAVEAARAAWHAEPAA>pQR1752ATGGGAAAGTTTCTTAAGAAACACTACATAATGGCACCTGGACCAACACCAGTCCCAAACGATATTTTAACAGAAGGAGCGAAGGAAACAATACACCACAGAACACCTCAGTTTGTTTCCATAATGGAAGAGACCCTCGAAAGTGCAAAGTACATCTTTCAGACAAAACACAACGTGTACGCCTTTGCTTCCACAGGAACTGGCGCTATGGAAGCGGCGGTGGCGAATCTTGTGAGCCCTGGAGACAAAGTGATCGTGGTTGTGGCTGGAAAGTTCGGTGAAAGATGGAGAGAGCTCTGTCAGGCTTACGGTGCTGATATCGTAGAAATCGCCCTCGAATGGGGAGACGCGGTCACACCTGAACAGATCGAAGAGGCTCTCAACAAAAACCCCGATGCGAAGGTCGTCTTCACCACCTACAGTGAAACATCGACGGGTACAGTCATAGACCTCGAAGGAATTGCCAGAGTCACGAAGGAAAAAGACGTTGTTCTTGTGACAGACGCTGTCAGCGCTCTTGGAGCAGAACCACTGAAGATGGATGAATGGGGTGTGGATCTCGTTGTCACAGGTTCACAGAAGGGTTTGATGTTACCTCCAGGACTGGCGCTCATCTCTCTCAACGACAAAGCGTGGGGGCTCGTGGAAAAATCCAGATCTCCAAGGTACTACTTCGATCTGAGGGCCTACAGGAAATCTTACCCCGACAATCCTTACACCCCCGCAGTAAACATGATATACATGTTGAGAAAGGCTCTTCAGATGATAAAAGAGGAAGGCATAGAAAACGTATGGGAAAGGCACAGAATACTGGGAGACGCAACAAGAGCAGCGGTGAAAGCACTTGGACTGGAACTCCTCTCGAAAAGACCGGGAAACGTTGTAACAGCCGTGAAAGTGCCTGAGGGCATCGATGGAAAACAGATTCCCAAGATCATGAGAGACAAGTACGGTGTGACCATCGCCGGTGGACAGGCTAAACTCAAGGGAAAAATATTCAGGATAGCACACCTCGGATACATGTCACCTTTCGACACCATAACTGCCATTTCCGCTCTTGAATTAACCTTGAAGGAACTCGGTTATGAGTTCGAACTCGGAGTCGGTGTTAAGGCAGCCGAAGCTGTCTTCGCTAAAGAATTCATTGGGGAGTGA>pQR1755MGKFLKKHYIMAPGPTPVPNDILTEGAKETIHHRTPQFVSIMEETLESAKYIFQTKHNVYAFASTGTGAMEAAVANLVSPGDKVIVVVAGKFGERWRELCQAYGADIVEIALEWGDAVTPEQIEEALNKNPDAKVVFTTYSETSTGTVIDLEGIARVTKEKDVVLVTDAVSALGAEPLKMDEWGVDLVVTGSQKGLMLPPGLALISLNDKAWGLVEKSRSPRYYFDLRAYRKSYPDNPYTPAVNMIYMLRKALQMIKEEGIENVWERHRILGDATRAAVKALGLELLSKRPGNVVTAVKVPEGIDGKQIPKIMRDKYGVTIAGGQAKLKGKIFRIAHLGYMSPFDTITAISALELTLKELGYEFELGVGVKAAEAVFAKEFIGE>pQR1755ATGACTCAGATTTTTAATTTTAGCGCCGGTCCAGCAATGCTGCCGGTTGAAGTACTGCGTCGTGCTGAACAGGAATTGTGTAATTGGAATGGCCTGGGCACATCGGTTATGGAAATCAGCCACCGCAGTAAAGAGTTTATGCAGGTTGCCGCTGAATCCGAACAGGATCTGCGTGATTTGCTGAAAATCCCCTCCAACTACAAAGTGCTCTTTTGCCACGGCGGTGCTCGTGCGCAATTCGCCGCAGTGCCGTTAAATCTTCTGGGCGAACGCTCAACGGCCGACTACATCGACGGCGGGTATTGGGCGCACAGCGCAATCAATGAAGCAGAAAAATACTGCACGCCTAACGTGATTGACGTGAAAATGCGCGTGGGCGAACTGCGTGGCATTAAGCCGATGCGTGAATGGAAATTGTCTGATGACGCGGCGTTTGTGCATTACTGCCCGAATGAAACCATCGACGGTATTGCGATCGAAGAAGAGCCGGACTTTGGCGATAAAATTGTGGTCGCCGACTATTCTTCCAGCATCCTGTCTCGTCGTATTGATGTCAGCCGTTACGGCGTGATCTATGCCGGTGCGCAGAAAAATATCGGCCCTGCCGGCCTGACGCTGGTTATCGTACGTGAAGATTTGCTGGGCAAGGCGCGCCGTGAGCTGCCATCGATTCTGGATTACCAGGTTCTGGCGGACAATGACTCCATGTTTAACACGCCACCGACCTTTGCCTGGTACCTGTCCGGTATGGTCTTCAAATGGCTGAAAGAGTACGGCGGTCTGGCTGAAATGGAAAAACGTAACCAGGAGAAGGCTGACCTGCTGTATAGCGCGATTGACGGTAACGATTTCTATCGTAATGACGTTGCGGTAGCGAACCGTTCTCGCATGAATGTGCCATTCCTGTTGGCGGATTCTGCGCTGGATAAAGTCTTCCTGGAAGAATCAGTCGCTGCAGGTCTGCACGCGCTGAAAGGCCATCGCGTAGTAGGCGGCATGCGTGCCTCTATCTACAATGCGATGCCGTTGGAAGGCGTGAAAGTGCTGACGGAATTTATGGCTGACTTCGCTCGTCGCCACGGTTGAMTQIFNFSAGPAMLPVEVLRRAEQELCNWNGLGTSVMEISHRSKEFMQVAAESEQDLRDLLKIPSNYKVLFCHGGARAQFAAVPLNLLGERSTADYIDGGYWAHSAINEAEKYCTPNVIDVKMRVGELRGIKPMREWKLSDDAAFVHYCPNETIDGIAIEEEPDFGDKIVVADYSSSILSRRIDVSRYGVIYAGAQKNIGPAGLTLVIVREDLLGKARRELPSILDYQVLADNDSMFNTPPTFAWYLSGMVFKWLKEYGGLAEMEKRNQEKADLLYSAIDGNDFYRNDVAVANRSRMNVPFLLADSALDKVFLEESVAAGLHALKGHRVVGGMRASIYNAMPLEGVKVLTEFMADFARRHG"}
+{"text": "To produce synthetic nucleotides of notifiable dengue virus (1\u20134 types), Japanese encephalitis, yellow fever and Zika flaviviruses. These notifiable flaviviruses, particularly dengue and Zika, are problematic mosquito-borne infections in the Philippines, as well as in those countries with tropical and subtropical climates.An algorithmic design formulation of overlap extension \u2013 polymerase chain reaction (OE-PCR) was performed to propagate 50\u201360 oligomer lengths of select notifiable flaviviral RNAs to DNA nucleotides via the two-step process of OE-PCR.Algorithmic OE-PCR design formulation efficiently produced 253\u2013256\u00a0bp of notifiable flaviviruses. Comparing the newly designed algorithmic OE-PCR with existing executable programs demonstrated it to be efficient and useful in generating accurate sequences of synthetic flaviviral nucleotides.The efficiently and accurately produced novel synthetic nucleotides of notifiable dengue virus 1\u20134,\u00a0Japanese encephalitis, yellow fever and Zika flaviviruses using OE-PCR is useful in understanding the dynamics of flaviviral species and holds potential for the development of synthetic nucleotide-based immunogens. Dengue virus (1\u20134), Japanese encephalitis and Zika fevers are notable mosquito-borne infections that continue to be problematic in the Philippines and its southeast Asian neighbors. Flaviviral yellow fever affects other tropical countries. This study aimed to effectively and efficiently synthesize flaviviral nucleotides through a predictively accurate algorithmic overlap extension-polymerase chain reaction design. The cost-saving arithmetic formulation has enabled the advanced production of 253\u2013256\u00a0bp flaviviral nucleotide lengths. The newly synthesized and sequenced products are now ready for further experimentation and open the door for immunological exploration.  Dengue and Zika fevers, including Japanese encephalitis (JEV), are particularly burdensome mosquito-borne infections in the Philippines and are also selectively reported in Japan . The chaThe OE-PCR in this experiment was performed via two processes: the first one is using the reference or original OE-PCR protocol ,12 and t1.Preparation of the first-stage primer mixed solution(A) Both ends primer  2.5\u00a0\u03bcl(B) Central primer (2\u20135) 0.5\u00a0\u03bcl(U) Deionized water (DW) 18\u00a0\u03bcl2.Preparation of reaction solution for the first-step PCR(A) First-stage primer mixed solution \u00d7 6\u00a0\u03bcl(A) GoTaq 12.5\u00a0\u03bcl(U) DW 6.5\u00a0\u03bcl3.First-stage PCR reaction(A) 95\u00b0C 2\u00a0min21(C) 72\u00b0C - 2\u00a0min4.Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1\u00a0\u03bcl(B) Both ends primer (stock solution) 1\u00a0\u03bcl\u00a0\u00d7 2(C) GoTaq 12.5\u00a0\u03bcl(D) 9.5\u00a0\u03bcl of DW5.Second-step PCR reaction(A) 95\u00b0C 2\u00a0min(A) (95\u00b0C - 1\u00a0min \u2192 72\u00b0C - 2\u00a0min) \u00d7 35-times (C) 72\u00b0C - 2\u00a0min1. Preparation of the first-stage primer-mixed solution(A) Both ends primer  2.5\u00a0\u03bcl(B) Central primer (2\u20135) 0.5\u00a0\u03bcl(U) DW 193\u00a0\u03bcl(A) GoTaq 12.5\u00a0\u03bcl(U) DW 6.5\u00a0\u03bcl3. First-stage PCR reaction(A) 95\u00b0C 2\u00a0min(A) (95\u00b0C - 1\u00a0min \u2192 48\u00b0C - 1\u00a0min \u2192 72\u00b0C - 1\u00a0min) \u00d7 20(C) 72\u00b0C - 2\u00a0min4. Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1\u00a0\u03bcl(B) Both ends primer (stock solution) 1\u00a0\u03bcl \u00d7 2(C) GoTaq 12.5\u00a0\u03bcl(D) 9.5\u00a0\u03bcl of DW5. Second-step PCR reaction(A) 95\u00b0C 2\u00a0min(B) (95\u00b0C. -1\u00a0min \u2192 72\u00b0C. -2\u00a0min) \u00d7 25(C) 72\u00b0C. -2\u00a0minDepicted in In the second PCR, the most outside oligomers work for PCR primers and amplified the whole size of target DNA.An electrophoresis of OE-PCR products was done for PCR product-size prediction after amplification of both OE-PCR and pathogen-specific PCR. Single product was confirmed on 1.5% agarose gel electrophoresis with 1\u00a0\u00d7 Tris/Borate/EDTA (TBE) buffer and UV-visualized with 0.7 micro-g/ml-ethidium bromide . The PCRThe following notable flaviviruses,\u00a0namely dengue (4 types), YF, JEV and Zika, were specifically processed for production of artificial DNAs. The algorithmic formulated OE-PCR was subsequently carried out in two-step cycles, with each cycle's DNA products depicted via UV-illuminated bands following gel electrophoresis . In the DNA sequencing was carried out in similar steps as previously described ,7 Eurof.Approaches to determine efficiency and cost\u2013effectiveness of the current algorithmic OE-PCR tool versus the existing programs are presented in It must be noted that the price of oligomers varies greatly depending on the length in base and according to the viral species involved. Owing to this, an algorithm was designed to achieve the maximum length within the lowest cost range in base size. Unfortunately, this is difficult to realize using the currently available reported design algorithms. In the stated design algorithms, the difficulty lies in how to control the length of the oligomer. This is because the setting of overlapping region of the oligomer has been designed first. With the current study, the basic length has been decided to begin with; coupled with a lowered priority on the thermodynamic stability of the overlapping region. This has resulted in fewer problems in the conduct of OE-PCR.The ongoing dilemma of largely problematic and imperfectly developed flaviviral immunogens prompted us to responsibly contribute on the need to develop an efficient algorithmic OE-PCR program design for synthetic nucleotide production of select notifiable dengue (1\u20134), JEV, YF and Zika flaviviruses. To this end, we developed an OE-PCR program that can be utilized as reference tool in the furtherance of flaviviral research. It can be deduced that the program formulation was found to be demonstrably reproducible, cost-effective and efficient in yielding substantial nucleotides of select notifiable flaviviral DNAs, which can be sourced out at the databases of DDBJ/NCBI with the respective accession numbers of LC227563, LC227561, LC227562, and LC227568 (dengue 1\u20134); LC227560 (JEV); LC227567 (YF); and LC227589 (Zika). The OE-PCR is a reliable method that can be utilized when designed and accurately predicted using algorithmic manipulation.\u00a0Henceforth, we finally conclude that the DNA primer design using algorithmic OE-PCR is a valuable tool in producing synthetic nucleotide products of pathogenic species of flaviviruses.in vivo would result in pathological or immunological response.The novel nucleotide sequences generated from this study can be utilized for studies looking to further understand the genomic diversity of a variety of flaviviral strains, including dengue (1\u20134), YF, JEV and Zika. In addition, clinical trials involving initial utilization of whole nucleotide sequence followed by booster immuno-stimulation of antigenic protein may serve as a starting point for generating long-term immune responses for flaviviral infections such as dengue and Zika, where trials for DNA-based vaccines are ongoing, yet faced with challenging dose-dependency concerns. Further experiments should be conducted to determine whether administration of synthetic nucleotides Box 1.\u2002flavi |2|1: ATGTATGCCGATGACACCGCAGGATGGGATACAAGAATCACACTAGAAGACCTAAAAAAT: 60flavi |2|2: TTGTGTTCTCCTTCCATGTGGTTTGTTACCATTTCTTCATTTTTTAGGTCTTCTAGTGT: 59flavi |2|3: CATGGAAGGAGAACACAAGAAACTAGCCGAGGCCATTTTCAAACTAACGTACCAAAACA: 59flavi |2|4: TGTGCCTCTTGGTGTTGGTCTTTGCACACGCACCACCTTGTTTTGGTACGTTAGTTTG: 58flavi |2|5: CAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGAGGTAGTGGA: 59flavi |2|6: TGGTGAAAGTATTGAGTCCATAGGTGCCAACTTGTCCACTACCTCTTTGGTCTC: 54flavi |3|1: ATGTATGCTGATGACACAGCAGGCTGGGACACAAGAATCACTGAGGATGACCTTCAAA: 58flavi |3|2: TGGTGGGGAGCCATCTGTTCCGTGATCAGTTCCTCATTTTGAAGGTCATCCTCAGT: 56flavi |3|3: TGGCTCCCCACCACAAGATCCTAGCCAAAGCCATTTTCAAACTAACCTATCAAAACAA: 58flavi |3|4: TCCCCGCGGTGTGGGTCTGAGGACTTTCACCACTTTGTTTTGATAGGTTAGTTTG: 55flavi |3|5: CACCGCGGGGAGCGGTGATGGATATCATATCCAGGAAAGACCAAAGAGGTAGTGGA: 56flavi |3|6: TGGTGAATGTGTTCAAACCATATGTTCCAACTTGTCCACTACCTCTTTGGTCTT: 54flavi |5|1: ATGTATGCAGATGACACAGCCGGATGGGACACAAGAATAACAGAGGATGATCTTCAGAA: 59flavi |5|2: CATGTTCAGGTTCCATGATGTCAGTGATTTTGGCCTCATTCTGAAGATCATCCTCTGT: 58flavi |5|3: CATGGAACCTGAACATGCCCTATTGGCCACGTCAATCTTTAAGCTAACCTACCAAAACA: 59flavi |5|4: GTTCCATTTTTCGCTGGTCTCTGCACCCTTACTACCTTGTTTTGGTAGGTTAGCTTA: 57flavi |5|5: AGCGAAAAATGGAACCGTGATGGATGTCATATCCAGACGTGACCAGAGAGGAAGTGG: 57flavi |5|6: TGGTGAAGGTGTTTAAGCCATAGGTTCCAACCTGTCCACTTCCTCTCTGGTCA: 53flavi |8|1: ATGTATGCTGATGACACAGCTGGTTGGGACACAAGAATAACAGAAGATGACCTGCACA: 58flavi |8|2: TGTGTTCAGGGTCCATTTGCTGTATGATCTTTTCCTCATTGTGCAGGTCATCTTCTG: 57flavi |8|3: AATGGACCCTGAACACAGGCAGTTAGCGAACGCTATATTCAAGCTCACATACCAAAACA: 59flavi |8|4: TGCCCGTTGGAGTCGGTCGTTGAACTTTGACCACTTTGTTTTGGTATGTGAGCTTG: 56flavi |8|5: ACTCCAACGGGCACGGTAATGGATATTATATCTAGGAAAGACCAAAGGGGCAGT: 54flavi |8|6: TGGTGAATGTATTCAGGCCATAAGTTCCCAGTTGTCCACTGCCCCTTTGGTC: 52flavi |15|1: ATGTACGCAGATGACACTGCTGGCTGGGACACCCGCATTAGTAAGTTTGATCTGGAGAA: 59flavi |15|2: GTGCCCTTCCTCCATTTGGTTGGTAATCAGAGCTTCATTCTCCAGATCAAACTTACT: 57flavi |15|3: TGGAGGAAGGGCACAGAACTCTGGCGTTGGCCGTGATTAAATACACATACCAAAACAA: 58flavi |15|4: TTTTTCCTCCTTCAGCTGGTCTGAGAACCTTCACCACTTTGTTTTGGTATGTGTATTTA: 59flavi |15|5: CAGCTGAAGGAGGAAAAACAGTTATGGACATCATTTCAAGACAAGACCAGAGAGGGAG: 58flavi |15|6: TGGTGAATGTGTTGAGAGCATAAGTGACAACTTGTCCACTCCCTCTCTGGTCTTGTC: 57flavi |25|1: TTCTACGCGGATGACACCGCTGGATGGGACACGCGCATCACAGAGGCAGACCTTGAT: 57flavi |25|2: TGTGATGTGGGCTCATGTAGTTCAAGATCTCCTGTTCATCATCAAGGTCTGCCTCTG: 57flavi |25|3: ATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAGAACA: 58flavi |25|4: TTCCCTCCTGGGGCTGGTCTCAACACTTTCACCACTTTGTTCTTGTATGTCATTTCC: 57flavi |25|5: GCCCCAGGAGGGAAAGCCTACATGGATGTCATAAGTCGACGAGACCAGAGAGGAT: 55flavi |25|6: TGGTGATGGTGTTCAGAGCATAAGTCACTACCTGCCCGGATCCTCTCTGGTCTCGT: 56flavi |27|1: ATGTACGCTGATGACACCGCCGGATGGGACACTAGAATTACCAGAACTGATTTAGAAAA: 59flavi |27|2: GGTGTTCACCGTCTAGGAGCTCCAGCACCTTGGCTTCATTTTCTAAATCAGTTCTGGT: 58flavi |27|3: CCTAGACGGTGAACACCGCATGCTCGCTCGAGCCATAATTGAACTGACTTACAGGCACA: 59flavi |27|4: CTTTCCTTCTGCTGCAGGTCTCATGACCTTGACCACTTTGTGCCTGTAAGTCAGTTCA: 58flavi |27|5: TGCAGCAGAAGGAAAGACCGTAATGGACGTGATATCAAGAGAAGATCAAAGGGGGAG: 57flavi |27|6: TCGTGAAAGTGTTAAGGGCATAAGTGACCACCTGTCCACTCCCCCTTTGATCTTCT: 56This project aimed to develop synthetic nucleotides using overlap extension-polymerase chain reaction (OE-PCR), particularly of flaviviral organisms that are notorious for their ability to cause both animal and human diseases.A method is provided for generating template designs that could help to address epidemics caused by select flaviviral dengue, Zika and related species.Expansion of OE-PCR as a tool in developing templates for viral studies is presented, addressing concern regarding the lack of reported commercial production of artificial nucleotides of these flaviviruses.This research has delivered a reproducibly effective and efficient algorithmic OE-PCR design that is suitable for yielding substantial nucleotides of these flaviviruses.These synthetic nucleotides could pave the way for future collaborative research exploring the possibility of generating synthetic flaviviral nucleotide-based immunogens."}
+{"text": "Tmem119 gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous Tmem119 in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11b+CD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the Tmem119-EGFP and Tmem119-CreERT2 lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice.Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell types. We have targeted the recently discovered microglia-specific  Tools that specifically label and manipulate only microglia are currently unavailable, but are critically needed to further our understanding of this cell type. Complementing and significantly extending recently introduced microglia-specific immunostaining methods that have quickly become a new standard in the field, we generated two mouse lines that label and control gene expression in microglia with high specificity and made them publicly available. Using these readily accessible mice, the research community will be able to study microglia biology with improved specificity.Tie2, Runx1, Csf1r, Aif1, Lyz2, Itgam, Sall1, or CX3cr1  is highly and exclusively expressed in microglia in the brains of mice and humans  and Tmem119-CreERT2 (JAX#031820), where microglia express EGFP and CreERT2, respectively, while preserving endogenous Tmem119 expression. We demonstrate that EGFP is expressed throughout the brain and that the tag is confined to microglia only, without significantly labeling other brain macrophages. We further provide evidence that the inducible Cre is primarily active in microglia by crossing to the conditional fluorescent reporter mouse line Ai14. In these mice, we also observe activity in leptomeningeal cells that line the surface of the brain and penetrate deep into the brain ensheathing some large blood vessels. Finally, we demonstrate minimal to absent transgene expression in monocytes of the mice. These publicly available mouse lines provide valuable tools for the functional study of bona fide microglia.Recent advances in RNA sequencing and other cell profiling technologies have enabled the discovery of cell-type-specific signature genes . Among td humans . AlthougAll animal procedures were performed in accordance with MIT\u2019s animal care regulations. Overall, >30 animals of >10 litters were generated per line and mice were crossed to C57BL/6J up to generation N3. For all experiments, at least three independent mice were analyzed, which included both sexes and no apparent sex differences were observed.Tmem119 knock-in mice, donor DNA templates encoding ribosome-skipping peptide porcine teschovirus-1 polyprotein and EGFP (P2A-EGFP) or CreERT2 (P2A-CreERT2) were synthesized. These sequences were flanked by sequences of 55\u2013300 bp for EGFP and 1.5 kb for CreERT2 homologous to 5\u2032 and 3\u2032 regions around the Tmem119 stop codon. These templates were injected into fertilized mouse oocytes together with a single crRNA  that cuts at the stop codon.To generate AACTGGTCCTCCTGAAA-3\u2032 and 5\u2032-CAAAGCCTGTGAAGGGTGGG-3\u2032, respectively; * denoting phosphorothioate) and a reverse primer for a 55 bp right homology arm . Using these primers, large scale PCR with Takara PrimeStar was performed to obtain 40\u201360 \u03bcg of product. The PCR product was highly purified with the Qiagen PCR purification kit and subject to digestion of the antisense strand. Lambda exonuclease (New England Biolabs) was used to digest 20 \u03bcg of dsDNA at 37\u00b0C for 60 min. Complete digestion of dsDNA was confirmed by agarose gel electrophoresis and Sanger sequencing with sense- and antisense-binding primers.Donor DNA templates were generated by digesting pAAV-P2A-EGFP (sequence below) with XbaI and EcoRI (New England Biolabs) and inserting three gblocks  using Gibson cloning  according to the manufacturers\u2019 protocols. Resulting plasmids were purified and sequenced. For CreERT2, the highly purified dsDNA plasmid was directly used as donor DNA in injections. For EGFP, single strand DNA (ssDNA) was produced using PCR with forward primers for left homology arms between 55 and 300 bp  and heated to 95\u00b0C for 5 min. Heated mixtures were cooled to room temperature for 10 min and 30 ng/\u03bcl EnGen Cas9 protein (New England Biolabs) was added. Mixtures were incubated at 37\u00b0C for 15 min to form Cas9 to crRNA-tracrRNA complexes. Final concentrations of 5 ng/\u03bcl donor DNA (P2A-EGFP ssDNA or P2A-CreERT2 plasmid dsDNA) and 10 ng/\u03bcl recombinant RAD51 protein were added. Mixtures were kept on ice until use, when they were incubated at 37\u00b0C for 15 min followed by centrifugation at 10,000 rpm for 1 min to prevent clogging of the micropipette.Mixtures for injection of zygotes were prepared freshly on the morning of the day of injection. Briefly, water to a final volume of 100 \u03bcl was mixed with final concentrations of 10 mInjection of mixtures was conducted using standardized protocols of the transgenics facility in zygotes obtained from pure C57BL/B6N mice (Taconic).GCCTCTGTCACTTAAGTTGG) and P2A-R (GCTTCAGCAGGCTGAAGTTA). A second amplicon of \u223c 2.5\u20133.5 kb (CAGTGTCGGAAGCGGAGCTA) and 3\u2032ofRHA-R (GAAAGAGGAAGCTAGAAGGG). Both amplicons were purified and then sequenced using primers spanning the entire length using Sanger sequencing (GENEWIZ). Trace files were aligned to in silico assemblies and analyzed using Snapgene.Founder mice were genetically examined by amplifying sequences spanning the 5\u2032 and 3\u2032 junction and including the entire inserted transgene. Specifically, high-quality DNA was obtained from ear punches using a tissue DNA extraction kit according to the manufacturer\u2019s instructions (Macherey-Nagel). One amplicon spanning \u223c1.5 kb  at 95\u00b0C for 30 min. The lysed tissue solution was neutralized using an acidic buffer . PCR using primers WT-F GTCAGGAGGAGGCCCAGGAA, EGFP-F CTGCTGCCCGACAACCACTA, CreERT2-F ACCGCCTACATGCGCCCACT, and Common-R GTTTCCTGGGGTGCACCAGA yielded products of 400 bp for the wild-type allele and 320 bp for the EGFP or CreERT2 allele.To genotype mice, ear tissue was prepared using an alkaline buffer  mice were obtained from JAX (stock 007914) and maintained in house. Homozygous Ai14 mice were bred to heterozygous Tmem119-EGFP+/\u2212 mice were dissected and snap-frozen in liquid nitrogen. Highly pure RNA was isolated using an RNeasy purification kit (Qiagen) following the manufacturer\u2019s instructions. Equal amounts of RNA (1.5 \u03bcg per sample) were reverse transcribed using an iScript Advanced cDNA synthesis kit (Bio-Rad). Resulting cDNA was diluted 1:50 in ultrapure water. Quantitative PCR (qPCR) was conducted using SsOAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX96 real-time system. Primers used were exon-spanning whenever possible and of the following sequences: Tmem119-F CCTTCACCCAGAGCTGGTTC, Tmem119-R GGCTACATCCTCCAGGAAGG, GAPDH-F GCCTTCCGTGTTCCTACC, GAPDH-R CCTCAGTGTAGCCCAAGATG, b-actin-F CTAAGGCCAACCGTGAAAAG, b-actin-R ACCAGAGGCATACAGGGACA. Differential gene expression analysis was performed using built-in software for the Bio-Rad CFX96 real-time system.Brain hemispheres from adult Tamoxifen  was dissolved in corn oil at 20 mg/ml under agitation for several hours in the dark and kept at room temperature for 2\u20133 d. Using needles from Harvard Apparatus (52-4025), separately housed adult animals were fed via oral gavage for 3 d. Needles were rinsed between days and syringes were replaced. At time of administration, the mice weighed \u223c19\u201326 g and received 250\u2013400 \u03bcl of the 20 mg/ml tamoxifen solution, corresponding to 0.2 mg/g body weight. Postnatal day (P)2 mice weighing \u223c2 g received 5 \u03bcl of the 20 mg/ml solution, corresponding to 50 \u03bcg/g body weight per day for 3 consecutive days. The health of the mice was monitored daily. At least 7 d week after the last dose, mice were killed by transcardiac perfusion.Adult mice were deeply anesthetized and perfused with 25 ml PBS followed by 25 ml 4% paraformaldehyde (PFA) in PBS. Early postnatal mice (P3) were perfused with 8 ml PBS and 8 ml PFA. Brains were surgically removed and postfixed in the fixative at 4\u00b0C for 24 h. Fixed brains were washed once in PBS and sliced into 100-\u03bcm-thick sagittal slices using a Leica VT1000S. Slices were washed twice in PBS, permeabilized in 1.2% TX100 in PBS for 15 min, washed twice in PBS, and subject to incubation in blocking solution . Blocked sections were incubated with primary antibodies for IBA1 , GFP , TMEM119 , Olig2 , NeuN , GFAP , or CD163  for 24 h at 4\u00b0C. Primary antibody incubation was followed by three washes in PBS and incubation with species-matched and Alexa fluorophore-conjugated secondary antibodies raised in goat  for 2 h. DAPI (1:10.000) was included in a washing step or secondary antibody incubation. Slices were washed three times in PBS and mounted and coverslipped using VECTASHIELD H-1000 mounting medium.m Sodium citrate, pH 8.5) for 5 min, followed by incubation in the retrieval buffer at 80\u00b0C for 30 min. Sections were cooled to room temperature and washed twice in PBS. Blocking and immunostaining were then conducted as described for other antibodies in the immunofluorescence staining and imaging methods section.For immunostaining of CD163, an antigen retrieval step was included. Briefly, vibratome slices were washed twice in PBS, incubated in a retrieval buffer  were counted manually.For imaging, slides were scanned on an Olympus FluoView FV1000 fixed stage confocal microscope (high-power magnifications) or Olympus BX61 epifluorescence microscope  using built-in software. For coexpression analysis in the z-stacks were acquired at 20\u00d7 magnification with 2\u00d7 digital zoom and 1.2 \u03bcm step size. Images were imported as stacks into Neurolucida software. Cell bodies in the middle of the stacks with complete process arbors were selected and manually traced. Raw trace files were imported into Neurolucida explorer software and convex hull area, number of processes and total process length were determined using the convex hull analysis and branched structure analysis functions. Rendered traces were exported as monochrome vector graphics. Cell body areas were separately determined in FIJI using the polygon tool.For microglia morphology analysis, g for 5 min at 4\u00b0C, resuspended in 10 ml ice-cold HBSS, and pelleted again at 300 \u00d7 g for 5 min at 4\u00b0C. WBCs were resuspended in 500 \u03bcl ice-cold FACS buffer  and subject to staining. In parallel with the WBC enrichment, brains were rapidly dissected into 2 ml ice-cold HBSS and cerebella and brainstem were removed. Brains were minced into small pieces and transferred to a Dounce homogenizer containing 5 ml ice-cold HBSS with 20 \u03bcg/ml DNase I . Tissue chunks were homogenized with 15 loose and 15 more tight strokes and the homogenate was transferred to a 50 ml falcon tube through a pre-wet 70 \u03bcm strainer. The strainer was rinsed with HBSS to top off the volume of each sample to 10 ml. Filtered homogenates were transferred to 15 ml falcon tubes and spun at 300 \u00d7 g for 5 min at 4\u00b0C. Supernatants were carefully removed, and pellets resuspended in 10 ml of ice-cold 40% Percoll in HBSS. Samples were spun at 500 \u00d7 g for 30 min at 4\u00b0C with full acceleration and deceleration. Myelin and debris from the supernatant were carefully removed and pellets resuspended in 10 ml ice-cold HBSS. Following another spin at 300 \u00d7 g for 5 min at 4\u00b0C, the supernatant was removed and the microglial pellet resuspended in 1 ml FACS buffer .Microglia and blood monocytes for single-cell suspensions for flow cytometry were prepared as follows. Mice were deeply anesthetized with isoflurane and transcardially perfused with ice-cold HBSS. During perfusion, 2 ml of whole blood was collected from the right atrium into tubes containing 40 \u03bcl of 10% (w/v) EDTA. The 2 ml of whole blood were added to 40 ml of red blood cell lysis buffer  and incubated for 10 min at room temperature to lyse red blood cells. The suspension containing lysed RBCs and white blood cells was spun down at 300 \u00d7 m EDTA) and spun down at 300 \u00d7 g for 5 min at 4\u00b0C. Pellets were resuspended and incubated with 1:200 Mouse Fc Block  on ice for 15 min. Samples were incubated with 1:200 rat anti CD45-APC/Cy7  and rat anti CD11b-PE/Cy5  at 4\u00b0C. Tubes were topped off to 2 ml with ice-cold FACS buffer and microglia pelleted at 300 \u00d7 g for 5 min at 4\u00b0C. Supernatants were removed and microglia resuspended in 500 \u03bcl. Resuspended microglia were filtered through corning strainer polystyrene tubes . Flow cytometry data were acquired on Aria II, Fortessa HTS, and LSRII HTS, and analyzed using FlowJo.Suspensions of white blood cells and microglia were transferred to 2 ml Eppendorf microcentrifuge tubes, and a small fraction of sample was removed for single-color controls. To stain dead cells, live/dead violet  was added and incubated for 5 min on ice. Tubes with live/dead-stained cells were topped off to 2 ml with FACS buffer . Not assuming Gaussian distribution of the data, a nonparametric test (Mann\u2013Whitney) was used to compare median expression in both groups.Quantitative data from the qPCR, immunofluorescence, and microglia morphology experiments were analyzed using GraphPad prism. Testing for normality was not possible because pAAV-P2A-EGFPCCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAGTGTCTAGACTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAAACCGGCTAGAGGATCCTTCGAAACCGGTGCTAGCAGCGCTGTTAACGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGCGCGCCCCTGCAGGGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGAGCGCTGCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTLHAGCGGCCGCACGCGTTTAATTAAGTGTCTAATTGGACAGGGAGATGGCTCAGGTGTGGAAAGCCAATATAGCGGTGCATGCTTGCAAGGCCAGGAGTAATGATGGGGACATAGGAGGATTCTGGGAGCCTGGCCGGATAACCTATCCCCTAACACATAAGCACCAGGTCCCACCCAGTAAGAGGCCCTGCCTCGAAGAAGAAGTGGAGGGTCCCTGAGGAGGAAGGGCATTTGGGGTTATCCTCTGGTCTCCAAATGCATCCCTCCTCCCACACAAACACACAAAGACACTTTCACTTGTATCTGATGCCTGGTTAGCCATGAGCGTAAGCTAGGATTGTGAATTCAGCCACCCTGGTACAGCAACTATTGAGTGCTTAGTGTATACTCTGGGTTCGAAACACCGAGGACAGAAATGAATAAGATGCAGTTTCTGTCCTCAGGACCACCAGAAGGAAACCAGGACGGGTGGCATCCTGTGTAGACAGACCCCGGAGTGTGTGCGTGTTGCTTAGCTAAGTGATTTTCTGGAGTGTGTTCTGGCTCCCCGGCCGTCCTCCCGAACTAGCAGACCACTCGTGCTGCCGGAGTCCTGGAGCGACAGGAGACTCAGCCTGACTTCTCTCTTAACTCTCCCTCCGCGCAGGTTCCATAGCTCAACATGGTCCCCTGGTTCCTCCTGTCTCTGCTGCTACTTGCGAGGCCTGTGCCTGGGGTGGCCTACTCTGTGTCACTCCCGGCCTCCTTCCTGGAGGATGTAGCCGGCAGCGGGGAAGCTGAGGGTTCTTCAGCCTCTTCCCCGAGCCTGCCGCCGCCTGGGACTCCAGCCTTCAGTCCCACACCGGAGAGACCCCAGCCCACAGCTCTGGACGGCCCCGTGCCACCCACCAACCTCCTGGAAGGGATCATGGATTTCTTCCGGCAGTACGTGATGCTCATCGCGGTGGTGGGCTCGCTGACCTTCCTCATCATGTTCATAGTCTGCGCCGCCCTCATCACGCGCCAGAAGCACAAGGCCACAGCCTACTACCCATCCTCGTTCCCTGAAAAGAAGTATGTGGACCAGAGAGACCGGGCTGGGGGACCCCGTACCTTCAGCGAGGTCCCTGACAGGGCACCTGACAGCCGGCATGAAGAAGGCCTGGACACCTCCCATCAGCTCCAGGCTGACATTCTGGCTGCTACCCAGAACCTCCGGTCTCCAGCTAGAGCCCTGCCAGGCAATGGGGAGGGAGCAAAGCCTGTGAAGGGTGGGTCGGAGGAGGAGGAGGAAGAGGTGCTCAGCGGTCAGGAGGAGGCCCAGGAAGCCCCAGTATGTGGGGTCACTGAAGAGAAGCTGGGGGTCCCAGAGGAGTCGGTCTCAGCAGAGGCTGAAGGGGTTCCTGCCACCAGTGAGGGCCAAGGGGAAGCAGAAGGGTCTTTCTCCTTAGCCCAGGAATCCCAGGGAGCAACTGGTCCTCCTGAAAGTCCCTGTGCCTGCAACAGAGTCTCTCCCAGTGTCP2A-CreERT2TGTGCCTGCAACAGAGTCTCTCCCAGTGTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATCTCGAGCCATCTGCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCTGACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGGCGGCGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACAGCTTGACATGCCCCAGAACTGCTGGGACCCGAATGTP2A-EGFPTGTGCCTGCAACAGAGTCTCTCCCAGTGTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACATGCCCCAGAACTGCTGGGACCCGAATGTRHACATGCCCCAGAACTGCTGGGACCCGAATGTTGGGTCCTTGAGGGTCACCTCTTTGGTCAAGAAAGGCATTCAGCTCTAACTGCTCCTTGATACCACGTGGCTTGGCCATTGCTGGTGCCAAGGCTGACCCCGAACTGGCAGAGCCGATGCCCTCTGGTGCACCCCAGGAAACATCTCCCCAAGTTCCAGCGCCCTTAATGACTCTTGCCACCCTGGGGGCTTCACCCTAACGCACCACTTCTCTGGAAGGGGAAGGCCAGACACATGCCAGTTGGGGCTGCATGAGGCAGTCCTCAGAGCAGAAGGGGACCAGGCCAGAGGCCACCTGTGACGGGGCAAACTGCATCTCGGCTGTGGAGACCAGAGGGGCTGTTAGATTTGGAAGACATCAATGACTGGGCCTGCGGCGCAGCCCGTGTCTGGTAATACCAGGGACGGCAGAGGCGTTTGCATCTTCCCATCACCTGCAATGTCGCTGTCACTCTGCCCCTGTTCAGTGGACTTGATTAGTTAGGAAACTTCTGGAAGGGGCCCCCTACTTTATATCACAGAGTTTGCCCTAGACACCCCGTGGAAACACAAACTCAAAATCAAGTGGCTTTAGGAGCCGCTGTGCCCCTCCACAAGCTGACATGGCTACTCTAAGGGTTCCTGCTGGGCTGGCTTTGCTACGCTTTCCTCAAGCTGCTTTCTTATTACCAGGATGCCTCACAGCTACAAAGTCCAATCTCACAGCACCCGCACTGGAAAATACTGTTCTCCATTTTTTTCCAAGGCACCCTGCTTTATGATTGGCTCAGAGTTGGAATATGGGATGCAGGGCGTCTGGCTCTCTCAAGTGTCAAGCAACCCTGGCTTCCGCATGTGGGGCGAGGGGAGTGAGCAGAACTCTTCTCTGTCAGTCCACGAAGTAGACTAGAAGCCAACGGGTGCCTGGGGGCAACTGACGGTCTGGATTCTGACGCCCAGCCTGGAGCAGGGGCCTGGCCCATCCTATGCTCACACAGTGGTCTGGCAGCCTGAGCCCACACAACTCCTCAGTCCTTGACAATGCTTAGGCTCTGTTCTGAGGGACTCCCCACACCTCCATTCAGGGCTCCCCGAGTCCTCAGCTCTTCGGATGTGGATATATGACACACTAGCATAATAAATCTTGATCTGGCTTGAGTCTCTCTGGATTATCTGCACAGGTCTGCAGGAATGGGGTGTTCATGGGCTGGGGGCAGCATTGGGGGGAGGGCATCAGCCAGGTCACAGAGCTGAGACTGTGTGTGGGTGCTCACAGGTGTTGGACCCACGTACCTGAGGAGTCCTGGAGGTGGTGTGTCAAACGTACAGACACTTTGCTGCCTGGCTCTGCAGCTGGTGCAGCCATGCCACAGTCACTTCTTTGTGCTGTGGGACAAAGCTGGGGCAAAGTCATGGCTGCCAATTCCCTACAGATGGGTGAGAACTGACTGAGCCTCAGGGCGATATCAAGCTTATCGATAATCAACCTCTTmem119 gene to generate such mouse lines , into the stop codon of murine Tmem119  animals B. This ie 3\u2032 UTR C,D. AddiTmem119 is currently unknown, both its temporally distinct pattern and high expression level suggest functional importance of this gene . We further examined TMEM119 protein expression using immunofluorescence and found no difference between WT and Tmem119-EGFP+/\u2212 knock-in mice  Together, these data support the conclusion that Tmem119 knock-in mice were generated with precision.Although the function of endogenous his gene . We thusTmem119-EGFP+/\u2212 knock-in mice , number of processes , convex hull area , and total process length  are comparable between microglia from control and knock-in mice. These data suggest that the genetic modification does not affect basic microglial properties.To further test whether the genetic engineering could affect the properties of microglia, we examined basic morphologic features of reconstructed immunostained microglia in WT and -in mice H,I. UsinTmem119-EGFP line, we used confocal microscopy on brain slices. Native EGFP fluorescence and antibody-enhanced signal were visible across the brain in sagittal brain sections prepared from P25 Tmem119-EGFP+/\u2212 mice . We determined that all CD11b+CD45lo microglia  corresponding to non-microglial cells express EGFP  to all parenchymal microglia (IBA1+) revealed very high efficiency of recombination (+IBA1+) among all tdTomato-labeled cells (tdT+) in different parenchymal regions, and found high specificity close to 100% .In this study, we addressed the critical need for transgenic mouse lines that specifically label or control microglia, but not other closely related cell types with overlapping signature gene expression . We targTmem119 out through insertion of EGFP or CreERT2 into the Tmem119 ORF and instead chose to preserve expression by using a polycistronic knock-in approach with ribosomal skipping peptide P2A +/\u2212 mice show tdTomato expression in IBA1-expressing microglia, indicating that this line is suitable for Cre-dependent manipulation of genes and transgene expression in microglia without affecting other major neuronal or glial cell types in the CNS (Cx3cr1 mouse line (Tmem119-CreERT2 line a highly useful tool for the conditional study of microglia, while also suggesting potential application for leptomeningeal cells.Our experiments using  the CNS . In this the CNS K,L. The use line , and miguse line N\u2013P. Thisuse line , which muse line Q\u2013V. Althuse line Q\u2013S. Togeuse line . TogetheTmem119-EGFP and Tmem119-CreERT2; Ai14 mice revealed complete absence and minimal expression, respectively (Tmem119-EGFP knock-in mice was expected based on previous observations, tdTomato expression in 3% blood monocytes of the Tmem119-CreERT2; Ai14 mice was rather surprising. Similar to other brain macrophages discussed above, blood monocytes may express Tmem119 at very low, almost undetectable levels, which may nonetheless be sufficient to cause occasional all-or-none Cre recombination events. Together, these data render the Tmem119 knock-in lines powerful tools to investigate microglia in health and disease without significant confounds from monocyte contribution.Examination of EGFP and tdTomato expression in blood monocytes of ectively . While, Tmem119-EGFP line might be of great value (Tmem119-EGFP line may enable cell sorting without the need for antibodies, possibly even at early developmental stages before the onset of TMEM119 appearance on the membrane, as well as in vivo imaging approaches. Furthermore, the absence of EGFP fluorescence in blood monocytes renders this line uniquely useful for the study of microglia in brain disorders that involve monocyte infiltration. The Tmem119-CreERT2 line will significantly facilitate conditional control of gene expression selectively in microglia, especially in cases when monocytes, but not leptomeningeal cells, could likely confound interpretations. Compared with constitutive Cre lines, which cumulatively recombine throughout development and thus deny temporal control, the CreERT2 line will allow precise control of gene expression to examine the role of microglia in temporally restricted developmental processes. In this capacity, dose-dependency of CreERT2 and potentially resulting mosaic expression of recombined alleles using low doses may allow the study of candidate genes in adjacent microglia populations sharing the same microenvironment. Beyond the application of Tmem119 knock-in and other currently available mouse lines, genetic approaches such as targeting other microglia-signature genes, engineering highly specific artificial promoters and regulatory regions, and using intersectional split-Cre approaches hold great promise to produce even more specific tools to study microglia in the future.During the past decade, available microglia lines have been instrumental in advancing our understanding of microglia. These lines remain powerful tools to study microglia function. At the same time, it is widely acknowledged that ontogenetic differences, as well as differences in localization and microenvironment between parenchymal microglia and their closely related cells from the monophagocytic system warrant more discerning observation and manipulation . Recentlat value B. In add"}
+{"text": "Abs targeting the HA head of influenza viruses are often associated with protection from influenza virus infections. These Abs typically have limited breadth, since mutations frequently arise in HA head epitopes. New vaccines targeting the more conserved HA stalk domain are being developed. Abs that target the HA stalk are protective in animal models, but it is unknown if these Abs exist at protective levels in humans. Here, we completed experiments to determine if Abs against the HA head and stalk were associated with protection from naturally acquired human influenza virus infections during the 2015\u20132016 influenza season. in vitro antibody-dependent cellular cytotoxicity activity. In passive transfer experiments, sera from participants with high HAI activity efficiently protected mice, while sera with low HAI activity protected mice to a lower extent. Our data suggest that HA head Abs are more efficient at protecting against H1N1 infection than HA stalk Abs.Seasonal influenza viruses are a major cause of human disease worldwide. Most neutralizing antibodies (Abs) elicited by influenza viruses target the head domain of the hemagglutinin (HA) protein. Anti-HA head Abs can be highly potent, but they have limited breadth since the HA head is variable. There is great interest in developing new universal immunization strategies that elicit broadly neutralizing Abs against conserved regions of HA, such as the stalk domain. Although HA stalk Abs can provide protection in animal models, it is unknown if they are present at sufficient levels in humans to provide protection against naturally acquired influenza virus infections. Here, we quantified H1N1 HA head- and stalk-specific Abs in 179 adults hospitalized during the 2015\u20132016 influenza virus season. We found that HA head Abs, as measured by hemagglutinin inhibition (HAI) assays, were associated with protection against naturally acquired H1N1 infection. HA stalk-specific serum total IgG titers were also associated with protection, but this association was attenuated and not statistically significant after adjustment for HA head-specific Ab titers. We found slightly higher titers of HA stalk-specific IgG1 and IgA Abs in sera from uninfected participants than in sera from infected participants; however, we found no difference in serum IMPORTANCE Abs targeting the HA head of influenza viruses are often associated with protection from influenza virus infections. These Abs typically have limited breadth, since mutations frequently arise in HA head epitopes. New vaccines targeting the more conserved HA stalk domain are being developed. Abs that target the HA stalk are protective in animal models, but it is unknown if these Abs exist at protective levels in humans. Here, we completed experiments to determine if Abs against the HA head and stalk were associated with protection from naturally acquired human influenza virus infections during the 2015\u20132016 influenza season. Seasonal influenza viruses cause annual epidemics worldwide. Although seasonal influenza vaccines usually provide moderate protection against circulating strains, vaccine effectiveness can be low when there are antigenic mismatches between vaccine strains and circulating strains , 2. Addiin vitro (\u2013Antibody (Ab)-mediated immunity is important for protecting against influenza virus infections . The vir5\u2013in vitro \u201317.Conventional influenza vaccines effectively elicit HA head-reactive Abs but not HA stalk Abs . As a reHA stalk Abs protect animals from group 1 and group 2 influenza A virus infections , 23\u201329. 23\u201330\u2013Despite the recent interest in developing new HA stalk-based vaccines, the amount of HA stalk Abs required to protect humans from influenza virus infections and influenza-related disease has not been established. A recent human pH1N1 challenge study demonstrated that HA stalk Ab titers are associated with reduced viral shedding but are not independently associated with protection against influenza infection . While h(This article was submitted to an online preprint archive .)We analyzed sera collected from 179 participants enrolled in a hospital-based study during the 2015\u20132016 influenza season . Adults We quantified serum titers of HA head-specific Abs against the predominant 2015\u20132016 H1N1 strain using hemagglutination inhibition (HAI) assays . HAI assWe next quantified relative titers of H1 stalk IgG Abs using enzyme-linked immunosorbent assays (ELISAs) coated with headless H1 proteins . SimilarAlthough both HAI titers and HA stalk IgG titers were associated with H1N1 protection in unadjusted models , only HAWe completed several experiments to validate our HA stalk IgG data. Headless H1 proteins are engineered to possess only the HA stalk domain and not the HA globular head domain . We compSome HA stalk Abs mediate protection through nonneutralizing mechanisms that involve processes such as ADCC . IgG1 anWe next evaluated serum HA stalk IgA Abs, since IgA Abs can be important for controlling respiratory infections. For example, mucosal IgA potently reduces the risk of influenza transmission events in guinea pigs in a dose-dependent manner  and suppin vitro and in vivo -reporter influenza viruses  and HA head (EM-4C04). For these assays, we incubated HA-expressing 293T cells with serum and then added human peripheral blood mononuclear cells (PBMCs). We then measured CD107a (LAMP1) expression on CD3\u2212 CD56+ NK cells by flow cytometry. CD107a is a sensitive NK cell degranulation marker whose expression levels strongly correlate with cytokine production and cytotoxicity by NK cells in response to Ab-Fc receptor engagement  and uninfected individuals that had low (\u226440) HAI titers (abbreviated as uninfected HAIlow). We also passively transferred pooled sera from infected individuals, all of whom had low (\u226440) HAI titers (abbreviated as infected HAIlow). For these experiments, equal volumes of human sera were transferred for each experimental condition. Mice were challenged with a sublethal dose of H1N1 4 h after serum transfer, and body weights were monitored for 15\u2009days  was larger than the effect size of HA stalk Ab-associated protection (14.2% reduced risk of infection for every 2-fold increase in titer). In our study, HAI titers were independently associated with protection in adjusted models; however, HA stalk Abs were not. However, the effects of both HAI and HA stalk Ab titers were only slightly attenuated in our adjusted model, and it is possible that our relatively small sample size limited our ability to detect an independent association between HA stalk titers and protection.There are several limitations to our study. Since our sample size was relatively small, we only evaluated the contribution of Abs to the HA head and stalk. Larger studies will be required to independently evaluate other immune correlates of protection. For example, it will be important for future studies to evaluate the relationship between HA head and stalk Ab-associated protection and neuraminidase (NA) Ab-associated protection. Recent studies have shown that NA Abs are associated with protection in an H1N1 challenge cohort , and NA de novo Ab responses to H1N1 infection, which could convolute the analyses of Ab types associated with protection. While this is a possibility, it is less of a concern since we found that all infected individuals have very low HAI titers. If our infected participants were making de novo Ab responses, we would anticipate that some of them would have high HAI titers to the infecting H1N1 virus. In addition, Ab titers do not typically increase as days from symptom onset to blood specimen collection increases , we cannot exclude that some participants in our studies were infected for a prolonged period of time before being admitted to the hospital. This raises the possibility that some participants have already mounted ncreases , which sin vitro neutralization titers  contributed to protection in these experiments.It is interesting that n titers , but notn titers , were ashead Abs \u20139, whereead Abs \u2013. It shouead Abs \u2013, which cead Abs \u2013, and theead Abs \u2013 and D. ITaken together, our findings provide important new insights into the prevalence and functionality of HA head and stalk Abs in humans. Future studies that tease out the interdependence of HA head and stalk Abs, as well as Abs and T cells against other viral antigens, will be useful in guiding the development of new universal influenza vaccine antigens.in vitro neutralization assays, ADCC assays, and passive transfers) were completed at the University of Pennsylvania using deidentified sera.During the 2015\u20132016 influenza season, adult (\u226518\u2009years) patients hospitalized for treatment of acute respiratory illnesses at the University of Michigan Hospital in Ann Arbor, MI, and Henry Ford Hospital in Detroit, MI, were prospectively enrolled in a case test-negative design study of influenza vaccine effectiveness. All participants provided informed consent and were enrolled \u226410\u2009days from illness onset during the period of influenza circulation (January to April of 2015 and 2016). Participants completed an enrollment interview and had throat and nasal swab specimens collected and combined for influenza virus identification. Influenza vaccination status was defined by self-report and documentation in the electronic medical record and Michigan Care Improvement Registry (MCIR). When available, clinical serum specimens collected as early as possible after hospital admission were retrieved; all specimens were collected \u226410\u2009days from illness onset based on the enrollment case definition. Studies involving humans were approved by the Institutional Review Boards of the University of Michigan and University of Pennsylvania. All experiments  ethyl chloromethyl ketone (TPCK)-treated trypsin, HEPES, and gentamicin. Virus was isolated from the infected MDCK cells 3\u2009days later. We extracted viral RNA and sequenced the HA gene of A/HUP/04/16.Viruses possessing A/California/07/2009 HA and NA or A/HUP/04/2016 HA and NA were generated by reverse genetics using internal genes from A/Puerto Rico/08/1934. Viruses were engineered to possess the Q226R HA mutation, which facilitates viral growth in chicken eggs. Viruses were grown in fertilized chicken eggs, and the HA gene was sequenced to verify that additional mutations did not arise during propagation. We isolated the A/HUP/04/2016 virus from respiratory secretions obtained from a patient at the Hospital of the University of Pennsylvania in 2016. For this process, deidentified clinical material from the Hospital of the University of Pennsylvania Clinical Virology Laboratory was added to Madin-Darby canine kidney (MDCK) cells  in serum-free medium with 2HCO3, 300\u2009mM NaCl, and 20\u2009mM imidazole, and then eluted using pH 8 buffer containing 50\u2009mM Na2HCO3, 300\u2009mM NaCl, and 300\u2009mM imidazole. Purified protein was buffer exchanged into phosphate-buffered saline . Following purification, the headless HA stalk proteins were biotinylated using the Avidity BirA-500 kit (no. BirA500) and stored in aliquots at \u221280\u00b0C. Plasmids encoding the recombinant chimeric (c6/H1) HA were provided by Florian Krammer (Mt. Sinai). The detailed protocol for expression of this protein is published elsewhere  in 5-ml polypropylene columns , washed with pH 8 buffer containing 50\u2009mM Nalsewhere . In briePlasmids encoding the human MAb EM-4C04, 70-1F02, and CR9114 IgG1 isotypes were provided by Patrick Wilson at the University of Chicago. The heavy-chain constant regions for IgG2, IgG3, and IgA (sequences listed below) were synthesized as a gBlock by IDT and cloned into the pSport6 vector containing the heavy chain of CR9114. All MAbs were expressed in 293T cells and purified 4 days postinfection using NAb protein A/G spin kits  for the IgG isotypes or using peptide M agarose  for the IgA isotype.The IgG2 sequence is CGCATGATGCGTCGACCAAGGGTCCTAGCGTTTTCCCGCTCGCACCTTGTAGTCGGAGCACCTCCGAATCTACGGCGGCGCTCGGATGTCTGGTTAAGGATTACTTTCCTGAACCTGTTACTGTATCTTGGAATTCAGGAGCACTGACATCTGGTGTACATACTTTTCCAGCGGTTTTGCAGTCATCTGGTCTTTATTCCCTGTCCAGTGTGGTAACAGTACCATCCTCAAACTTTGGAACTCAGACCTATACCTGCAATGTGGACCACAAGCCATCCAATACAAAAGTCGATAAGACTGTCGAGCGGAAGTGCTGTGTCGAATGCCCTCCCTGCCCCGCTCCGCCGGTTGCAGGGCCAAGTGTATTTCTTTTTCCACCAAAACCAAAAGATACGCTTATGATATCTCGCACGCCTGAAGTAACCTGCGTAGTCGTTGATGTAAGTCACGAGGATCCCGAAGTTCAATTCAATTGGTATGTAGATGGCGTTGAAGTGCATAATGCAAAGACCAAACCTAGAGAAGAACAATTCAATAGTACCTTTCGCGTGGTTAGCGTACTCACAGTCGTCCACCAGGATTGGCTGAATGGGAAGGAGTACAAATGCAAGGTCTCTAACAAAGGTCTTCCGGCCCCCATAGAAAAAACGATCAGTAAGACCAAGGGGCAGCCCAGAGAGCCACAGGTTTATACGTTGCCTCCGTCTCGCGAGGAAATGACTAAAAACCAGGTCAGCCTGACTTGTTTGGTGAAAGGGTTTTACCCGAGCGATATTGCTGTGGAATGGGAGAGTAACGGGCAACCGGAGAACAATTACAAAACGACACCGCCCATGCTTGATAGTGATGGTTCCTTCTTCTTGTACAGCAAGTTGACGGTTGATAAATCCAGGTGGCAGCAAGGAAATGTTTTCTCTTGTTCAGTGATGCATGAGGCGCTCCACAACCATTATACGCAAAAATCACTCTCACTTTCACCGGGGAAATGAAGCTTGAGCAGGGCCT.The IgG3 sequence is CGCATGATGCGTCGACCAAAGGGCCGTCAGTCTTTCCCTTGGCGCCGTGCTCCAGGAGTACCAGCGGCGGCACCGCGGCGTTGGGATGTCTTGTCAAGGATTATTTTCCCGAACCCGTCACCGTAAGCTGGAACAGTGGGGCATTGACGTCTGGCGTTCATACTTTTCCGGCAGTACTTCAGAGTTCCGGCCTTTATTCTTTGTCAAGCGTTGTTACCGTACCATCCAGTAGCCTTGGCACCCAGACCTACACCTGTAATGTTAATCACAAACCAAGTAACACCAAGGTTGATAAGAGGGTTGAGCTTAAAACACCGCTTGGTGACACAACCCATACGTGTCCAAGATGTCCGGAGCCGAAGAGTTGTGATACCCCGCCGCCGTGTCCTCGCTGTCCGGAACCAAAGAGCTGTGATACCCCCCCACCTTGTCCCAGATGTCCTGAACCGAAATCATGTGACACGCCACCACCTTGCCCAAGATGTCCCGCGCCAGAGCTGCTGGGTGGGCCCAGCGTATTTCTTTTTCCACCCAAACCGAAGGATACCCTTATGATAAGCAGGACTCCCGAGGTTACCTGCGTGGTGGTTGACGTAAGTCACGAAGACCCCGAAGTCCAATTCAAATGGTATGTTGATGGGGTCGAAGTACACAACGCGAAGACTAAACCGAGAGAGGAACAGTATAATAGCACATTCCGGGTTGTTTCCGTACTTACAGTACTTCATCAGGACTGGCTTAATGGCAAGGAGTACAAGTGCAAAGTCAGTAACAAGGCACTCCCTGCTCCGATTGAAAAGACAATATCAAAGACGAAAGGTCAACCCAGAGAGCCGCAGGTCTACACACTCCCTCCGTCCAGAGAAGAGATGACGAAAAACCAAGTTTCATTGACGTGCCTCGTTAAAGGATTCTACCCAAGCGACATAGCTGTTGAGTGGGAGAGCAGCGGCCAGCCTGAGAACAATTATAATACTACCCCCCCCATGCTCGACTCTGATGGTAGTTTTTTTCTGTACTCCAAGCTGACGGTAGACAAAAGTAGATGGCAGCAAGGCAACATCTTCAGTTGCTCTGTTATGCACGAGGCGTTGCACAACCGATTCACACAGAAGTCACTGAGCCTGTCTCCGGGTAAATGAAGCTTGAGCAGGGCCT.The IgA sequence is CGCATGATGCGTCGACTTCTCCAAAAGTGTTTCCCCTCAGTTTGTGTTCCACTCAACCGGATGGTAACGTGGTGATTGCTTGTCTCGTGCAAGGTTTTTTCCCACAGGAACCGCTGAGTGTTACATGGTCAGAGTCAGGCCAAGGTGTAACCGCGCGCAACTTTCCCCCTTCACAGGACGCTAGTGGCGATCTGTATACTACCTCCTCTCAGCTCACTCTTCCCGCCACACAATGCCTCGCTGGGAAATCTGTAACCTGCCACGTTAAACATTACACTAATCCATCACAGGACGTTACCGTGCCGTGCCCTGTACCATCCACGCCGCCTACGCCGTCACCGTCAACTCCTCCTACTCCCTCACCCTCTTGTTGTCACCCGCGCCTCTCTCTTCACAGACCGGCCTTGGAGGACCTTCTCCTTGGGTCTGAGGCGAATTTGACTTGCACGCTCACGGGGTTGCGGGACGCTAGTGGGGTTACGTTTACATGGACACCTTCATCAGGGAAGTCTGCCGTTCAGGGCCCCCCAGAGCGCGATTTGTGCGGGTGTTACAGCGTATCTTCTGTGCTGCCTGGGTGCGCTGAGCCCTGGAATCACGGCAAAACGTTTACCTGCACCGCTGCTTACCCAGAGAGCAAAACCCCTCTGACGGCTACATTGTCCAAGTCAGGCAACACATTTCGCCCCGAAGTCCACCTCTTGCCACCTCCATCCGAAGAACTCGCCCTGAACGAACTCGTGACGCTGACGTGCCTTGCACGCGGCTTTTCCCCGAAAGACGTTCTCGTCCGGTGGCTTCAAGGTTCTCAGGAACTCCCACGGGAGAAGTACCTGACCTGGGCTTCACGCCAGGAACCTTCACAAGGGACGACCACTTTCGCAGTCACGTCAATTCTGAGAGTTGCCGCTGAGGACTGGAAGAAGGGAGATACTTTCAGTTGTATGGTAGGTCACGAAGCACTGCCGCTGGCATTTACGCAGAAAACCATCGATCGGCTTGCCGGAAAGCCTACTCATGTTAACGTTTCCGTAGTGATGGCGGAGGTAGATGGCACATGTTACTGAAGCTTGAGCAGGGCCT.Serum samples were pretreated with receptor-destroying enzyme  followed by hemadsorption, in accordance with WHO recommended protocols . HAI titHeadless HA ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates  coated with 0.5\u2009\u03bcg/well of streptavidin . We completed total IgG headless HA ELISAs with all serum samples and isotype headless HA ELISAs with serum samples that had sufficient volumes. Biotinylated headless HA protein was diluted in biotinylation buffer containing 1\u00d7 Tris-buffered saline , 0.005% Tween , and 0.1% bovine serum albumin  to 0.25\u2009\u03bcg/ml, and 50\u2009\u03bcl was added per well and incubated on a rocker for 1 h at room temperature. Each well was then blocked for an additional 1 h at room temperature using biotinylation blocking buffer containing 1\u00d7 TBS , 0.005% Tween 20 , and 1% bovine serum albumin . Each serum sample was serially diluted in biotinylation buffer , added to the ELISA plates, and allowed to incubate for 1 h at room temperature on a rocker. As a control, we added the human CR9114 stalk-specific MAb, starting at 0.03\u2009\u03bcg/ml, to verify equal coating of plates and to determine relative serum titers. Peroxidase-conjugated goat anti-human IgG , peroxidase-conjugated mouse anti-human IgG1 , peroxidase-conjugated mouse anti-human IgG2 , peroxidase-conjugated mouse anti-human IgG3 , or peroxidase-conjugated goat anti-human IgA  was incubated for 1 h at room temperature on a rocker. Finally, SureBlue TMB peroxidase substrate  was added to each well, and the reaction was stopped with the addition of 250\u2009mM HCl solution. Plates were extensively washed with PBS  and 0.1% Tween 20  between each step using a BioTek 405 LS microplate washer. Relative titers were determined using a consistent concentration of the CR9114 MAb for each plate and reported as the corresponding inverse of the serum dilution that generated the equivalent optical densities (OD). Each type of ELISA  was performed twice.Chimeric HA ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates . HA proteins were diluted in PBS  to 2\u2009\u03bcg/ml and coated at 50\u2009\u03bcl per well overnight at 4\u00b0C. Plates were blocked using an ELISA buffer containing 3% goat serum , 0.5% milk , and 0.1% Tween 20  in PBS  1\u00d7 for 2 h at room temperature. Each serum sample was serially diluted in the ELISA buffer (starting at 1:100 dilutions), added to the ELISA plates, and allowed to incubate for 2 h at room temperature. As a control, we added the human CR9114 stalk-specific MAb, starting at 0.03\u2009\u03bcg/ml, to verify equal coating of plates and to determine relative serum titers. Peroxidase-conjugated goat anti-human IgG  next was incubated for 1 h at room temperature. Finally, SureBlue TMB peroxidase substrate  was added to each well, and the reaction was stopped with the addition of 250\u2009mM HCl solution. Plates were extensively washed with PBS  and 0.1% Tween 20  between each step using a BioTek 405 LS microplate washer. Relative titers were determined using a consistent concentration of the CR9114 MAb for each plate and reported as the corresponding inverse of the serum dilution that generated the equivalent OD. Each ELISA was performed twice.Competition ELISAs were performed on 96-well Immulon 4HBX flat-bottom microtiter plates . HA proteins were diluted in 1\u00d7 Dulbecco\u2019s PBS  to 2\u2009\u03bcg/ml and coated at 50\u2009\u03bcl per well overnight at 4\u00b0C. Plates were blocked using the biotinylation blocking buffer, described earlier, for 2 h at room temperature. Each serum sample was serially diluted in biotinylation buffer (starting at 1:10 dilution), added to the ELISA plates, and allowed to incubate for 1 h at room temperature before adding human 70-1F02 MAb  that haIn vitro neutralization assays were completed using a subset of samples. We excluded samples that had limited amounts of sera. Plasmids encoding pH1N1 viruses possessing genes encoding eGFP in place of most of the PB1 gene segment were provided by Jesse Bloom at The Fred Hutchinson Cancer Research Center. The eGFP segment retained the noncoding and 80 terminal coding nucleotides, allowing this segment to be efficiently and stably packaged into the virions. Detailed protocols for the reverse genetics, expression, and in vitro neutralization assays using the recombinant viruses have been published elsewhere  24 h before transfection. 293T cells were then transfected using 20\u2009\u03bcl Opti-MEM , 1\u2009\u03bcl Lipofectamine 2000 , and 500\u2009ng plasmids encoding the HA gene from A/California/07/09 per well and incubated at 37\u00b0C for approximately 30 h before performing the ADCC assay. Approximately 12 h before performing the ADCC assay, frozen PBMCs from four separate donors (obtained through the University of Pennsylvania Human Immunology Core) were thawed at 37\u00b0C and then washed 3\u00d7 using 15 ml of warmed complete RPMI medium  . Each aliquot of PBMCs was then transferred to a 50-ml conical tube and rested overnight in 23 ml of complete RPMI medium at a 5\u00b0 angle, with the cap loosened to allow for gas exchange. On the day of the assay, serum samples were diluted in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% FBS  at a 1:10 dilution. As a control for this assay, the human CR9114 HA stalk-specific MAb was included at a concentration of 5\u2009\u03bcg/ml to ensure efficient activation of ADCC. Transfected 293T cells were loosened by pipetting and transferred to a 96-well U-bottom plate  and spun down for 1 min at 1,200 rpm, and the medium was flicked out. The serum/MAb dilutions were transferred to the plates containing the transfected 293T cells and were mixed with the transfected cells by gentle pipetting and incubated at 37\u00b0C for 2 h. PBMC aliquots were combined, spun down, and counted, and a master mix of 2e7 cells/ml was set up using complete RPMI medium. Aliquots of the PBMC master mix were set up for the live/dead and unstained control wells. Phycoerythrin-conjugated mouse anti-human CD107a  was added at a 1:50 dilution. Brefeldin A  was added to 10\u2009\u03bcg/ml. Monensin  was added to 5\u2009\u03bcl per 1\u2009ml of PBMC master mix concentration. An aliquot of 200\u2009\u03bcl was made, phorbol myristate acetate  was added to a 5\u2009\u03bcg/ml concentration, and ionomycin  was added to a 1\u2009\u03bcg/ml concentration. Serum/cell suspensions were spun down at 1,200 rpm for 1 min, and medium was flicked out. The PBMC master mix and the aliquots for the various controls were plated at 50\u2009\u03bcl per well and mixed gently by pipetting, followed by incubation at 37\u00b0C for 4 h. Cells were then stained in the following manner. Live/dead fixable near-infrared stain  was diluted 1:50 in DPBS  and 1% bovine serum albumin  for 30 min in the dark at 4\u00b0C. Human FcR blocking reagent  was diluted 1:25 in DPBS  and 1% bovine serum albumin  and incubated in the dark for 10 min at 4\u00b0C. Alexa Fluor 647-conjugated mouse anti-human-CD3  and BV421-conjugated mouse anti-human CD56  were diluted 1:200 in DPBS  plus 1% bovine serum albumin  and incubated in the dark for 30 min at room temperature. Cells were then fixed using 10% paraformaldehyde  diluted in MilliQ water for 6 min at room temperature. Cells were extensively washed with DPBS  and 1% bovine serum albumin  between each step. Cells were stored overnight at 4\u00b0C in 100\u2009\u03bcl/well of DPBS  and 1% bovine serum albumin . Flow cytometry was performed using and LSRII . Compensation controls were set up using anti-mouse Ig\u03ba beads  and run for each antibody for every experiment, and voltages were adjusted accordingly. All data were analyzed in FlowJo  by gating on single cells that were CD3+/mFcgR alpha chain \u2212/\u2212) that were provided by Jeffrey Ravetch at the Rockefeller University (high (>40 HAI titer), uninfected HAIlow (\u226440 HAI titer), and infected HAIlow (\u226440 HAI titer), and then heat treated for 30 min at 55\u00b0C. Serum or sterile PBS was then transferred into mice by intraperitoneal injection. Four hours posttransfer, mice were bled by submandibular puncture, anesthetized using isoflurane, and challenged intranasally using 50\u2009\u03bcl of sterile PBS containing a sublethal dose (9e4 50% tissue culture infective dose units) of A/California/07/09. ELISAs were run on the sera collected from each animal to verify that the passive transfer was successful. Mice were weighed on the day on infection and then daily for 15 days postinfection. Weight loss was reported as percent weight loss relative to the starting weight of each mouse. For the passive transfer normalized by volume, two independent experiments were performed using a mix of male and female humanized FcR mice for a total of 6 mice per group per experiment. For the passive transfer normalized by HA antibody titer, a single experiment was performed using a mix of male and female humanized FcR mice for a total of 6 mice per group, since we had limited amounts of sera available for this study. One-way analysis of variance (ANOVA) was performed for each day postinfection between groups using GraphPad Prism software.All mouse experiments were reviewed and approved by the IACUCs of the Wistar Institute and University of Pennsylvania. All passive transfer experiments were performed in humanized FcR mice .Fisher\u2019s exact tests, one-way ANOVAs, and Welch\u2019s"}
+{"text": "Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100\u2013200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3'UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes . In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells. Via the target-directed miRNA degradation process, RNAs can induce degradation of miRNAs by binding with extensive complementarity. Here, the authors show Serpine1 mRNA as one such RNA that can control the levels of the endogenous miRNA miR-30b/c-5p by modulating miRNA degradation. Targets are bound through base paring between the miRNA and their miRNA responsive elements (MREs), usually located in the 3\u2032 untranslated region (3\u2032UTR)2. To act as such, any MRE usually presents complementarity to bases 2\u20137 (the seed) of miRNAs; however, other sequences, usually located near the miRNA 3\u2032 end, may also form additional base pairs and thus participate in target recognition. Due to the low levels of complementarity between miRNAs and their RNA targets, from hundreds to thousands RNAs could interact with the same miRNA sequence, as demonstrated by high-throughput experimental studies4. For the interaction with their targets to take place, miRNAs must be loaded onto Argonaute proteins (AGO) and form the core of the RNA-induced silencing complex (RISC). Within RISC, miRNAs induce silencing by target destabilisation and/or translational repression6. Computational methods, such as TargetScan7 and others8, are able to predict miRNA targets and their MREs based on seed type hierarchy (8-mer\u2009>\u20097-mer\u2013m8\u2009>\u20097-mer\u2013A1\u2009>\u20096-mer) and on sequence conservation of orthologous mRNAs as found by comparative genome analysis. Usually, target expression changes slightly when miRNA levels are perturbed10; however, the resulting phenotypic effect can be profound as targets often converge towards the same pathway or biological process.MicroRNAs (miRNAs) are an evolutionarily conserved class of small (about 18\u201322 nt long) non-coding RNAs that function in post-transcriptional regulation of gene expression11 and the target-directed miRNA degradation (TDMD) mechanism12. The ceRNA theory postulates that endogenous targets compete with each other for binding to a shared miRNA; therefore, a sudden change in the expression of a competing endogenous target  might influence miRNA activity on other targets13. Most evidence in favour of the ceRNA hypothesis comes from over-expression approaches, so that the impact of ceRNAs on miRNA-mediated mechanisms in physiological settings is still debated16. In the TDMD mechanism, the RNA target (the TDMD target) promotes degradation of its miRNA18, accompanied by post-transcriptional modification of the miRNA sequence, i.e., tailing (addition of nucleotides at the 3\u2032 end) and trimming (shortening)19, and unloading from AGO20. Studies performed using artificial targets showed that extended complementarity to miRNAs 3\u2032 regions combined with a central bulge of\u2009\u2264\u20095 nt, promotes miRNA degradation21. However, TDMD molecular basis and physiological role are still obscure. Endogenous RNA targets implicated in TDMD and the role they play in modulating miRNA activity need to be further investigated, especially in non-neuronal cells. So far, the evidence for accelerated miRNA decay comes from studies on viral targets  and on artificial transcripts, both characterised either by a central bulge or by perfect complementarity24. Indeed, it has been shown that, in physiological conditions, miRNA decay can be accelerated by a rapid change in gene expression , suggesting the existence of a post-transcriptional mechanism able to control miRNA levels. However, precise molecular details remain obscure. We and others have recently shed light on the dynamics of miRNA decay in mammalian cells by using new tailored approaches based on in vivo RNA labelling28. In our study, different pools of miRNAs were identified on the basis of their decay pattern: \u201cslow\u201d miRNAs, very stable (T1/2\u2009>\u200924\u2009h), and apparently downregulated through dilution by cell division; and \u201cfast\u201d miRNAs, quickly turned over in the cell (T1/2 from 4 to 14\u2009h). Intriguingly, we found a significant association between miRNA type of decay (\u201cfast\u201d or \u201cslow\u201d) and number and expression levels of targets bearing an additional complementarity to miRNA 3\u2032 ends (3C-targets27), as required for TDMD. On these bases, we hypothesised that TDMD is a general mechanism, with specific endogenous targets able to control the dynamics of degradation of their miRNAs.Intriguingly, target:miRNA interactions have been suggested to act as a bidirectional control mechanism, with targets in turn affecting miRNAs activity. Two mechanisms have been reported: the competing endogenous RNA (ceRNA) hypothesisIn this study, we provide evidence that the endogenous target Serpine1 is able to control the levels of the two miRNAs miR-30b-5p and -30c-5p by inducing their degradation during cell cycle re-entry of quiescent fibroblasts stimulated with serum. Following an in-depth investigation of the interaction between Serpine1 and miR-30b/c, we describe TDMD as a post-transcriptional mechanism that controls levels and activity of miRNAs.29) on top of the canonical seed pairing  low, with 3C-scores between \u22120.01 and -0.03; (ii) mid, scoring between \u22120.03 and \u22120.05; and (iii) high, with 3C-score lower than \u22120.05 , 18,856 pairs were found, with 1083 falling in the 3C-high class ; and (b) the max contribution (over the entire time course) of each single target to the total pool of 3C-targets of a given miRNA . A few 3C-targets stood out in the analysis , a very high 3C-score for miR-30b-5p and -30c-5p, and a significant contribution to the 3C-target pool .Using the TargetScan database as a source of miRNA:target pairs, we identified endogenous RNAs that could trigger miRNA degradation and focused our analysis on targets presenting a 3\u2032 pairing contribution . Using the IsomiRage tool to map isomiRs32, we found that 3\u2032 tailing of both miR-30b/c had substantially increased, mirroring their downregulation patterns  belong to two different genetic units and, therefore, are not co-transcribed   and 30b-5p  . To this aim, a titration curve was obtained using synthetic miR-30 RNA oligonucleotides and a Serpine1 plasmid DNA template  Fig.\u00a0, suggest80) Fig.\u00a0. To rule80) Fig.\u00a0. In MRE-80) Fig.\u00a0. We also80) Fig.\u00a0, with a 80) Fig.\u00a0. In MRE-80) Fig.\u00a0. While t80) Fig.\u00a0. The lev80) Fig.\u00a0, further18, four mismatches in the 3\u2032 end were sufficient to nearly abolish TDMD  fused to Serpine1 3\u2032UTR Fig.\u00a0. We used15. Conversely, TDMD suppression (as in MRE-KO cells) should result in increased miRNA activity. Using a highly sensitive reporter system to measure miR-30c activity at single-cell level , we isolated miR-30 targets based on: (i) predictions by computational approach ; (ii) experimentally supported interactions (n\u2009=\u20091037 for miR-30\u2009s) obtained by High-throughput Sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP-from TarBase7.035); and (iii) miRNA:target chimaeras (n\u2009=\u20091037 for miR-30c-5p), obtained by sequencing of Covalent Ligation of Endogenous Argonaute-bound RNAs  but significantly more repressed in mutant cells  and IV [genes repressed in mutant cells at late (8h-10h-12h) time-points] were significantly enriched with miR-30 direct targets  and of the circadian clock , which in turn is coupled with the cell cycle40. In summary, the Serpine1:miR-30b/c interaction profoundly affects cell behaviour by increasing sensitivity to apoptosis and by accelerating the G1/S transition during cell cycle re-entry of quiescent cells.We next investigated whether the increased miR-30 activity observed when the Serpine1:miR-30b/c interaction is lost, might also cause phenotypical alterations Fig.\u00a0. Previoulls Fig.\u00a0, it appells Fig.\u00a0. Analysills Fig.\u00a0. BrdU inlls Fig.\u00a0, suggestlls Fig.\u00a0. Throughlls Fig.\u00a0, which mlls Fig.\u00a0. In partlls Fig.\u00a0. Notablyets Fig.\u00a0. In part18. Finally, it only affects two members (miR-30b and 30c) of the conserved miR-30 family that are not co-transcribed, thus coupling them post-transcriptionally while uncoupling them from the other three miR-30 members (miR-30a/d/e). All miR-30s can interact with Serpine1 via their seed sequence , but only miR-30b/c present an extended 3\u2032 end complementarity (3C pairing) to Serpine1 transcript. Therefore, as previously observed in vitro and with artificial targets20, 3C pairing is essential in the case of a TDMD mechanism triggered by endogenous targets. Accordingly, Serpine1 has stronger effects on miR-30c than on miR-30b, as miR-30c forms additional 3\u2032 base pairings with Serpine1. The importance of 3C pairing is highlighted also by the fact that it is required in reconstitution experiments. Additional mismatches can be tolerated close to the bulge, but completely impair miRNA degradation if located in proximity of miRNA 3\u2032 end. Complementarity is not the only requisite for TDMD, which also presents some context-dependent quantitative requirements. Therefore, we performed absolute measurements of miRNA and target expression (copies per cells) and computed the corresponding target-per miRNA value (TPM) to investigate the stoichiometry of their interaction. Upon serum stimulation of quiescent cells, Serpine1 is strongly induced so to largely exceed the levels of miR-30b/c (TPM\u2009>\u200910) and very rapidly halve their levels . No changes in transcription or processing are concomitantly observed  and in range with. the number of miRNA copies (about 70\u201380\u2009cpc for miR-30c and 140 for miR-30b). Nevertheless, miR-30b and miR-30c are subjected to TDMD also in growing cells, as in MRE-KO cells the levels of the two miRNAs almost doubled. Therefore, endogenous TDMD takes place even for targets that are present at relatively low levels of expression, provided that they are in a stoichiometric range with their cognate miRNAs (TPM\u2009\u2265\u20091). The reason being that every time the miRNA interacts with the target, it is irreversibly subtracted from the miRNA pool by degradation16. It is worth mentioning that in reconstitution experiments, where targets are provided ectopically, TDMD requirements differ slightly as Serpine1-MRE acts mostly on miR-30c-5p and, in order to do so, must reach higher levels of expression (at least ~1000\u2009cpc). In these experiments, miRNA levels were analysed at 36\u2009h post infection. It is conceivable that a longer time interval is necessary to observe a significant reduction in miR-30b abundance, as this is expressed at relatively higher levels than miR-30c. It should be also stressed that, at low MOI, some cells were uninfected and did not ectopically express Serpine1-MRE (as determined by monitoring RFP). This might explain why ectopically expressed Serpine1 triggered TDMD at higher levels (at least ~1000 cpc) when compared to endogenously expressed Serpine1 (170\u2009cpc).Here, we provide molecular evidence that in mammalian cells a target-directed miRNA degradation mechanism exists and can control miRNA activity and cell functions. We found hundreds of targets that are potentially capable of TDMD. Focusing our study on Serpine1, we provide a proof-of-principle characterisation of TDMD at endogenous levels of expression, as opposed to acute expression of artificial targets or ectopic over-expression. Indeed, we show that under physiological conditions  Serpine1 post-transcriptionally regulates two closely related miRNAs, miR-30b, and miR-30c, by inducing their degradation. Degradation promoted by Serpine1 is characterised by some peculiar features. It involves specific post-transcriptional modifications at the miRNA 3\u2032 end, with higher-than-normal levels of adenylation but no uridylation involved, as previously reported17. In their study, the NREP transcript promotes degradation of miR-29b in neuronal cells. The NREP:miR-29b and Serpine1:miR-30b/c pairs share the same complementarity features. Significantly, NREP is almost exclusively expressed in the brain (where TDMD is very effective) and imparts a spatial restriction to miR-29b expression in the cerebellum. Loss of the NREP:miR-29b interaction generates an in vivo phenotype characterised by impaired motor functions in fish and mice. A relevant role for TDMD in animal behaviour has thus been claimed. However, the impact of NREP on shared miR-29 targets was not investigated. Endogenous targets involved in TDMD are expected to influence miRNA-mediated repression. By investigating the Serpine1:miR-30 case, we provide compelling evidence in favour of such hypothesis. MRE-KO cells show a significant increase in miR-30 activity, as confirmed using both a synthetic reporter directly monitoring miRNA activity (miR-sensor) and endogenous targets identified by prediction algorithms or experimental approaches. In growing conditions (at steady-state), effects on endogenous targets are mild (with a ~5% increase in repression) and no major changes in phenotype are observed. Gene expression alterations observed in this context are likely to be a consequence of clone adaptation to the new (increased) levels of miR-30b/c. On the contrary, MRE-KO cells show remarkable phenotypes when exposed to acute stresses. In particular, upon serum stimulation mutant cells fail to properly regulate miR-30b/c levels, enter more quickly into S-phase and display an increased mitotic and cell death rate. In this context, strong (~20%) and specific repression of miR-30 targets is observed, with a certain degree of specificity towards miR-30b/c preferential targets (as opposed to shared miR-30 targets). Indeed, two independent studies mapping miRNA:target interactions suggest that miR-30b/c preferentially bind those targets that possess supplementary pairing in the 3\u2032 region37. In future, it would be worth trying to identify such specific targets and characterise them in serum-stimulated cells , as they might help clarify miR-30b/c function in cell cycle re-entry. In conclusion, we have provided evidence that in mammalian cells endogenous targets can control miRNA levels post-transcriptionally and thus modulate miRNA activity on all their targets.How many endogenous TDMD targets do exist? We chose to study Serpine1 as it was one of the top hits in a list of candidate targets displaying ideal structural characteristics: a high-affinity seed (8-mer or 7-mer-m8), a central bulge (\u2009>\u20092 and\u2009<\u20095\u2009nts) and a high 3C-score (high degree of complementarity at the 3\u2032 end). Based on our preliminary observations, we can anticipate that at least 1000 target:miRNA pairs  present molecular characteristics that make them suitable for TDMD. While this work was under revision, a different case of endogenous TDMD was provided by Bitetti and co-workers17) or impart temporal-restriction to miRNA activity in dynamic settings (regulation in time\u2014as for Serpine1:miR-30b/c during cell cycle re-entry).We can no longer consider miRNA targets as being all equal. The way has now been opened to the identification of new endogenous TDMD targets that might be involved in different physiological or pathological processes. TDMD confers a further level of flexibility to miRNA regulation. It can restrict miRNA expression to a specific subset of cells  and statistics were produced using JMP 12 (SAS) software. Microsoft Excel was used to generate bar graphs with average and standard deviation (s.d.) or standard error mean (s.e.m.) of repeated experiments. The number of replicates and the statistical tests used are indicated in the figure legends.Mouse 3T9 fibroblasts a kind gift of Bruno Amati. Cells were cultured in DMEM (Dulbecco\u2019s modified Eagle\u2019s medium) supplemented with 10% fetal bovine serum (FBS), 100\u2009U/ml penicillin and 100\u2009mg/ml streptomycin. Cells were routinely checked for mycoplasma contamination and resulted negative. 3T9 cells were induced to quiescence in a serum-deprived medium (0.1% FBS) for 3 days. Cell cycle re-entry was stimulated by adding fresh medium with 10% FBS. For cell cycle analysis, cells were treated with 33\u2009\u00b5M 5-bromo-2\u2032-deoxyuridine (BrdU) for 30\u2009min, harvested by trypsinisation, and ethanol-fixed. Staining was performed with anti-BrdU primary antibody  and anti-mouse FITC-conjugated secondary antibody . DNA was stained with overnight incubation with 2.5\u2009\u00b5g/ml propidium iodide (Sigma). Samples were acquired on a FACS-CALIBUR (BD Biosciences) flow cytometer and analysed using FlowJo 10 software. Live time-lapse analysis of mitosis and apoptosis during cell cycle re-entry was performed with a high-content screening station  equipped with a microscope incubation chamber, imaging cells in bright field with a 20\u2009\u00d7\u2009/NA 0.45 objective (Nikon Eclipse TE2000-E inverted microscope) every 15\u2009min starting at 12\u2009h after serum stimulation and reconstructed with ImageJ software. For each sample, mitosis and cell deaths were manually annotated at each time frame: ~200 cells were followed (in three independent replicas) and data reported as number of mitosis (or cell deaths) occurring every 100 cells.Cells were grown in normal conditions or under starvation, and treated or not with 1\u2009\u03bcM of doxorubicin (Sigma) for 48\u2009h to induce apoptosis. After treatment, adherent as well as detached cells were harvested and labelled with Annexin-V-APC  for 45\u2009min. Next, cells were labelled with propidium iodide  and immediately analysed by MACSQuant Flow cytometer (Miltenyi). Data analysis was performed by FlowJo software (TreeStar).Cells were plated in 96-well plate and analysed at 24, 48, 72, and 96\u2009h by Cell Titre GLO (Promega) following the manufacturer\u2019s protocol., and RNA was extracted with the miRNeasy Micro Kit  and then eluted in 14\u2009\u03bcl of RNase-free water. 4sU-RNA quantification was conducted with Qubit\u00ae (Life Technologies).Starting from 40\u2009\u00b5g of total RNA diluted in 100\u2009\u00b5l of RNase-free water, 100\u2009\u00b5l of biotinylation buffer  and 50\u2009\u00b5l of EZ-link biotin-HPDP  were added and incubated for 3\u2009h at room temperature (RT). Unbound biotin-HPDP was removed by adding chloroform/isoamyl alcohol (24:1) and purifying the mix using MaXtract high-density tubes (Qiagen). RNA was precipitated by adding NaCl (5\u2009M) at 1/10 volume and isopropanol at equal volume. Biotinylated RNA was resuspended in water , quantified with a Nanodrop 1000 spectrophotometer (to keep track of the yield after the first phase of the procedure) and then purified using 50\u2009\u00b5l of Dynabeads MyOne Streptavidin T1 (Invitrogen). Before mixing with RNA, beads were washed twice in wash buffer A  and once in wash buffer B (100\u2009mM NaCl) and then resuspended in 100\u2009\u00b5l of buffer C  to a final concentration of 5\u2009\u03bcg/\u03bcl. RNA was added in an equal volume and rotated at RT for 15\u2009min. Beads were washed three times with wash buffer D . RNA was eluted from the beads in 100\u2009\u00b5l of 10\u2009mM EDTA in 95% formamide . The eluted fraction was diluted in TRIzol\u00ae LS Reagent IsomiRage workflow, as previously described32. Raw data together with detailed description of the procedures are available in GEO database (GSE104650). Normalisation was performed with the library size (reads-per-million) and then (only for the serum stimulation experiments) further corrected for serum-induced RNA amplification as previously reported26.Total RNA, including small species, was isolated through the miRNeasy Mini Kit (Qiagen). When the expected yield was\u2009<\u20091\u2009\u00b5g, the miRNeasy Micro Kit (Qiagen) was used. Small RNA sequencing (sRNA-seq) libraries were prepared using 1000\u2009ng of total RNA with the Illumina TruSeq\u2122 Small RNA Kit, following the manufacturer\u2019s instructions. Sequencing was performed on an Illumina HiSeq 2000 at 50\u2009bp single-read mode and 20 million read depth (8\u00d7). Sequencing quality was checked in the FASTQC report, considering only experiments with Q30 or above . Analysis was performed with the q value relative to the control was lower than 0.05 and whose maximum expression was higher than RPKM of 1. For the identification of genes differentially expressed in MRE-KO cells upon serum stimulation  and then 1000\u2009ng were purified with Ribozero rRNA removal kit (Illumina). Libraries were generated with the TruSeq RNA Library Prep Kit v2 (Illumina). Next, sequencing was performed on an Illumina HiSeq 2000 at 50\u2009bp single-read mode and 50 million read depth (3\u00d7). Reads were aligned to the mm9 mouse reference genome using the TopHat aligner (version 2.0.6) with default parameters. Differentially expressed genes . In particular, miR-30c-5p and miR-30b-5p were detected using miScript Primer Assay MS00001386 and MS00011725. Primary transcripts of miRNAs (pri-miRNAs) were evaluated by quantitative PCR on total RNA. RT-qPCR was performed using SuperScript\u00ae VILO cDNA Synthesis Kit (Life Technologies cat.no. 11754050) and Fast SYBR green master mix (Life Technologies). About 25\u2009ng of cDNA were used as the input to detect pri-miRNAs, and 5\u2009ng to detect mRNAs. Primer pairs were designed using computer-assisted primer design software (Primer3), preferentially in the 500\u2009bp upstream of the sequence of the mature miRNA according to mm10 genome. The complete list of RT-qPCR primers used in this study is in Supplementary Table\u00a0Absolute quantification of miR-30c was performed by droplet-digital PCR on a QX200 system (Bio-Rad), following the manufacturers\u2019 instructions. Briefly, 10\u2009ng of RNA was reverse-transcribed with miRCURY LNA Universal RT microRNA PCR kit (Exiqon). Then, ddPCR reactions were prepared replacing Exiqon PCR master mix with QX200 EvaGreen ddPCR Supermix (Bio-Rad) and diluting 1:4 Exiqon PCR primer mix for miR-30c-5p (cat. 204783). Serial dilutions of cDNA (0.4/0.2/0.1/0.05\u2009ng) were used as to measure assay linearity. Droplets were generated and PCR amplification performed following EvaGreen cycling conditions. Finally, we read the droplets on a QX200 droplet reader. We typically obtained ~20,000 droplets for each sample.Mouse miRNA targets were downloaded from TargetScan 6.2 . 3C-targets were selected according to criteria outlined in Fig.\u00a034. SgRNAs were subsequently cloned in PX458 vector and sequence-verified. 3T9 fibroblasts were co-transfected with two PX458 vectors (sgRNA_1 and sgRNA_2) harbouring single sgRNAs sequence (5\u2009\u00b5g of DNA for each construct). At 24\u2009h and 48\u2009h post-transfection, cells were harvested for FACS sorting. The top 2%\u20133% of GFP\u2009+\u2009cells were sorted as individual cells into 96-well plates. After 2 weeks, 96-well plates were duplicated and deletions in clonal cells were screened by PCR , using the CRISPR Design ToolgAAGCAAGCTGTGTCAAGGGAsgRNA_1(-) Sense: cacccAntisense: aaacTCCCTTGACACAGCTTGCTTgTCTCCCAGTGGGGGGGCCCTsgRNA_2 (\u2009+\u2009) Sense: cacccAntisense: aaacAGGGCCCCCCCACTGGGAGANote that the efficient transcription from the U6 promoter requires a 5\u2032 g.Serpine1 protein expression in MRE-KO clones was analysed by western blot .KpnI-NotI sites at the extremities. After homologous recombination in BJ5183-AD-1 cells (Agilent), positive recombinants (isolated by PacI digestion) were used to produce adenoviruses in 239Ad cells. Viruses were finally concentrated with Adeno-X maxi purification kit (Takara).In order to re-express miR-30 MRE, recombinant adenoviruses were produced using the AdEasy System (Agilent), following manufacturers\u2019 instructions. Briefly, gene synthesis (GenScript) generated a mCherry-Serpine1 (3\u2032UTR) construct (see below for the complete sequence) that was cloned into pShuttle vector (Agilent) by adding mCherry-Serpine 3\u2032UTR construct (underlined with a solid line the miR-30 MRE):ACGCGTAGAGTGTAGGTGACTTGTTTACAGAGCTCCAGCTTTTTTCGACCCACAAACTTTTTTCATTTGGAAAGGGTGTAAGAAAAGTCGGACGTGTGTGTGCCTGGCTCTTCGTCCCCAGTCTCCCAGTGGGGGGGCCCTGGGGAGATTCCAGGGGTGTGATTGAATATTTATCTCTTGCTCTTGTATGTTTGTTGGGGAGAAGAAGCACTTTTAAGGAAAATGCTTCTTATTTAAACCGTGGCATACGGCATCCCATTTGGGGTCTGCATCCCTGTATGTCAGGGGTGCATCACTCCACAAACCTGCCCCTCTGGGTAGCCTCGTGATGGGGCTCACACTGCCGCCTAGTGGCAGCCGAACACACCCTTACCCGGTCCCTCCCTCCCTCCCCCCCCCCCCCCCCCCCCCCCGTGGCTCTTTTCCTTAGGGACCTTGCCAAGGTGATGCTTGGCAACCCACGTTAAAGGAAGGGGGGAAAAAAGATTAGATGGAAGAGAGAGAGATTTGAGAGAGGGCAAAGTGGTTTCAAATTTTTCCAAGGCATTCAGAAGCAGAGAGGGAAAAGGGGCTGTGTGACCTAACAGGACAGAACTTTCTCCAATTACTGGGTGAGTCAGAGCTGCACTGGTGACTCACTTCAATGTGTCATTTCCGGCTGCTGTATGTGAGCAGTGGACACGTGGGGGGGCGGGGGGGGGATGAAAGAGACAGCAGCTCCTGGTCAACCACCTTAGTTAGATAATCTTTTTTGAAAGCTTCCTAGCTGGAGGTATGATCAGAAAACCAATTTACTGAAAAACTGCACAAGAAGGTACGGTGAATGTAATTTCCTAGCAGGCCACTCTGCATCTGTTATGTCTCCACCGGAAAAAAAATAATCATGTTGGTGTTTTTGCTTTTCTCTCTCTCCCTCTTTCTCTCTGATTTTTTTTTCCTCTCTTTTCATTATGCACTGGACAGCCACACACCGTGTACCCATAGGGCCCCAAATGTGGGGTCACATGGTCTTGAATTTTGTTGGTTACATATGCCTTTTTGTTGTTGTTTGTCTTCACTTTTGATATATAAACAGGTAAATATGTTTTTTAAAAAATACTAAATATAGAGAATATGCAAACAAAAGCGGCCGC-3\u20325\u2032-GGTACCCGCCACCATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGCTCGAGCAGTGGGAAGAGACGCCTTCATTTGGGACGAAACTGGAGATGTTATAAGCAGAAACTCTGAAGAAAAGGATTATTTAAAGGACTCTATGGGGAGAAAGAGAAGGCAACTCCTCCTTACCCCCCACACTGGTAATCTTTCCAACCAGCATCCCAGACCTCGGACTCTTGAAGGGAAAAGAGTCTAACTCCCTCCTCCCTAGGGATTCCTACCCCACAAAGGTCTCATGGACCATAGAACTCACAGTACCTGGATCTGCCCAGCATGCCCTTTGGACCCAGTTCCCACCGAGGCCCCAGCAGAGTGGAGGGCACAACACTTTCATTCAGCAAAATCGTTTGTGTTCCAGTCACACTGTGGGCACCTCTTGCATCGCCTGCCATTGCTGTGGAGGGTGGCCATGGGCCAAAGGAAAAAGCACTGTCCTATCTCAAGGTCCACTGTGGAAATGTCCACCTTGCCCACCTCCAAGGGGCAACGGATAGACAGATCAAATGGTGGCCCAATAGCGAGCCTTCTCCCTGCTCCCTCCCTTGACACAGCTTGCTTATGTTATTTCKpnIGGTACC MluIACGCGT SacIGAGCTC Not1GCGGCCGC In order to produce other adenoviral constructs, the following oligonucleotides were subcloned into MluI-SacI sites flanking the miR-30 MRE:Ad-RFP-SE1(MUT)FW: cgcgtACTCTGAAAGTGAAAAGCCAATGgagctREV: cCATTGGCTTTTCACTTTCAGAGTaAd-RFP-SE1(SEED)FW: cgcgtGATATTGTCCTGACTTGTTTACAgagctREV: cTGTAAACAAGTCAGGACAATATCamiR-30_MRE in MRE-KO cells, we cloned MRE\u2009+\u2009~20 surrounding bases (Supplementary Table\u00a0EcoRI-XhoI sites that were put after the stop codon of a eGFP cloned in a pcDNA3.1(\u2009+\u2009) mammalian expression vector (Thermo Fisher Scientific).In order to re-express different variants of Serpine1Vectors were transfected in MRE-KO cells with Lipofectamine 3000 reagent (Thermo Fisher Scientific). Cells  were collected for analysis after 48\u2009h.XbaI/XmaI) into the 3\u2032UTR of a destabilised GFP (dGFP) in a bidirectional lentiviral vector (BdLV_1370), which also expresses \u0394NGFR. The vector . Data analysis was performed by FlowJo software (TreeStar): fluorescent levels of \u0394NGFR and dGFP for each cell were log transformed and divided in 100 bins (each of 1000 cells) based on \u0394NGFR level. The mean \u0394NGFR or dGFP expression was calculated in each bin and repression folds were calculated, at similar \u0394NGFR mean expression, as the ratio: [dGFP(miR-30)/[dGFP(control)].Sequences harbouring four complementary repeats for miR-30c-5p or a control sequence (see below) were purchased from TwinHelix and cloned (miR-30c-5p sensor sequence:TCTAGATAAGCTGAGAGTGTAGGATGTTTACACGATGCTGAGAGTGTAGGATGTTTACAACGCGTGTCGACGCTGAGAGTGTAGGATGTTTACATCACGCTGAGAGTGTAGGATGTTTACACCCGGGControl (scrambled) sensor sequenceFW TCTAGAGGAGCTCCACCGCGGTGGCATCREV CCGGGATGCCACCGCGGTGGAGCTCCThttp://www.ncbi. nlm.nih.gov/geo/) under the accession number GSE104650. The 'serum stimulation data set', which contains miRNA expression analysis of WT cells, was previously submitted under accession number GSE72655. Data within the manuscript is available from the authors upon reasonable request.The small RNA sequencing (sRNA-seq) and the RNA-sequencing data set for this study have been submitted to the NCBI Gene Expression Omnibus (GEO; Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Data 1Supplementary Data 2Supplementary Data 3"}
+{"text": "EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the  Accumulating evidence indicates an ever-expanding role for RNAs in regulating most aspects of cell biology that range from small non-coding RNAs to messenger RNAs (mRNAs). The more traditional role of mRNAs as \u201conly messengers\u201d is changing and new knowledge is emerging showing how the encoding sequences are taking on more diverse functions and can influence the activity of the encoded proteins. Most, if not all aspects of mRNAs, and this is presumably true also for non-coding RNAs, are governed by interactions with cellular proteins. These ribonucleoproteins (RNP) complexes control RNA metabolism and form scaffolds to orchestrate and organize protein networks and complex functional units . Thus, iEBNA1 mRNA (EBNA1 mRNA translation and thereby the production of EBNA1-derived antigenic peptides for the major histocompatibility (MHC) class I pathway, allowing EBV-infected cells to evade the immune system [EBNA1 mRNA-NCL interaction represents an interesting target for developing drugs that aim to induce an immune response against EBV-related diseases. It also serves as a broader model for developing techniques required to study structured RNA-protein interactions. Here, we use the NCL-EBNA1 mRNA interaction to illustrate how different compounds binding to the same G4 have specific effects on the interaction with NCL in cellulo. This illustrates a so far unknown role of G4 structures in mediating specific interactions with proteins, indicating that particular G4-protein interactions can be targeted specifically. We also report how to generate quantitative data using proximity ligation to verify the capacity of different G4 ligands to prevent NCL-EBNA1 mRNA interactions and their role in controlling EBNA1 mRNA translation.G-quadruplexes (G4) are non-canonical nucleic acid structures based on the stacking of several G-quartets, further stabilized by cations positioned in the central channel of the G4 helix ,8. Thesee system . As EBV l-glutamine, 100 U/mL penicillin, and 100 \u00b5g/mL streptomycin. H1299 transient transfections were performed using Genejuice reagent  according to manufacturer\u2019s protocol. All cells were cultured at 37 \u00b0C with 5% CO2. For drug treatments, 105 B95-8 cells were incubated with 5 \u00b5M of PhenDC3 [The human lung carcinoma cell line H1299 and the EBV-producing marmoset B-cell line B95-8 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM v/v) ethanol at 4 \u00b0C. After rehydration in PBS for 30 min, samples were permeabilized with PBS 0.4% Triton X-100, 0.05% CHAPS for 10 min at room temperature, and pre-treated with hybridization buffer  for 30 min at room temperature. Samples were then incubated overnight with 50 ng of an EBNA1-digoxigenin DNA probe (5\u2032 CTTTCCAAACCACCCTCCTTTTTTGCGCCTGCCTCCATCAAAAA-digoxigenin 3\u2032) in a humidified chamber at 37 \u00b0C. To avoid secondary structures, the probe was diluted in 5 \u00b5L of water, denaturated at 80 \u00b0C for 5 min, chilled on ice for 5 min, and resuspended in 35 \u00b5L of hybridization buffer. After hybridization, samples were serially washed for 20 min with 2X SSC 10% formamide, hybridization buffer (twice), 2X SSC, and PBS. Washes were carried out at room temperature, except with hybridization buffer (37 \u00b0C). Samples were saturated with PBS 3% BSA for 30 min and incubated with a mouse anti-digoxigenin  for 2 h at room temperature. A goat anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to Alexa Fluor\u00ae 568 (Sigma) was used to detect immunocomplexes (1 h at 37 \u00b0C) and DAPI was used for nuclear counterstaining under standard conditions. For IF, fixed samples were saturated with PBS 3% BSA for 30 min, incubated with rabbit polyclonal antibody anti-NCL  for 2 h and goat anti-rabbit Ig secondary antibody conjugated to Alexa Fluor\u00ae 488 (Sigma) for 1 h at 37 \u00b0C. DAPI was used for nuclear counterstaining.H1299 cells were plated on 12-mm-diameter coverslips in 24-well plates and transfected with 200 ng of EBNA1 construct . At 24 hThe sequence of EBNA1 cDNA (GenBank: MG021311.1) is as follows (the GAr-encoding sequence which forms G4 is highlighted in cyan):GGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGAGGAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGGGCAGGAGGGGCAGGAGCAGGAGGAGGGGCAGGAGCAGGAGGGGCAGGAGCAGGAGGTGGAGGCCGGGGTCGAGGAGGCAGTGGAGGCCGGGGTCGAGGAGGTAGTGGAGGCCGGGGTCGAGGAGGTAGTGGAGGCCGCCGGGGTAGAGGACGTGAAAGAGCCAGGGGGGGAAGTCGTGAAAGAGCCAGGGGGAGAGGTCGTGGACGTGGTGAAAAGAGGCCCAGGAGTCCCAGTAGTCAGTCATCATCATCCGGGTCTCCACCGCGCAGGCCCCCTCCAGGTAGAAGGCCATTTTTCCACCCTGTAGGGGAAGCCGATTATTTTGAATACCACCAAGAAGGTGGCCCAGATGGTGAGCCTGACATGCCCCCGGGAGCGATAGAGCAGGGCCCCGCAGATGACCCAGGAGAAGGCCCAAGCACTGGACCCCGGGGTCAGGGTGATGGAGGCAGGCGCAAAAAAGGAGGGTGGTTTGGAAAGCATCGTGGTCAAGGAGGTTCCAACCAGAAATTTGAGAACATTGCAGAAGGTTTAAGAACTCTCCTGGCTAGGTGTCACGTAGAAAGGACTACCGATGAAGGAACTTGGGTCGCCGGTGTGTTCGTATATGGAGGTAGTAAGACCTCCCTTTACAACCTCAGGCGAGGAATTGCCCTTGCTATTCCACAATGTCGTCTTACACCATTGAGTCGTCTCCCCTTTGGAATGGCCCCTGGACCCGGCCCACAACCTGGCCCACTAAGGGAGTCCATTGTCTGTTATTTCATTGTCTTTTTACAAACTCATATATTTGCTGAGGGTTTGAAGGATGCGATTAAGGACCTTGTTATGCCAAAGCCCGCTCCTACCTGCAATATCAAGGCGACTGTGTGCAGCTTTGACGATGGAGTAGATTTGCCTCCCTGGTTTCCACCTATGGTGGAAGGGGCTGCCGCGGAGGGTGATGACGGAGATGACGGAGATGAAGGAGGTGATGGAGATGAGGGTGAGGAAGGGCAGGAGTGAATGTCTGACGAGGGACCAGGTACAGGACCTGGAAATGGCCTAGGACAGAAGGAAGACACATCTGGACCAGACGGCTCCAGCGGCAGTGGACCTCAAAGAAGAGGGGGGGATAACCATGGACGAGGACGGGGAAGAGGACGAGGACGAGGAGGCGGAAGACCAGGAGCTCCGGGCGGCTCAGGATCAGGGCCAAGACATAGAGATGGTGTCCGGAGACCCCAAAAACGTCCAAGTTGCATTGGCTGCAAAGGGGCCCACGGTGGAACAl-lysine 0.01% (Sigma) for 30 min and air-dried for 5 min in 24-well plates. B-cells were then resuspended in PBS, plated on pre-treated coverslips and incubated for 2 h at room temperature. After a wash with PBS for 5 min, cells were fixed with PBS 4% paraformaldehyde for 20 min and re-washed with PBS for 10 min. Following fixation, both cell lines were processed for in situ hybridization according to the rISH-IF protocol. Samples were then saturated with PBS 3% BSA for 30 min and incubated for 2 h at room temperature with a mix of primary antibody containing the mouse anti-digoxigenin and the rabbit anti-nucleolin previously described. Subsequently, PLA was carried out using the Duolink PLA in situ kit (Sigma), anti-rabbit plus and anti-mouse minus probes (Sigma) following the manufacturer\u2019s protocol. H1299 cells were plated and fixed as previously described. For experiments using B95-8, 12-mm-diameter coverslips were coated with poly-Samples were examined with an LSM 800 confocal laser microscope . ImageJ  was usedEBNA1 mRNA translation and, as a consequence, by minimizing the production of antigenic peptides for the major histocompatibility (MHC) class I pathway [EBNA1 mRNA and this is essential for suppressing the translation of the EBNA1 mRNA and the production of antigenic peptides  was originally conceived to detect proteins in close proximity. For this, one pair of primary antibodies raised in two different species is used to target the two proteins of interest C. AfterwEBNA1 mRNA interactions in situ using either transfected cells or EBV-infected cells. PLA complexes are depicted as white dots and each dot represents an interaction between NCL and EBNA1 mRNA. In line with rISH-IF and IF results, interactions were uncovered in or at the close vicinity of the nuclear compartment of EBNA1-transfected cells  \u2248 0.47 \u00b5M); DC50 (PhenDC3) \u2248 0.26 \u00b5M. This was further confirmed by the thermal stabilization values (\u0394Tm) measured by the FRET-melting assay on EBNA1 G4 , and anti-G4 antibodies [EBNA1 mRNA [An estimated 2\u20133% of cancers are associated with EBV, making GAr-based EBNA1 immune evasion an important target for therapeutic approaches against EBV-related cancers ,21. TherBNA mRNA , we treaeraction A. This iin vitro ,22. In ain vitro , while Pin vitro . Altogeteraction C. It has 20.5 \u00b0C . Althoug 20.5 \u00b0C . Howevertibodies , whereasNA1 mRNA . All theProtein-mRNA interactions influence multiple aspects of cellular function. However, in spite of progress experienced over the last decades, the study of these interactions has been restricted to assays technically demanding and unable to provide accurate information at subcellular levels. In this context, PLA represents an attractive alternative to overcome these limitations, opening new avenues for the study of protein-mRNA interactions in cellulo. We provided evidences that PLA can be coupled to quantitative analysis and be successfully employed to screen molecules interfering with target protein-mRNA interactions. Additionally, the approach described here can be adapted to other technologies, like flow cytometry, for medium to high throughput drug screening. This highlights the potential of mRNA-protein PLA as a tool for efforts focused not only on targeting specific G4 structures, but more generally for drug development based on disruption of specific protein-RNA structure complexes."}
+{"text": "The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10 Specifications tableValue of the data\u2022The finding that PoBChE is abundant in porcine milk \u2022The set of polyproline-rich peptides in PoBChE tetramers is different from the set in human BChE tetramers \u2022Are excess polyproline-rich peptides toxic to cells? Does BChE incorporate polyproline peptides from degraded cells because degraded cells are a convenient source? If excess polyproline-rich peptides are toxic to cells, BChE might be protecting the organism by scavenging these peptides.\u2022The BChE tetramer incorporates not only short polyproline-rich peptides, but also long proteins that contain a polyproline-rich region. An example is the C5 variant of human BChE whose tetrameric structure includes a 60\u202fkDa lamellipodin protein \u2022The ability of BChE subunits to assemble into stable, long-lived tetramers by binding the polyproline-rich region of a protein, suggests that BChE could serve as a delivery vehicle for any protein that has been engineered to include a polyproline-rich tag.1We present all the polyproline-rich peptides associated with PoBChE tetramers that were identified by mass spectrometry. In addition we show the complete amino acid sequence of the full-length protein that donated each polyproline-rich peptide, the location of the polyproline-rich peptide within the full-length protein sequence, and the abundance of the polyproline-rich peptide relative to PoBChE peptides. A brief description is given of the function of the donor protein and its location within a cell.22.1PoBChE was purified from defatted porcine milk (1200\u202fmL) by affinity chromatography on procainamide-Sepharose 2.2The Hupresin-purified PoBChE was reduced in volume from 7.3\u202fmL to 0.17\u202fmL in a Centricon YM-30 spin filter. After the buffer was changed to 10\u202fmM ammonium bicarbonate pH 8, the PoBChE was deglycosylated with 1\u202f\u00b5l of PNGaseF for 1\u202fh. Noncovalently bound polyproline-rich peptides were released from PoBChE by denaturing the protein in a boiling water bath for 5\u202fmin. The denatured 170\u202f\u00b5g of PoBChE protein in 170\u202f\u00b5L of 10\u202fmM ammonium bicarbonate pH 8 was digested with 2\u202f\u00b5g of trypsin  for 20\u202fh at 37\u202f\u00b0C in a humidified chamber. Particles that could clog the small diameter tubing in the Ultra High Pressure Liquid Chromatography column were removed by centrifuging the digest for 30\u202fmin at 14,000\u202frpm in a microfuge. A 10\u202f\u00b5L aliquot from the top of the centrifuged digest was transferred to an autosampler vial. The protein concentration in the digest was estimated at 1\u202f\u00b5g/\u00b5L.2.3Sus Scrofa proteins using the Paragon algorithm from Protein Pilot (AB Sciex) (The protocol for liquid chromatography tandem mass spectrometry (LC\u2013MS/MS) is described in detail in B Sciex) .Table 1AImage 1VSAKPPQALSFAKRLQQLIEALKGTAFSSGRSYYETETTDLQELPAS.>sp|P08001|ACRO_PIG Acrosin OS=Sus scrofa GN=ACR PE=1 SV=5 MLPTAVLLVLAVSVAARDNATCDGPCGLRFRQKLESGMRVVGGMSAEPGAWPWMVSLQIFMYHNNRRYHTCGGILLNSHWVLTAAHCFKNKKKVTDWRLIFGANEVVWGSNKPVKPPLQERFVEEIIIHEKYVSGLEINDIALIKITPPVPCGPFIGPGCLPQFKAGPPRAPQTCWVTGWGYLKEKGPRTSPTLQEARVALIDLELCNSTRWYNGRIRSTNVCAGYPRGKIDTCQGDSGGPLMCRDRAENTFVVVGITSWGVGCARAKRPGVYTSTWPYLNWIASKIGSNALQMVQLGTPPRPSTPAPPVRPPSVQTPVRPPWYFQRPPGPSQQPGSRPRP27 residues PAPPPAPPPPPPPPPPPPPPPPPPPQQ MW 2648.4.https://www.gtexportal.org/home/gene/ACR The human acrosin gene is expressed in testis and to a small extent in mammary breast tissue, lung, spleen, adipose tissue, and tibial artery.Acrosin is the major proteinase present in the acrosome of mature spermatozoa. It is a typical serine proteinase with trypsin-like specificity. It is stored in the acrosome in its precursor form, proacrosin. The active enzyme functions in the lysis of the zona pellucida, thus facilitating penetration of the sperm through the innermost glycoprotein layers of the ovum. The mRNA for proacrosin is synthesized only in the postmeiotic stages of spermatogenesis. In humans proacrosin first appears in the haploid spermatids. Image 2SPRAPAPPPSGALLPEPGQRCEAVSSSPPPPPCAQNPLHPSPSHSACKEPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDHKLPNTKIRSGGPASAAGGPPGRPNGGPPAL.>XP_003131596.1 PREDICTED: homeobox protein Hox-B4 [Sus scrofa] MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESSFQPEAGFGRRAACTVQRYAAC21 residues RDPGPPPPPPPPPPPPPPPGL MW 2069.1.The HOXB4 gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on human chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. Intracellular or ectopic expression of this protein expands hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion.Image 3SPPFQLTKPGLWSTLHGDAWGPERKGTAPPERQEQRHSLPRHPYPYPAPAYPSRTPWPRLVPAAPPGPGPXPPGAESHGCPPATRPPGSDLRESRVQRSRMDSSVSPAATTACVPYAPSRPPALPGTTTSSSSSSSNTGLRGVEPSPGIPGADHYQTPALEVSSHQGRLGPSAHSSRKPFLAAPAATPHLSLPPGPPSPPPPPCPRLLRPPPPPAWLKGPACRAAREDGEILEELFFGAEGRPRPPPPPLPHREGFLGPPAPRFSVGTQDSHTPPTPPTTSSSSSNNGSHSSSPTGSVSFPPPPYLARSMDPLPRPPSPTLSPQDPPLAPLSLALPPAPPSSCHQNTSGSFRRPESPRPRVSFPKTPEVGPGPSPGPLNKAPQPVPSRVGELPARGPRLFDFPPTPLEDQFEEPAEFKILPDGLANIMKMLDESIRKEEEQQQQEAGVVPPPPLKEPFASLQPPFPTDTAPATTAATTAATTTATQEEEKKPPPALPPPPPLAKFPPPPQPQPPPPPLPPPASPASLLKSLASVLEGQKYCYRGTGAAVATRPGPLPTTQYSPGPPSGATAPPPTSAAPSAQGSPQPSASSSSQFSTSGGPWARERRAGEEPAPGPTTPAPPPPPLPLPPARSESEVLEEISRACETLVERVGRGATDPADPADTADPVDTGAERLLPPAQAKEEAGGASAVAAAAAGPGSSKRRQKEHQKEHRRHRRACKDSVGRRPREGRAKAKAKAPKEKSRRVLGNLDLQSEEIQGREKARPDLGGASKAKPPTAPAPLPAPAPSTQSTPPSAPVPGKKAREEAPGPPGVSRADMLKLRSLSEGPPKELKIRLIKVESGDKETFIASEVEERRLRMADLTISHCAADVVRASKNAKVKGKFRESYLSPAQSVKPKINTEEKLPREKLNPPTPSIYLESKRDAFSPVLLQFCTDPRNPITVIRGLAGSLRLNLGLFSTKTLVEASGEHTVEVRTQVQQPSDENWDLTGTRQIWPCESSRSHTTIAKYAQYQASSFQESLQEEKESEDEESEEPDSTTETPPSSAPDPKNHHIIKFGTNIDLSDAKRWKPQLQELLKLPAFMRVTSTGNMLSHVGHTILGMNTVQLYMKVPGSRTPGHQENNNFCSVNINIGPGDCEWFAVHEHYWETISAFCDRHGVDYLTGSWWPILDDLYASNIPVYRFVQRPGDLVWINAGTVHWVQATGWCNNIAWNVGPLTAYQYQLALERYEWNEVKNVKSIVPMIHVSWNVARTVKISDPDLFKMIKFCLLQSMKHCQVQRESLVRAGKKIAYQGRVKDEPAYYCNECDVEVFNILFVTSENGSRNTYLVHCEACARRRSAGLQGVVVLEQYRTEELAQAYDAFTLAPASTSR.>XP_005657086.1 PREDICTED: LOW QUALITY PROTEIN: lysine-specific demethylase 6B [Sus scrofa] MHRAVDPPGARTAREAFALGGLSCAGAWSSCPPHPPPRSAWLPGGRCSASIGQPPLSAPLPPSHGSSSGHPNKLYFAPGTPNPRPLHGKLESLHGCVQALLREPAQPGLWEQLGQLYESEHDSEEAIRCYHSALRYGGSLAELGPRIGRLQQAQLWNFHAGSCQHRPKVLPPLEQVWNLLHLEHKRNYGAKRGGPPVKRAAEPPVVQPVPPAALSGPSGEEGLSPGGKRRRGCNSEQTGLPPGL28 residues PLPPPPLPPPPPPPPPPPPPPPLPGLAT MW 2737.5.Histone demethylase specifically demethylates \u05f3Lys-27\u05f3 of histone H3, thereby playing a central role in histone code . Demethylates trimethylated and dimethylated H3 \u05f3Lys-27\u05f3 . Plays a central role in regulation of posterior development, by regulating HOX gene expression (PubMed:17851529). Involved in inflammatory response by participating in macrophage differentiation in case of inflammation by regulating gene expression and macrophage differentiation (PubMed:17825402). Plays a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression by acting as a link between T-box factors and the SMARCA4-containing SWI/SNF remodeling complex.Image 4PPRVQLPVSLDLPLFPSIMMQPVQHPALPPQLALQLPQMDTLSADLTQLCQQQLGLDPNFLRHSQFKRPRTRITDDQLKILRAYFDINNSPSEEQIQEMAEKSGLSQKVIKHWFRNTLFKERQRNKDSPYNFSNPPITVLEDIRIDPQPSSLEHYKSDASFSKRSSRTRFTDYQLRVLQDFFDTNAYPKDDEIEQLSTVLNLPTRVIVVWFQNARQKARKSYENQAEAKDNEKRELTNERYIRTSNMQYQCKKCNVVFPRIFDLITHQKKQCYKDEDDDAQDESQTEDSMDATDQVVYKHCTVSGQTEAAKNAPVAAASSGSGASTPLLPSPKPEPEKTSPKPEYPTEKPKQSDPSPPSQGTKPALPLASTSSEPPQAAAAQPQPQPPKQPQLIGRPPSASQTPIPSSPLQISMTSLQNSLPPQLLQYQCDQCTVAFPTLELWQEHQHMHFLAAQNQFLHSPFLERPMDMPYMIFDPNNPLMTGQLLSGFLTQMPPQNASSQTPASATVAASLKGNWDDKEDTNCSEKEGGNSGEDQHRDKRXRTTITPDKLEILYEKYLLDSNPTRKMLDHIAREVGLKKRVVQVWFQNTRARERKGQFRAVGPAQSHKRCPFCRALFKAKSALESHIRSRHWNEGKQAGYSLPPSPLIATEDGGESPQKYIYFDYPSLPLTKIDLSSENELASTVSTPVSKTAELSPKNLLSPSSFKAECSEDVENLNAPPAEAGYDQNKPDFDETSSINTAISDATTGDEGNAEMESTTGSSGDVKPALSPKEPKTLDTLAKTATTPTTEVCDEKFLFSLTSPSIPFNDKDGDHDQSFYITDDPDDNADRSETSSIADPSSPNPFGSSNPFKSKSNDRPGHKRFRTQMSNLQLKVLKACFSDYRTPTMQECEMLGNEIGLPKRVVQVWFQNARAKEKKFKINIGKPFMINQSGTEGTKPECTLCGVKYSARLSIRDHIFSKQHISKVRETVGSQLDREKDYLAPTTVRQLMAQQELDRIKKASDVLGLAVQQPSMMDSSSLHGISLPAAYPGLPGLPPVLLPGMNGPSSLPGFPQNSNTLTPPGAGMLGFPTSATSSPALSLSSAPTKPLLQImage 5SGQQTEPQNKESEKKQTKPNKVKKIKEEELEATKPEKHPKKEEKISSALSVLGKVVGETHVDPSQLQALQNAIAGDPASFLGGQFLPYFIPGFASYFTPQLPGTVQGGYLPPVCGMESLFPYGPTMPQTLAGLSPGALLQQYQQYQQNLQDSLQKQQKQQQQEQPQKPGQAKTSKGESEPPQNASDASETKEDKSTATESTKEEPQLESKSADFSDTYVVPFVKYEFICRKCQMMFTDEDAAVNHQKSFCYFGQPLIDPQETVLRVPVSRYQCLACDVAISGNEALSQHLQSSLHKEKTIKQAMRNAKEHVRLLPHSVCSPNPNTTSTSQSAASSNTYPHLSCFSMKSWPNILFQASARRAASSPSSPPSLSLPSTVTSSLCSTSGVQTSLPTESCSDESDSELSQKLEDLDNSLEVKAKPASGLDGNFNSIRMDMFSV.>XP_005663076.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger homeobox protein 4 [Sus scrofa] METCDSPPISRQENGQSTSKLCGTAQLDNEVPEKVAGMEPDRENSSTDDNLKTDERKSEVLLGFSVENAAATQVTSAKEIPCNECATSFPSLQKYMEHHCPNARLPVLKDDNESEISELEDSDVENLTGEIVYQPDGSAYIIEDSKESGQNAQTGANSKLFSTAMFLDSLASAGEKSDQSASAPMSFYPQIINTFHIASSLGKPFTADQAFPNTSALAGVGPVLHSFRVYDLRHKREKDYLTSDGSAKNSCVSKDVPNNVDLSKFDGCVSDGKRKPVLMCFLCKLSFGYIRSFVTHAVHDHRMTLNEEEQKLLSNKCVSAIIQGIGKDKEPLISFLEPKKSTSVYPHFSTTNLIGPDPTFRGLWSAFHVENGDSLPAGFAFLKGSAGTSGSAEQPLGITQMPKAEVTLGGLSSLVVNTPITSVSLSNASSESSKMSESKDQENDCERPKESNALHPNGECPVKSEPTEAGDEDEEDAYSNELEDEEVLGELTDSIGNKDFPLLNQSISPLSSSVLKFIEKGPSSSSASVTDDAEKKKPTAAVRASGGVANSYGIGGKDFAEASASKDGATAAHSSEPARGDEDSSATPHQHGFTPSAPGTPGPGGDGSPGSGIECPKCDTVLGSSRSLGGHMTMMHSRNSCKTLKCPKCNWHYKYQQTLEAHMKEKHPEPGGSCVYCKTGQPHPRLARGESYTCGYKPFRCEVCNYSTTTKGNLSIHMQSDKHLNNVQNLQNGNGEQVFGHSAPAPNTSLSGCGTPSPSKPKQKPTWRCEVCDYETNVARNLRIHMTSEKHMHNMMLLQQNMKQIQHNLHLGLAPAEAELYQYYLAQNIGLTGMKLENPGDPQLMLNPFQLDPATAAALAPGLVNNELPPEIRLASGQLMGDDLSLLTAGELSPYISDPALKLFQCAVCNKFTSDSLEALSVHVSSERSLPEEEWRAVIGDIYQCKLCNYNTQLKANFQLHCKTDKHMQKYQLVAHIKEGGKSNEWRLKCIAIGNPVHLKCNACDYYTNSVDKLRLHTTNHRHEAALKLYKHLQKQEGAVNPESCYYYCAVCDYSTKVKLNLVQHVRSVKHQQTEGLRKLQLHQQGLAPEEDNLSEIFFVKDCPPNELETASLGARTCEDDLLEQQLRAPSEEQSEETEGASRPTAVAEDDEKDTSERDNNEGKNSNKDTGIITPEKELKVSVAGGTQPLLLAKEEDVATKRSKPTEDSKFCHEQFYQCPYCNYNSRDQSRIQMHVLSQHSVQPVICCPLCQDVLSNKMHLQLHLTHLHSVSPDCVEKLLMTVPVPDVMMPNSLLLPAAASEKSERDTPAAITAEGPGKYSGESPMDDKSMAGLDDSKAIMEIKSEEQKPTKEPTEASEWNKNSSKDGKISDPLQDQLSEQQKRQPLSVSDRHVYKYRCNHCSLAFKTMQKLQIHSQYHAIRAATMCNLCQRSFRTFQALKKHLEAGHPELSEAELQQLYASLPVNGELWAESETMAQDDHALEQEMEREYEVDHEGKASPVGSDSSSIPDDMGSEPKRTLPFRKGPNFTMEKFLDPSRPYKCTVCKESFTQKNILLVHYNSVSHLHKLKKVLQEASSPVPQETNSSTDNKPYKCSICNVAYSQSSTLEIHMRSVLHQTKARAAKLEPSSHVVSGHSAANVSSPGQGMLDSMSLAGVSSKDTHLDAKELNKKQTPELISAQPAHHPPQSPAQIQMQLQHELQQQAAFFQPQFLNPAFLPHFPMTPEALLQFQQPQFLFPFYIPGTEFSLGPDLGLPGSAAFGMPGMTGMAGSLLEDLKQQIQTQHHVGQTQLQILQQQAQQYQATQPQLQSQKPQQQPQPQPQQQQASKLLKQEQTTLASAECPIVKDIPSFKEAEEMAKKQDKPKQEVXSEGEGLKEGKDEKKQKSSEPSILPPRIASGARGNAAKALLENFGFELVIQYNENRQKVQKKGKSGEGESTEKLECGTCGKLFSNVLILKSHQEHVHGQFFPYGALEKFARQYREAYDKLYPISPSSPETPPPPPPPPPLPPAPPQPASLGPVKLPSTVSTPIQAPPP22 residues TPPPPPPPPPPPPPPPPPPPSA MW 2121.1.18 residues TPPPPPPPPPPPPPPSSL MW 1764.9.May play a role in neural and muscle differentiation. May be involved in transcriptional regulation.Image 6PMPVHEPLSPQQLQQQQDMYNKKIPSLFEIVVRPTGQLAEKLGVRFPGPGGPPGPMGPGPNMGPPGPMGGPMHPDMHPDMHPDMHPDMHPDMPMGPGMNPGPPMGPGGPPMMPYGPGDSPHSGMMPPIPPAQNFYENFYQQQEGMEMEPGLIGDTEDYGHYEELPGEPGEHLFPEHPLEPDSFSEGGPPGRPKPGAGVPDFLPSAQRALYLRIQQKQQEEEERARRLAESSKQDRENEEGDPGNWYSSDEDEGGSSVTSILKTLRQQTSSRPQASGGELSSSGLGDPRLQKGHPTGGRLADPRLSRDPRLSRHAEASGGSGPGDTGPSDPRLARSLPTPRPKGGLHSSPGGPSGSKGSGPPPAEEEEGERALREKAVNIPLDPLPGHPLRDPRSQLQQFSHIKKDVTLSKPSFARTVLWNPEDLIPLPIPKQDAVPPVPVALQSMPALDPRLHRTTTTSGPPNPRQRPGTSTDPSASGSNLPDFELLSRILKTVNATGPSAAPGPGDKPSDPRVRKTPTDPRLQKPADSATSSRAAKPGSTEVSPSASPSGESSPPATAPYDPRVLAAGGLGQGSGSGQSSVLSGISLYDPRTPNAGGKATEPAADTGTQPKGPEGNGKSAATKAKEPPFVRKSALEQPESGKPGADGGAAAATDRYNSYNRPRPKATPAAAASGTPPPEGASPQPGVHNLPVPTLFGTVKQAPKTGSGSPFAGNSPAREGEQDAGSLKDVFKGFDPTASPFCQ.>XP_005664683.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger CCCH domain-containing protein 4 [Sus scrofa] MEAAPGTPPPPPSESPPPPSPPLPSTPSPPPCSPDACPATPHLLHHRLPLPDDREDGELEEGELEDDGAEETQDTSGGPERSRKEKGDKHHSDSDEEKSHRRLKRKRKKEREKEKRRSKKRRKSKHKRHASSSDDFSDFSDDSDFSPSEKGHRKYREYSPPYAPSHQQYPPSHTTPLPKKAYSKMDSKGYSMYEDYENEQYGEYEGDEEEDMGKDDYDDFTKELNQYRRAKEGSSRGRGSRGRGRGYRGRGSRGGSRGRGMGRGSRGRGRGSMGGDHPEDDEDFYEEEMEYGESEEPMGDEDYDDYSKELNQYRRSKDGRGRGLSRGRGRGSRGRGKGMGRGRGRGGSRGGMNKGGMNEDDDFYDEDMGDGGGGGGSYRRSDHDKPHQQSDKKGKVICKYFVEGRCTWGDHCNFSHDIELPKKRELCKFYITGFCARAENCPYMHGDFPCKLYHTTGNCINGDDCMFSHDPLTEETRELLDKMLADDAEAGAEDEKEVEELKKQGINPLPKPPPGVGLLPTPPRPPGPPAPTSPNGRPLQ19 residues GGPPPPPPPPPPPPGPPQM MW 1806.9.NCBI and UniProt have ZC3H4 zinc finger CCCH-type containing protein 4 (Homo sapiens).This gene encodes a member of a family of CCCH (C-x8-C-x5-C-x3-H type) zinc finger domain-containing proteins. These zinc finger domains, which coordinate zinc finger binding and are characterized by three cysteine residues and one histidine residue, are nucleic acid-binding. Other family members are known to function in post-transcriptional regulation.Image 7GRTPPTLLSTLQYPRPSSGTLASASPDWAGPGARLRQQSSSSKGDSPELKPRAVHKQGPSPVSPNALDRTAAWLLTMNAQLLEDEALGPDPPHRDRLRSKEELSQAEKDLAVLQDKLRISTKKLEEYETLFKCQEETTQKLVLEYQARLEEGEERLRRQQEDKDIQMKGIISRLMSVEEELKKDHAEMQAAVDSKQKIIDAQEKRIASLDAANARLMSALTQLKERYSMQARNGISPTNPTKLQITENGEFRNSSNC.>XP_003353684.3 PREDICTED: LOW QUALITY PROTEIN: disabled homolog 2-interacting protein-like isoform 1 [Sus scrofa] MSAGGSARKSTGRPSYYYRLLRRPRLQRQRSRSRSRTRPARESPQERPGSRRSLPGSLSEKSPSMEPSAATPFRVTGFLSRRLKGSIKRTKSQPKLDRNHSFRHILPGFRSAAAAAAAASAADNERSHLMPRLKESRSHESLLSPSSAVEALALSMEEEVVIKPVHSSILGQDYCFEVTTSSGSKCFSCRSAAERDKWMENLRRAVHPNKDNSRRVEHILKLWVIEAKDLPAKKKYLCELCLDDVLYARTTGKLKTDNVFWGEHFEFHNLPPLRTVTVHLYRETDKKKKKERNSYLGLVSLPAASVAGRQFVEKWYPVVTPNPKGGKGPGPMIRIKARYQTITILPMEMYKEFAEHITNHYLGLCAALEPILSAKTKEEMASALVHILQSTGKVKDFLTDLMMSEVDRCGENEHLIFRENTLATKAIEEYLKLVGQKYLQDALGEFIKALYESDENCEVDPSKCSAADLPEHQGNLKMCCELAFCKIINSYCVFPRELKEVFASWRQECSSRGRPDISERLISASLFLRFLCPAIMSPSLFHLLQEYPDDRTARTLTLIAKVTQNLANFAKFGSKEEYMSFMNQFLEHEWTNMQRFLLEISNPETISNTAGFEGYIDLGRELSSLHSLLWEAVSQLEQSIVSKLGPLPRILRDVHTALSTPGSGQLTGTNDLASTPGSGSSSISAGLQKMVIENDLSGLIDFTRLPSPTPENKDLFFVTRSSGVQPSPARSSSYSEANEPDLQMANGGKSLSMVDLQDARALDGEAGSPAGPDALAADGQVPTAQLVAGWPARAAPVSLAGLATVRRAGQTPTTPGTSEGAPGRPQLLAPLSFQNPVYQMAAGLPLSPRGLGDSGSEGHSSLSSHSNSEELAAAAKLGSFSS;ATAAAAEDLGRRPGELARRQMSLTEKGGQPTVPRQNSAGPQRR16 residues IDQPPPPPPPPPPAPR MW 1668.9.Functions as a scaffold protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways.Image 8LALSEDTEPSSSESRTGLCSPEDNSLTPLLDEVAAPEGRAATVPRGRGRSRGDSSRSSASELRRDSLTSPEDELGAEVGDEAGDKKSPWQRREERPLMVFNVK.>XP_005668832.1 PREDICTED: protein FAM171A2 [Sus scrofa] MPPPSGPSVLARLLPLLGLLLGGASRAPGKSPPEPPSPQEILIKVQVYVSGELVPLARASVDVFGNRMLLAAGTTDSEGVATLPLSYRLGTWVLVTAARPGFLTNSVPWRVDKLPLYASVSLYLLPERPATLILYEDLVHILLGSPGARSQPWVQFQRRAARLPVSSTYSQLWASLTPASTQQEMRAFPAFLGTDASSSGNGSWLELMPVAAVSVHLLAGNGTEVPLSGPIHLSLPVPSEPRALAVGTSIPAWRFDPKSGLWVRNGTGVIRKEGRQLYWTFISPQLGYWAAAMASPTSGLVTITSGIQDIGTYHTIFLLTILAALALLVLILLCLLIYYCRRRCLKPRQQHRKLQLSGPSDGNKRDQATSMSQLHLICGGPLEPAASGDPEAPPPGPLHSAFSSSRDLAASRDDFFRAKPRSASRPAAEPAGARGGEGAGLKGARSVEGPGGLEPGLEEYRRGPPGTATFLQEPPSPPPPFEHYLGHKGAAESKTPDFLLSQSVDQLERPPSLSQAGQLIFCGSIDHHSQVRHSYIDLQAGGGGRSTDASLDSGVDVHEARPARRRPLREERERAA17 residues AAAPPPPPPPPPPAPPR MW 1622.9.Image 9CPYSGAGFPPAPPPPPPPPLPGGPPVPPPPPGLPPPSHLNGYSHLGKKKRMRSFFWKTIPEEQVRGKTNIWTLAARQQHHYQIDTKTIEELFGQQEESAKSSPSRRGGPLNSSFREAREEITILDAKRSMNIGIFLKQFKKSPPSIVEDIHQGKSEHYGSETLREFLKLLPESEEIKKLKAFSGDVAKLSLADSFLHCLIQVPNYSLRIEAMVLKKEFLPSCSSLYTDMTILRTATKELMSCEELHSILHLVLQAGNIMNAGGYAGNAVGFKLSSLLKLADTKANKPGMNLLHFVAQEAQKKDAVLLNFSEKLHHVQEAARLSLDNTEAELHSLFVRTRSLKENIQRDGELCQQMEDFLQFAVEELSELERWKQELLAEAHTLIDFFCEDKDTVKLDECLQIFRDFCIKFNKAVKDNHDREVQELKQLQRLKEQEQKRRSWAAGELGFSRSSSENDVELLTKRGAEDPFLHSRPISPSHRPPNTRRSRLSLGASADRELLTFLESSTGNPEELKFNSLPRSCPRQAPPSRAWMESGEQRDQDSSQAHRLPASKDQEEATDPPSTWQSQLLAPRLEEPATALPRVRRSGVSILRKRNSEPLGLGPVRSPPLSPLALGIKEHELVTGLAQFDLQAPKGPEEPARLTMNDFSPMELMSVVGESPQAPRAPNDHRCEGLIPPCFSNEDLGNILLYVRAHAASRPYRESRAPSRSSFRKPSVKPLRNVPRPKPDEDKMCRSSSQGPESPEEAPRAPAAPSAPRGPAPVPSFARNTVASSSRCLRTDSPAVARPPGLTRTVSQRQLRAKGGPEEAAPKDGGALRRASSARGPRKGPELPEGPRAGSEASPKGRGAGERASVRLKDASRPALGKGLHPLRK.>XP_005666867.1 PREDICTED: FH2 domain-containing protein 1 [Sus scrofa] MHVMNCVSSVSDKGNGNIAPAAGFMIGQT14 residues PPPPSPPPPPPPPP MW 1366.7.Formin-homology-domain-containing protein FHOD1 is involved in cell migration and adhesion, acting as a regulator of stress fibers organization, maturation of integrin-based adhesion sites, and podosome-associated contractility.Image 10GGLTSPIFCSTKPKKLLKTSSFHLLRRRDPPFQTPKKLYAQEYEFEADEDKADVPADIRLNPRRLPDLVSSCRSRPALSPLGDIDFCPPNPGPDGPRRRGRKPTKAKRDGPPRPRGRPRIRPLEGPATAGPALASTPTDGAKKPRGRGRGRGRKAEEAGGTRLEPLKPLKIKLSVPKAGEGLGASSGEAVSGADPNSLDSSLTREKIEAKIKEVEEKQPEMKSGFMASFLDFLKSGKRHPPLYQAGLTPPLSPPKSVPPSVPARGLQPQPPSTPAVPHPPPAGAFGLGGALEAAESEGLGLGCPSPCKRLDEELKRNLETLPSFSSDEEDSVAKNRDLQESISSAISALDDPPLAGPKDTSTPDGPPLAADAAVPGPPPLPGLPSASSNGTPEPPLLEEKPPPSPPPVPTPQPPPPPPALPSPPPLVAPAPSSPPPQImage 11PPAPAPAPAPPALPSPPAPPPPPAAAAAPPPEEPAAPSPDDSEPPDARPLHLAKKQETAAVCGETDEEAGESGGEGIFRERDEFVIRAEDIPSLKLALQTGREPPPIWRVQKALLQKFTPEIKDGQRQFCATSNYLGYFGDAKNRYQRLYVKFLENVNKKDYVRVCARKPWHRPPVPVRRSGQAKGPSSSGGSSAPPPKAPAPPPKPETPDKMASEKPPEQTPETAVPEPPAPEKPSPPRLVEKEKEKERTPRGERPLRGERGTGGRQIRPDRGLTTGQPATSRLPKSRPTKVKAEPPPKKRKKWLKEAAGNASAGGGPPGSSSDSESSPGAPSEDERAVPGRLLKTRAMREMYRSYVEMLVSTALDPDMIQALEDTHDELYLPPMRKIDGLLNEHKKKVLKRLSLSPALQDALHTFPQLQVEQSGEGSPEEGAVRLRPAGEPYNRKTLSKLKRSVVRAQEFKVELDKSGYYTLYHSLHHYKYHTFLRCRDQTLAIEGGAEDLGQEEVVQQCMRNQPWLEQLFDSFSDLLAQAQAHSRCG.>XP_003127395.2 PREDICTED: proline-rich protein 12 isoform X1[Sus scrofa] MDRNYPSAGFGDPLGAGAGWSYERSAKASLVYGSSRTSHPETDILHRQAYAAPHPLQSYATNHHPAGLSGLFDTGLHHAGSAGPDASVMNLISALESRGPQPGPSASSLLSQFRSPSWQTAMHTPGPTELFISGALPGSSTFPSSSALSAYQHPASFGSRPFPVPSSLSLQDPPFSPPANGLLSPHDVLHLKPSQAPTVPSSLGFERLAGGGVLGPAGLGPAQTPPYRPGPPDPPPPPRHLPTQFNLLASSSAAAAAAAEQSSPQLYNFSGAAPGPPPPERALPRQDTVIKHYQRPASAQPPPPPPPAHALQHYLSCGGSYPSMGHRANLACSPLGGGEPSPGAGEPSKAGPSGATAGASGRAAGPEAAGGGGAGGGGGGYRPIIQSPGYKTGKGGYGAAAGGANRPPPPRSTATPKCQSLGGPAAAYATGKASGAGGAGGQAYSPGQPQGLLGPQAYGQGFGGGQAQDLSKGPSYSGGPQQPPNGPPPPGLATCQSYSPDQLQGQLYGVQGEPYPGPAAHSQGLPTASPSLSYSTGHSPALSGHGGGWGPSSLGGGGEASPSHIIRPLQSPPAPGRPPGVGSPGAPGKYLSSVLASAPFLAPPGAGSYAAGAGGYKGKGDGSELLAGPGGPPAERTEDEEFLIQHLLQAPSPPRTSGADGLVGEDGAADASKGLGGSGGAGGPPGTPYELAKEDPQRYHLQSVIRTSASLDEGATAALELGLGRLKEKKKGPERGGETPEGLATSVVHYGAGAKELGAFLQKSPPPPPPTAQSAQPTPHGLLLEAGGPDLPLVLPPPPPQLLPSVLSHAPSPSSSAPKVGVHLLEPAARDGAPPPPPPPPPPPMPLQLEAHLRSHGLEPGAPSPRLRPEESLEPPGAMQELLGALEPLPPGPGDTGVGPPTAEGKDPSGAYRSPSPQGTKAPRFVPLTSICFPDSLLQDEERSFFPTMEEMFGGGPADDYGKAGPPEDEGDPKAGAGPPPGPPAYDPYGPYCSSRASGAGPETPGLGLDPSKPPELPSTVNAEPLGLIQSGPHQA19 residues APPPPPPPPPPPPPASEPK MW 1863.0.20 residues LPPPPPPPPPPPPPPPPPPP MW 1975.1.Image 12NQANTELPKLSRDEQRGRGALLQDICKGTKLKKVTNINDRSAPILEKPKGSSGGYGPGAAALQPKGGLFQGGVPKLRPVGAKDGSENLAGKPALQVPSSRAAAPRPPVSTASGRPQDDTDSNRASLPELPRTQRPSLPDLSRPHATSSTGMKHSSSAPPPPPPGRRANAPPTPLAMHSNKAPAYNREKPLPPTPGQRLHPGREGPSAPPPVKPPPSPVNIRTGPSGQSLAPPPPPYRQPPGVPNGPSSPTNESAPELPQRHNSLHRKTPGPVRGLAPPPPTSASPSLQSNRPPPPARDPPSRGAAPPPPPPMIRNGARDAPPPPPPYRMHGSEPLSRGKPPPPPSRTPAGPPPPPPPPLRNGHRDSITTVRSFLDDFESKYSFHPVEDFPAPEEYKHFQRVYPSKTNRAARGAPPLPPILR.>NP_001231241.1 WAS/WASL-interacting protein family member2 Sus scrofa 19 residues MPIPPPPPPPPGPPPPPTF MW 1926.0.Plays an active role in the formation of cell surface protrusions downstream of activated PDGFB receptors. Plays an important role in actin-microspike formation through cooperation with WASL. May cooperate with WASP and WASL to induce mobilization and reorganization of the actin filament system.Image 13LTPVKCEDPQRVVPTVNPVKTNGTLLRNGGFPGAPNKIPNGDICCKPGSIVDKAPVQPLMHRPEKDRCPQAGPRERVRFNEKVQYHGYCPDCDTRYNIKNREVHLHSEPVHPPGKLPPQGPHHPPPPHLPPFPLENGGLGISHSNSFPPLRPATVPPPTAPKPQKTILRKSTTTTV.>XP_005655053.1 PREDICTED: proline-rich protein 16 [Sus scrofa] MTDSSKTDTLNSSSSGTTASSIEKIKVQANAPLIKPPAHPSAILTVLRK9 residue PNPPPPPPR MW 967.5.Regulator of cell size that promotes cell size increase independently of mTOR and Hippo signaling pathways. Acts by stimulating the translation of specific mRNAs, including those encoding proteins affecting mitochondrial functions. Increases mitochondrial mass and respiration.Image 14LLSAPAPNATSCPAEESWWSGLAIVIAVCCASLVFLTVLVIICYKAIKRKPLRKEENGTSVAEYPMTSSQSNKGVDVNSAVV.>XP_020955308.1 proline-rich membrane anchor 1 isoform X2 [Sus scrofa] MLLRDLVLRRGCCWPSLLLHCALHPLWGFVQVAHGEPQKSCSKVTDSCQHICQCR16 residues PPPPLPPPPPPPPPPR MW 1645.9.Required to anchor acetylcholinesterase (ACHE) to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses in brain. Also able to organize ACHE into tetramers."}
+{"text": "Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells.  For shRNA gene delivery, researchers explore viral or non-viral delivery vectors. While viral vectors are costly and often trigger immune responses or pose the risk of genomic vector integration, many non-viral delivery vectors involve non-nucleic acid helper functions that can be toxic to the cells.Here, we report a cloning-free method for the generation of shRNA-expressing dumbbell vectors. This PCR-based method uses a universal template and sequences coding for a specific shRNA are introduced by the PCR primers. This novel protocol produces size-minimized hairpin-template transcribing dumbbells, does not require any restriction or nicking endonucleases, and is high throughput compatible.5), and (3) the hsa-miR-30 precursor stem  in which the self-complementary sequence portions were separated from each other A\u2013S1C. Paequenced D and S1ENext, we aimed to PCR-amplify the universal template using primers that introduced the sequence coding for a published firefly luciferase-targeting shRNA.With the decision to use either a 5\u2032-phosphorylated forward primer, a 5\u2032-phosphorylated reverse primer, or two phosphorylated primers for PCR, only (1) plus-strand-derived dumbbells, (2) minus-strand-derived dumbbells, or (3) a mix of both can be generated  and S2. Employing the above protocol using either phosphorylated forward or reverse primers, we generated both plus- and minus-strand-derived luciferase- or lamin A/C-targeting dumbbells in separate reactions\u00a0 and 4. TThe protocol described here combines all the advantages of previously reported protocols for dumbbell vector production. It represents (1) a cloning-free protocol that (2) does not involve any restriction or nicking endonucleases, (3) employs an efficient intra-molecular ligation reaction, and (4) allows production of extremely small hairpin template-transcribing dumbbell vectors. The previously described gap-primer PCR protocol also involves an intra-molecular ligation but requires a cloning step for the generation of every new vector, and it is not suitable to generate hairpin template-transcribing vectors due to the presence of abasic sequence positions.We demonstrate the proof-of-principle that this new method can generate partly mismatched shRNA-expressing dumbbell vectors, indicating the technology might also be explored for the generation of miRNA-expressing dumbbells. Mismatches in dumbbell vectors were reported earlier and demonstrated not to impair vector activity.We observed that among the luciferase- or lamin A/C-targeting dumbbells, the minus- or plus-strand-derived dumbbell exhibited a stronger target gene knockdown activity, respectively. This difference might be assigned to differences with regard to the efficiency and accuracy of endogenous shRNA processing by Dicer, which depends on the sequence and structure of shRNA loops and stems. Here, we employed\u00a0the hsa-miR-30 stem, as miRNA stems were reported to facilitate shRNA processing and knockdown activities in most of the cases.In conclusion, this novel method efficiently generates size-minimized hairpin template-transcribing dumbbells in a short period of time and at low costs and can be explored for the parallelized production of shRNA or miRNA expression vectors for functional genomics screens or drug development.AGCTTCGCGCTCACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGTT-3\u2032; oligo UT2, 5\u2032P-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3\u2032; oligo UT3, 5\u2032P-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3\u2032; oligo UT4, 5\u2032-GATCCTTTGCCTACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGAAAACT-3\u2032. Bold indicates HindIII and BamHI compatible overhangs; underlined indicates dumbbell loop-forming tetranucleotide. The 5\u2032 phosphorylated oligos UT2 and UT3 were hybridized with the complementary oligos UT1 and UT4, respectively. The resulting UT1/UT2 and UT3/UT4 duplexes were ligated to form the universal template sequence bearing HindIII and BamHI-compatible 5\u2032 overhangs, which was subsequently cloned into pVax1 , yielding the universal template vector pVax1-UT  endonucleolytic cleavage followed by analytical agarose gel electrophoresis, which yielded the expected insert size of 262\u00a0bp, and by sequencing of the ligation sites  A and S1BpVax1-UT B. For clon sites C and S1DtgaaggctcctcagaaacagctcCGCGCTCACTGAGAAGATTT-3\u2032; FP_Lamin 5\u2032-tgaaagcccagatcgtcaccacccgcCGCGCTCACTGAGAAGATTT-3\u2032; reverse primers, RP_Luciferase 5\u2032-agagaggctcctcagaaacagctcTTTGCCTACTGAGAAGATTTTTCTGT-3\u2032. RP_Lamin 5\u2032-agagaagcccagatcgtcaccaccttTTTGCCTACTGAGAAGATTTTTCTGT-3\u2032.Luciferase- or lamin A/C-specific primers were synthesized by AITbiotech (Singapore) or IDT (Singapore). Uppercase letters indicate the universal template binding sites, and lowercase letters indicate the shRNA coding sequences in which the loop-forming nucleotides are underlined: forward primers, FP_Luciferase 5\u2032-Two blocking ODNs  were added to the PCR to suppress refolding and self-priming of the universal template strands: Block_1, 5\u2032-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3\u2032; Block_2, 5\u2032-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3\u2032.Primers for the quantification of luciferase and \u03b2-actin mRNA levels were synthesized by AITbiotech (Singapore). PCR forward primers are as follow: qPCR_FP_Luciferase 5\u2032-CGCTGGGCGTTAATCAAAGA-3\u2032; qPCR_RPb-actin 5\u2032-CTGGCACCCAGCACAATG-3\u2032. Reverse transcription and PCR reverse primers are as follows: qPCR_RP_Luciferase 5\u2032-GTGTTCGTCTTCGTCCCAGT-3\u2032; qPCR_RPb-actin 5\u2032- GCCGATCCACACGGAGTACT-3\u2032.For the generation of strand-specific dumbbell vectors, either the forward or the reverse primers were 5\u2032-phosphorylated. Each 50 pmol primer was incubated with 10\u00a0U T4 polynucleotide kinase  in the presence of 1\u00a0mM ATP at 37\u00b0C for 20\u00a0min followed by heat inactivation of the enzyme at 75\u00b0C for 10\u00a0min.Taq DNA polymerase (Invitrogen),\u00a01.0\u00a0\u03bcM of each primer and blocking ODNs, 0.2\u00a0mM of each 2\u2019-deoxyribonucleoside 5\u2019-triphosphate , 100\u00a0ng of HindIII/BamHI cleaved pVax1-UT, 5% v/v DMSO  in a reaction volume of 30\u201350\u00a0\u03bcL in 1\u00d7 Taq DNA polymerase buffer (Invitrogen). Linearization of pVAX1-UT usually improves the PCR yields but is not essential. Thermal cycling was carried out as follows: initial denaturation at 96\u00b0C for 5\u00a0mins 27 cycles of denaturation , annealing , and extension ; and final extension at 72\u00b0C for 10\u00a0mins. A 50-\u03bcL PCR reaction yielded about 10\u00a0\u03bcg DNA.PCR amplification of the universal template and appendage of shRNA encoding DNA was carried out using 1\u00a0U 2, and 200\u00a0mM Tris-HCl, pH 7.4), heat-denatured\u00a0at 96\u00b0C for 5\u00a0min followed by gradual cooling to room temperature to allow for intramolecular folding of plus- and/or minus-strand dumbbell vectors. The resulting DNA was concentrated using ethanol precipitation, pelleted by centrifugation, and resuspended in nuclease-free water.PCR products were purified through silica-membrane-based spin columns . Purified products were diluted to 400\u00a0\u03bcL in 1\u00d7 hybridization buffer , and 50 to 100\u00a0U CircLigaseII ssDNA ligase  in 1\u00d7 CircLigaseII reaction buffer at 60\u00b0C for 16 h, followed by heat inactivation of the ligase at 80\u00b0C for 10\u00a0min. Highest conversion yields were observed when ligating 6\u00a0\u03bcg DNA with 100\u00a0U CircLigase.1 to 6\u00a0\u03bcg (\u223c10 to 60 pmol) of DNA was incubated with 2.5\u00a0mM MnClAfter ligation, products were treated with 10\u00a0U of T7 DNA polymerase  at 37\u00b0C for 1\u00a0h followed by heat inactivation at 80\u00b0C for 10\u00a0min. Products were assessed on 10% native polyacrylamide gels or 1% agarose gels, stained with ethidium bromide post-electrophoresis, and/or purified using phenol-chloroform-isoamylalcohol (25:24:1) extraction (1\u00d7), chloroform-isoamylalcohol (24:1) re-extraction (3\u00d7), and ethanol precipitation.4 cells/well were seeded in a 96-well plate. Cells were co-transfected with 100\u00a0ng of luciferase expression plasmid pGL3  and 1.5 pmol or 0.5 pmol of either plus- or minus-strand dumbbell vector DNA using Lipofectamine 2000  and a reagent:DNA ratio of 1:2.5. For the positive control (pGL3 only), empty pVAX1  was used as feeder DNA to ensure all cells received the same quantity of DNA. 48\u00a0h post-transfection, cells were washed with sterile PBS and lysed in 20\u00a0\u03bcL passive lysis buffer  for 20\u00a0min, employing gentle shaking. 10\u00a0\u03bcL of lysate was treated with 50\u00a0\u03bcL of LARII reagent , and luminescence was quantified on the Biotek reader .HEK293T cells were maintained in DMEM  supplemented with 10% (v/v) fetal bovine serum  and 1% penicillin-streptomycin antibiotic solution . 24\u00a0h prior to transfection, 2\u00a0\u00d7 10HEK293T cells were cultivated and seeded in 96-well plates 24\u00a0h prior to transfection as described above. Cells were transfected with 0.1, 0.5, or 2.5 pmol dumbbell vector DNA or 3 pmol siGENOMELamin A/C control siRNA  using Lipofectamine 3000  according to the manufacturer\u2019s protocol. Medium was changed 24\u00a0h post-transfection, and cells were harvested after 48 h. For FACS analyses, the media was aspirated, and the cells were rinsed once with PBS before trypsinization with 50\u00a0\u03bcL of 1\u00d7 trypsin-EDTA (Gibco). Trypsinized cells were collected by centrifugation at 4,200\u00a0rpm for 6\u00a0min in 200\u00a0\u03bcL media. Pelleted cells were resuspended in 100\u00a0\u03bcL media, fixed and permeabilized with intracellular fixation and permeabilization buffer set  according to manufacturer\u2019s protocol prior to intracellular staining. To assess lamin A/C knockdown, cellular lamin A/C was stained by anti-lamin A+C antibody (ab133256) (1/200) and donkey anti-rabbit IgG H&Ls AF647 (ab150075) (1/200) . FACS was performed on LSRFortessa cell analyzer, and FACSDiva software v6.1.3  was used for the acquisition of the samples. FlowJo software V10.5.2  was used for data analyses.mfold and/or RNAfold.18Minimum free energy secondary structures of DNA and RNA were folded using the algorithms Diagrams represent mean values\u00a0\u00b1 SEM of three independent experiments.\u00a0The statistical analysis was performed using repeated one-way ANOVA with Tukey\u2019s post hoc multiple comparison\u2019s test (luciferase knockdown data) or using Student\u2019s t test (lamin A/C knockdown data). The GraphPad Prism version 6 software  was used for the statistical analysis. p values are as indicated.V.P. developed the concept. V.P., S.L.C., A.G., P.S.L., and G.S.X.T. designed the experiments. S.L.C., A.G., P.S.L., and G.S.X.T. carried out the experiments, and all authors analyzed the data. V.P., S.L.C., A.G., and P.S.L. wrote the manuscript.The authors declare no competing interests."}
+{"text": "Memory consolidation theory suggests that once memory formation has been completed, memory is maintained at a stable strength and is incapable of further enhancement. However, the current study reveals that even long after formation, contextual fear memory could be further enhanced. Such unexpected enhancement is possible because memory is dynamically maintained at an intermediate level that allows for bidirectional regulation. Here we find that both Rac1 activation and expression of \u03b12-chimaerin are stimulated by single-trial contextual fear conditioning. Such sustained Rac1 activity mediates reversible forgetting, and \u03b12-chimaerin acts as a memory molecule that reverses forgetting to sustain memory through inhibition of Rac1 activity during the maintenance stage. Therefore, the balance between activated Rac1 and expressed \u03b12-chimaerin defines dynamic long-term memory maintenance. Our findings demonstrate that consolidated memory maintains capacity for bidirectional regulation. Memory consolidation theory suggests that memory is maintained at a stable strength after formation. The authors show that memory is dynamically maintained at an intermediate level allowing a bidirectional regulation which is mediated by a balance between activated Rac1 and expressed \u03b12-chimaerin. Guided by such widely accepted theory, the study of memory maintenance is understandably devoted to the identification of learning-stimulated self-sustainable protein synthesis regulatory mechanisms5. Indeed, a number of candidate mechanisms for such a simple maintenance have been reported, including the autophosphorylation of calcium/calmodulin-dependent protein-kinase type II (CAMKII)7, and the persistent protein synthesis regulated via self-perpetuating prion-like translational factors such as the cytoplasmic polyadenylation binding protein-3 (CPEB-3)8. It is a common experience, however, when recalling an episode that happened some time ago, the memory is vivid at one time but vague at others. Such flexible recalling experiences seem to be counterintuitive to memory consolidation theory. In addition to common experiences, it is noteworthy that recent publications have reported a number of intriguing observations that appear inconsistent with the notion of stabilized memory. First, synaptic connections involved in maintaining LTM are highly dynamic5. Second, overexpression of the self-autonomous protein-kinase-M-zeta (PKM\u03b6)9 was reported to enhance memory long after memory formation, but mechanisms underlying such enhancement remain unexplained10.Memory consolidation theory suggests that a newly acquired labile memory is gradually consolidated or stabilized through gene-regulation-based persistent modifications in synaptic structures within neural circuits and the resulting consolidated long-term memory (LTM) is maintained at a stable strength11. The recently proposed concept of active forgetting describes an intrinsic mechanism, i.e., learning or training evokes active forgetting to accelerate the decay of a formed memory. Multiple mechanisms are attributed to forgetting, including through activation of Rac1 or Cdc42, as well as through neurogenesis11. Recent progress in study of forgetting reveals that suppression of forgetting, inhibition of Rac1-dependent forgetting in particular, is capable of prolonging an hour-long labile memory to days14 in a range of tasks from invertebrates to vertebrates16. Moreover, a study reports that LTM decayed away during infancy can be recovered in adulthood17, suggesting that neurogenesis-based forgetting does not lead to memory erasure. These observations got us interested in investigating whether forgetting mechanisms, Rac1-dependent forgetting in particular, play any roles in memory maintenance. One possible candidate molecule to inhibit Rac1 activity comes from recent studies in mice during developmental stages21. It reveals that the negative regulator of Rac1 activity, \u03b12-chimaerin, acts as a brake through its GTPase-activating function to constrain Rac1 activity in regulating hippocampal dendrite and spine morphogenesis22 for establishment of normal cognitive ability23. However, the roles of interaction between Rac1 activity and \u03b12-chimaerin in memory maintenance in adulthood remain to be unraveled.Forgetting, the flip side of memory consolidation, is also an important feature for different types of memoriesThe present study provides a novel mechanism for dynamic memory maintenance that affords an intuitive explanation as to why LTM is still capable of being enhanced or faded long after completing formation. By integrating genetic, pharmacological, and optogenetic manipulations during behavioral paradigms, we demonstrate that the interplay between learning-evoked Rac1-dependent forgetting and learning-induced expression of a memory molecule \u03b12-chimaerin enables consolidated contextual fear memory and is maintained at an intermediate level so that memory could be enhanced through expression of more \u03b12-chimaerin while attenuated through elevated Rac1 activity. Moreover, forgetting induced by enhanced Rac1 activity is reversible through suppression of Rac1 activity. Thus, our results reveal that consolidated memory is maintained through the learning-evoked Rac1-dependent reversible forgetting mechanism balanced by learning-evoked expression of a memory maintenance molecule, \u03b12-chimaerin.14, we were interested in studying whether Rac1-dependent forgetting mechanisms play roles in LTM maintenance. In an effort to investigate this issue, we assayed Rac1 activity in hippocampal extracts of mice trained in the contextual fear conditioning (CFC) during the maintenance period of memory. Wild-type (WT) mice were subjected to single- or three-trial CFC and were tested for memory retention at different time points   Fig.\u00a0. To avoiays Fig.\u00a0 and thenays Fig.\u00a0. Consideays Fig.\u00a0. Consistays Fig.\u00a0. In addiays Fig.\u00a0. Howeverays Fig.\u00a0.Fig. 1In15. Mice were bilaterally injected with adeno-associated viruses (AAVs), carrying mutant transgenes that encoded either dominant-negative Rac1 (Rac1-DN) to inhibit endogenous Rac1 activity or constitutively active Rac1 (Rac1-CA) to increase Rac1 activity, targeted to hippocampal excitatory neurons15  and AAV9-tetracycline response element (TRE)-Tandem dimer Tomato (tdTomato) to the CA1 region of WT mice  was assessed by the percentage of Rac1-GTP+/c-fos+ colabeled cells among c-fos+ cells (engram cells). We found that 79% of c-fos+ cells were labeled with activated Rac1 positive (Rac1-GTP+) at 24\u2009h following training  in CA1 engram cells and the control (tdTomato) mice were injected with AAV9-c-fos:tTA and AAV9-TRE-tdTomato  mice were injected bilaterally into the CA1 with a virus cocktail of AAVato Fig.\u00a0, top. Inato Fig.\u00a0, bottom.ato Fig.\u00a0 at day 4ato Fig.\u00a0. Next, wThe findings from the above two experiments led us to make a third prediction that the naturally faded memory for several days . Additional injections of ANI further supported the finding Fig.\u00a0. This ex23. For knockdown, we generated AAVs that carry a short hairpin RNA (shRNA) sequence22 that targets \u03b12-chimaerin (AAV-\u03b12 shRNA), fused to tdTomato, under the control of the cytomegalovirus (CMV) early enhancer/chicken \u03b2-actin (CAG) promoter  induced via a single tetanus (weak stimulation) at the Schaffer collaterals of the hippocampal CA1 region. Knockdown of \u03b12-chimaerin in slices expressing AAV-\u03b12 shRNA led to a reduction of potentiation and accelerated LTP decay Fig.\u00a0. In cont 14 Fig.\u00a0, because 14 Fig.\u00a0 while ac 14 Fig.\u00a0.Fig. 6\u03b1215. Thus, interplay between activated Rac1 and expressed \u03b12-chimaerin defines the intermediate level of memory during maintenance period and allows dynamic memory maintenance  were purchased from the Vital River Laboratory  and were housed in groups under standard conditions according to the Tsinghua University animal facility. Mice were maintained on a 12\u2009h light/dark cycle and were tested during the light phase of the cycle. All animal work complied with ethical regulations for animal testing and research, and was done in accordance with Institutional Animal Care and Use Committee (IACUC) approval by Tsinghua University and followed by all Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.All plasmids were constructed by standard molecular biology procedures and subsequently verified by double-strand DNA sequencing. The \u03b11-chimaerin and \u03b12-chimaerin were synthesized by the GENEWIZ company (sequence are showed below). The pAAV-CaMKII\u03b1-EGFP was a gift from Bryan Roth (Addgene plasmid #50469). The \u03b11-chimaerin and \u03b12-chimaerin were sub-cloned into AAV backbone pAAV-CaMKII\u03b1-EGFP using the BsrGI/EcoRI restriction enzymes.\u03b11-chimaerin (Sequence ID: NM_001206602.1)5\u2032-ATGCCATCCAAAGAGTCTTGGTCAGGGAGGAAAACTAATAGGGCTGCAGTTCACAAATCAAAACAAGAGGGCCGTCAGCAAGATTTATTGATAGCAGCCTTGGGAATGAAACTGGGTTCTCCAAAGTCGTCTGTGACAATCTGGCAACCTCTGAAACTCTTTGCTTATTCGCAGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3\u2032\u03b12-chimaerin (Sequence ID: NM_001822.5)5\u2032-ATGGCCCTGACCCTGTTTGATACAGATGAATATAGACCTCCTGTTTGGAAATCTTACTTATATCAGCTACAACAGGAAGCCCCTCATCCTCGAAGAATTACCTGTACTTGCGAGGTGGAAAACAGACCAAAGTATTATGGAAGAGAGTTTCATGGCATGATCTCCAGAGAAGCAGCCGACCAGCTCTTGATTGTGGCTGAGGGGAGCTACCTCATCCGGGAGAGCCAGCGGCAGCCAGGGACCTACACTTTGGCTTTAAGATTTGGAAGTCAAACCAGAAACTTCAGGCTCTACTACGATGGCAAGCACTTTGTTGGGGAGAAACGCTTTGAGTCCATCCACGATCTGGTGACTGATGGCTTGATTACTCTCTATATTGAAACCAAGGCAGCAGAATACATTGCCAAGATGACGATAAACCCAATTTATGAGCACGTAGGATACACAACCTTAAACAGAGAGCCAGCATACAAAAAACATATGCCAGTCCTGAAAGAGACACATGATGAGAGAGATTCTACAGGCCAGGATGGGGTGTCAGAGAAAAGGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3\u2032\u03b11-chimaerin was 5\u2032-GCTT TCAGCAATGTGTCAT-3\u203239. The \u03b12-chimaerin shRNA sequence binding to the mouse \u03b12-chimaerin was 5\u2032-GCACATGGCAGTCCTGAAA-3\u2032, whereas the negative control scrambled shRNA (ctrl shRNA) was 5\u2032-GTTCATATGTTCACCTATT-3\u203222. The U6 promoter sequence was 5\u2032- GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTT-3\u2032.The U6 promoter-driven \u03b11-chimaerin shRNA (\u03b11 shRNA), \u03b12-chimaerin shRNA (\u03b12 shRNA), or negative control scrambled shRNA (ctrl shRNA) constructs were synthesized (GENWIZ company) and sub-cloned into AAV backbone pAVV-CAG-tdTomato (Addgene plasmid # 59462) using the EcoRI/HindIII and EcoRV/HindIII restriction enzymes, respectively. The \u03b11-chimaerin shRNA sequence binding to the mouse 39. Keyhole limpet hemocyanin-coupled synthetic peptides for \u03b11-chimaerin was MPSKESWSGRKANR and keyhole limpet hemocyanin-coupled synthetic peptides for \u03b12-chimaerin was HDEKEATGQDGVSEKR. Anti-Rac1-GTP (active Rac1) monoclonal antibody was from the NewEast company (Cat. No. 26903). Anti-Rac1 antibody was from BD Transduction Laboratories (Cat. No. 610650). Anti-phospho-PAK1/2/3 antibody was from novusbio company (Cat. No. NB100-82131). Anti-Cleaved Caspase-3 antibody was from Cell Signaling (Cat. No. 9661). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cat. No. #7072) and HRP-conjugated goat anti-rabbit IgG (Cat. No. #7071) were from Cell Signaling Technology. Alexa Fluro\u00ae 488 affinipure donkey anti-mouse IgM was from the Jackson ImmunoResearch Laboratories company (Cat. No. #711-545-140). Actin antibody was from the Merck Millipore company (Cat. No. #MAB1501).The following antibodies were used: primary antibodies for \u03b11-chimaerin and \u03b12-chimaerin were raised in rabbits immunized with keyhole limpet hemocyanin-coupled synthetic peptides as previously reported15. ANI  was dissolved in 0.9% saline and the pH was adjusted with 1\u2009N HCl to 7.0\u20137.4. Mice were subcutaneously injected with 150\u2009mg per kg of ANI or an equivalent volume of saline immediately after training. This amount of ANI was shown to effectively inhibit cerebral protein synthesis in mice (~96%)40.Ehop016  was dissolved in a solution containing 1% dimethylsulfoxide/30% PEG/1% Tween-80. Mice were intraperitoneally injected with 20\u2009mg per kg of Ehop016 solution or an equivalent volume of saline. At the dose used, Ehop016 has high efficiency to block Rac1 activity9-c-fos:tTA (300\u2009nl of AAV9-c-fos:tTA) and 300\u2009nl of the AAV9-TRE-tdTomato into the CA1 . For the activation of Rac1 activity in CA1 engram cells, a virus cocktail AAV9-c-fos:tTA (300\u2009nl of AAV9-c-fos:tTA and 300\u2009nl of AAV9-TRE:photoactivatable Rac1 (PaRac1)-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) were injected into the mice CA1 . Following the injections, the needle stayed for 10\u2009min before slowly withdrawn and the wound sutured. After surgery, animals were allowed to recover for two weeks prior to the performance of all subsequent experiments.Mice were anesthetized with 0.2% sodium pentobarbital (5\u2009ml/kg) and fixed to a stereotaxic frame. Their body temperature was kept at 36\u2009\u00b0C by a heating pad and their skull was exposed. Furthermore, holes in the skull were drilled by a micromotor drill. The injection of the virus was performed using a 10\u2009ml nanofil syringe under controlled by the UMP3 and Micro4 system (WPI), with a speed of 50\u2009nl/min. The dorsal hippocampus injections were bilaterally targeted to \u22122.0\u2009mm AP, \u00b11.5\u2009mm\u2009ML, and \u22121.5\u2009mm DV; the DG injections were bilaterally targeted to -2.0\u2009mm AP, \u00b11.5\u2009mm\u2009ML, and \u22122.0\u2009mm DV. The viral volumes of the AAV-Rac1-CA, AAV-Rac1-DN, and AAV-control were 400\u2009nl for DG and 600\u2009nl for CA1. Mice were bilaterally injected with 1\u2009\u00b5l of either AAV-\u03b11-chimaerin, AAV-\u03b12-chimaerin, AAV-control, AAV-\u03b11-chimaerin shRNA, AAV-\u03b12-chimaerin shRNA, or AAV-control shRNA. For labeling of the CA1 engram cells, a virus cocktail AAV9-c-fos:tTA) and 500\u2009nl of the AAV9-TRE:PaRac1-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) into the CA1. Furthermore, they were bilaterally implanted with optical fibers  into the CA1 . Mice were allowed to recover for two weeks under the ON-Dox condition prior to the start of the behavioral experiments. The cohorts of mice used for the optogenetic engram stimulation, mice were fed regular food, without Dox, for 24\u201330\u2009h until contextual fear-conditioning training. After CFC, mice were returned to their home cage under the ON-Dox condition. Following behavioral testing, brain sections were examined to confirm efficient virus-mediated labeling of the target areas.Mice were bilaterally injected with a virus cocktail (500\u2009nl of AAV2) was measured at the tip of the fiber. A blue light from a laser was ON for 30\u2009s to stimulate the photoactivatable Rac1 for five trials, with trial intervals of 120\u2009s31. For all behavioral procedures, the freezing behavior was measured in light stimulated mice at 1.5\u2009h after stimulation. Successively, animals were put back to their home cage.Mice were intra-CA1 implanted with optic fiber after AAV injection. The optic fibers of mice were connected to a 473\u2009nm blue laser diode via a FC/PC adaptor. Light intensity  had a stainless-steel rod floor which was connected to a shock generator in a sound-attenuating box. In single-trial CFC sessions, mice were individually placed in the conditioning and freely explored the area for 3\u2009min. Then, mice were exposed to one footshock  and were returned to their home cage 30\u2009s after. In the three-trial CFC task, the footshock  was delivered at 180, 240, and 300\u2009s. Mice remained in the conditioning chamber for a total of 330 s. In only context or only shock training, mice were exposed in a context for 3\u2009min without shock or one footshock  in the absence of context. After that, mice were placed back in their home cage. During testing, mice were placed back in the conditioning chamber for 4\u2009min at different times . For all procedures, the animal freezing behaviors were monitored using a manufacturer\u2019s software.The single- and three-trial CFC procedures were based on those of Denny et al.Subject mice were individually placed in the three-chamber apparatus for 10\u2009min to habituate to the environment (day 1). On day 2, a 6-week-old male C57BL/6J mouse was placed in the left grid enclosure for familiarization. Successively, the test mouse was located in the center compartment of the three-chamber apparatus for 10-min social training. During experimental testing (1\u2009h and day 3), a novel 6-week-old male C57BL/6J mouse was placed in the right grid enclosure and the familiar mouse in the left grid enclosure. And then, test mouse was placed into the social chamber and was allowed to explore for 10\u2009min. The amount of time for the test mouse spent in close interaction was recorded using the Any maze system. The discrimination index (DI) was calculated using the following formula: (time exploring the novel mouse\u2009\u2212\u2009time exploring the familiar mouse)/(time exploring the novel mouse\u2009+\u2009time exploring the familiar mouse) * 100.15. For the testing phase, one of the objects was exchanged with a new one, and the time spent exploring the two objects was separately recorded using ANY-MAZE software. The DI was calculated using the following formula: (time exploring the novel object\u2009\u2212\u2009time exploring the familiar object)/(time exploring the novel object\u2009+\u2009time exploring the familiar object)\u2009\u00d7\u2009100. For the memory decay curve, time intervals between sampling and testing were 1 and 24\u2009h.For the habituation phase, individual adult male mice were placed in a chamber (50\u2009cm\u2009\u00d7\u200950\u2009cm\u2009\u00d7\u200940\u2009cm) and allowed to freely explore the context for 10\u2009min while being recorded by an overhead camera. This phase was also conducted as the open field test to assess the animal\u2019s basal locomotor activity indicated by the average speed and anxiety levels indicated by the percentage of time spent in the middle arena (30\u2009cm\u2009\u00d7\u200930\u2009cm). For the sampling phase, the animal was placed in the same chamber containing two different objects for 5\u2009min and allowed to explore the object. To saturate the odor left by the previous mice and to make the mice more relaxed, we placed standard animal beddings in the chamberIsolated hippocampi were homogenized in cell lysis buffer  with protease inhibitors. The protein concentration was determined using the BCA protein assay kit . For the detection of the levels of \u03b11-chimaerin, \u03b12-chimaerin, total Rac1 and actin, 20 \u03bcg of homogenates was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . Membranes were blocked in milk solution (5% milk in TBS and 0.1% Tween 20) for one hour at room temperature. Subsequently, membranes were individually incubated with primary antibodies against \u03b11-chimaerin , \u03b12-chimaerin , total Rac1 , and actin  overnight at 4\u2009\u00b0C. All the HRP-conjugated secondary antibodies were used at 1:2000 dilutions for membranes incubation at room temperature for one hour. For the relative level of the Rac-GTP assay, a GST-tagged PAK-PBD beads  were incubated with the lysate mix (300\u2009\u03bcg) for overnight at 4\u2009\u00b0C. The beads were washed three times with lysis buffer at room temperature. Then, bound proteins were eluted with loading buffer  and analyzed by western blotting. Image quantification analysis in bands of the western blots was calculated by the ImageJ software .Mice were anesthetized with 0.2% sodium pentobarbital (5\u2009ml/kg) and perfused intracardially with 30\u2009ml of 4% paraformaldehyde. The brains were fixed in 4% paraformaldehyde overnight at 4\u2009\u00b0C and serial coronal sections (30\u2009\u03bcm) were then taken throughout the hippocampus using a vibratome. The floating sections were collected and rinsed in a 0.1\u2009M phosphate-buffered saline (PBS) (pH 7.4) solution with 0.1% Triton X-100 and incubated with blocking solution (10% donkey serum and 0.1% Triton X-100) for 2\u2009h at room temperature. The slices were then incubated with the following primary antibodies at 4\u2009\u00b0C for overnight: anti-active Rac1 , anti-Cleaved Caspase-3 , and anti-phospho-PAK1/2/3 . Following three times rinsed (30\u2009min each) in the PBS solution, slices were incubated with the secondary antibodies conjugated with dyes. Then sections were washed three times with PBS solution and mounted on slides with antifade mounting medium . The images of the immunohistochemistry were captured on a Zeiss LSM 710.Cell counting of c-Fos-positive cells and Rac1-active cells was performed in the CA1 by utilizing imaging analysis function of Zeiss software (Zen blue 2.3). The quantification of the number of c-Fos-positive cells was performed by thresholding c-Fos immunoreactivity above background levels automatically. The quantification of the number of Rac1-GTP cells was performed by setting the threshold manually from 1000 to 4096. The quantification of the number of phospho-PAK cells was performed by setting the threshold manually from 692 to 4096. The quantification of the number of cells with 4\u2032,6-diamidino-2-phenylindole was performed by setting the threshold manually from 616 to 4096.2PO4, 1\u2009mM MgSO4, 2\u2009mM CaCl2, 26\u2009mM NaHCO3, and 10\u2009mM glucose) for at least 1\u2009h. Following the recovering at room temperature, the slices were transferred to the field excitatory post-synaptic potentials (EPSPs) recording chamber  with ACSF (flow rate 1.5\u2009ml/min) for at least 20\u2009min prior to the recording. Filed EPSPs were evoked from CA3-CA1 synapses through the stimulation of the Schaffer collateral axons and recorded in CA1 extracellular fEPSPs. The stimulation intensity was adjusted to elicit a 30% maximal response. The LTP protocol for the 100\u2009Hz consisted in 10\u2009\u00d7\u2009100\u2009Hz bursts (four pulses per burst) with a 200\u2009ms interval between bursts. Data acquisition and analysis were performed using the multielectrode MED64 hardware and software packages (Panasonic).Transverse hippocampal slices were prepared from 4-month-old mice. Animals were killed by decapitation in accordance to the institutional regulations. Hippocampi were sliced (300\u2009\u03bcm) by a VF-300 microtome (Precisionary Instruments) and kept in an interface chamber with oxygenated artificial cerebrospinal fluid  or two-way ANOVA where appropriate. The data are shown as the mean\u2009\u00b1\u2009SEM and NS indicates non-significance (p\u2009>\u20090.05). The significant levels were set to P\u2009=\u20090.05. Significant for comparison: *p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001; ****p\u2009<\u20090.0001. The specific statistical tests used and the details of p-values for each experiment can be found in Supplementary Table\u00a0Statistical analyses were performed in GraphPad Prism. All data were analyzed with an unpaired Further information on research design is available in the\u00a0Supplementary InformationTransparent Peer Review FileReporting Summary"}
+{"text": "Correction to: Nature Communications 10.1038/s41467-018-04901-6; published online 27 June 2018In the original version of the Supplementary Information file associated with this Article, the sequence \u20181x MS2 scRNA.b2\u2019 was incorrectly given as \u2018GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCGCACATGAGGATCACCCATGTGCTTTTTT\u2019 and should have read \u2018GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACATGAGGATCACCCATGTGCTTTTTTT\u2019. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article."}
+{"text": "Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics. Protein nanopores are emerging as sensors for a variety of biomolecules. Here the authors develop a nanopore based on the bacterial toxin ClyA, in conjunction with binding proteins for glucose and asparagine, to detect these biomolecules simultaneously from a variety of unprocessed, diluted body fluids. The ability to monitor the health of individuals that exchange information through the Internet without requiring human intervention would undoubtedly bring about a revolution in personalised healthcare. Over the past decades, the miniaturisation of electronic devices combined with recent advances in the wireless transmission of data and microfluidics have opened up new possibilities for making such devices. However, one key area that is preventing their breakthrough is the production of sensing elements for biologically relevant molecules, which can be easily interfaced with silicon-based electronics. Such elements have long lagged behind the electronic industry in terms of miniaturisation and cost reduction.1. However, such oxidase enzymes are not ubiquitous, and today\u2019s commercially available home diagnostic and implantable devices are entirely limited to one well-known example: the glucose sensor. However, despite the sensor has seen many improvements since it was first introduced in the early 1960s2, it still suffers from limitations including enzyme instability, complicated surface immobilisation procedures, baseline shifts, and does not tolerate well changes in micro-environmental factors such as changes in pH and ionic strength5.At the moment, biosensors that can be integrated in electronic devices rely mostly on the electric signal that is generated by the oxidation of a target analyte catalysed by an immobilised enzyme6. Nanopores can also detect polypeptide8 biomarkers. However, their use in diagnostic devices is complicated by the low, typically picomolar, concentration of such biomarkers in biological fluids. By contrast, metabolites are highly abundant in bodily fluids, and their variation in concentration is linked to many diseases9. Nanopores equipped with cyclodextrins10, cucurbiturils11, or cyclic peptides12 may recognise metabolites. However, the detection and quantification of such analytes directly from a biological sample cannot be easily implemented because the recognition elements lack selectivity.Nanopores, which are nanometre-sized water conduits spanning biological or artificial membranes, are emerging as a new class of biosensors. When a potential is applied, the flux of ions across an individual nanopore generates an electrical signal that allows direct interfacing with electronic devices. Most notably, nanopores can be integrated into electronic devices and are now used for low-cost, high-speed, and portable sequencing of DNA16. Here, using proteins from a protein family that in cells recognise an enormous variety of molecules, we show that ClyA nanopores can report the concentration of glucose and asparagine directly from samples of blood, sweat, and other bodily fluids. Conveniently, no sample preparation is required and the concentration of the metabolite can be monitored continuously.Recently, we have shown that the biological nanopore cytolysin A (ClyA) can be used to monitor the function of proteins when they are lodged inside the nanopore Fig.\u00a013\u201316. He17. GBP belongs to the class of substrate-binding proteins which, with minor structural differences, recognise hundreds of different metabolites in cells18. The structure of the GBP has been solved in the ligand-free state (open or apo form) configuration and in the closed, ligand-bound, configuration. Interestingly, NMR studies revealed that in solution, apo-GBP adopts both configurations even in the absence of glucose19. In our experimental setup, GBP (44\u2009nM) was added to the cis side of a ClyA-AS20 nanopore . Two current levels 19.In this work, we used a glucose-binding protein (GBP) that binds glucose with high affinity and specificitytrans side of the nanopore increased the number of events corresponding to the closed configuration  and opening  rate constants could be determined from the inverse of L1 and L2 dwell times 21. The dependence of the fractional time of GBP in the closed configuration with the glucose concentration fitted well to a Hill function with the Hill coefficient set to one  corresponded well to the value measured in bulk (2\u20135\u2009\u00d7\u200910\u22127\u2009M)23. A GBP variant (GBP-A213R) with reduced affinity for glucose25 showed only one single level in the absence of glucose . Extrapolation from the calibration curve , sweat (2\u2009\u00b5L), urine (200\u2009nL), and saliva (8\u2009\u00b5L) were added to the rve Fig.\u00a0 allowed 0\u2009nL, swe26, while its abnormal concentration in blood has been associated to Parkinson\u2019s disease27. Under \u221290\u2009mV, the blocked pore level of apo-SBD1 contained one main current level corresponding to the open protein configuration . As observed before for GBP, the binding constants measured by the nanopore were similar to the ones measured in bulk28. The percentage of bound fraction was used to extrapolate the concentration of asparagine in the trans solution  was markedly different than the open protein configuration of GBP (Ires%\u2009=\u200969.1\u2009\u00b1\u20090.2%). Thus, when GBP (44\u2009nM) and SBD1 (120\u2009nM) were simultaneously present in the cis solution, blockades corresponding to each protein adaptor could be readily distinguished , enzymes from Thermo Fisher Scientific, and lipids from Avanti Polar Lipids.Escherichia coli at the beginning of the gene . To maintain the open-reading frame, two extra bases were inserted after the NcoI site, resulting in an additional alanine residue after the starting methionine. For purification, a His6-tag was attached at the C-terminus of GBP, via a flexible glycine\u2013serine\u2013serine linker and the open-reading frame was terminated by two consecutive stop codons, followed by a Hind III restriction site (3\u2032-end). The attachment of the His6-tag was carried out in two consecutive PCR reactions. During the first PCR reaction, the GBP gene was amplified directly from a single BL21 (DE3) E. coli (Lucigen) colony using Phire Hot Start II DNA polymerase, 0.4\u2009\u00b5M dNTPs, 6\u2009\u00b5M of MS-Glu_F (TATATATCCATGGCTaataagaaggtgataaccctgtctgctgtg), and MS-Glu_r1 (gatggtgatgGCTGCTGCCtttcttgctgaattcagccaggttgtctttatctacgcc) primers in a 50\u2009\u00b5L reaction volume. The PCR reaction cycling protocol was as follows: pre-incubation step at 98\u2009\u00b0C for 30\u2009s and then 30 cycles of denaturation at 98\u2009\u00b0C for 5\u2009s and extension at 72\u2009\u00b0C for 1\u2009min. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen) and served as a template for the second PCR reaction, which used ~100\u2009ng of the purified PCR product amplified by Phire Hot Start II DNA polymerase using 0.4\u2009\u00b5M dNTPs, 6\u2009\u00b5M of MS-Glu_F, and MS-Glu_r2 (atatatataagctttcattaatggtGATGGTGATGGCTGCTGCCTTTCTTGCTGAATTC) primers in 300\u2009\u00b5L volume. The cycling protocol was the same as in the previous step. The resulting PCR product encoding for the His6-tagged GBP gene was purified with QIAquick PCR Purification Kit (Qiagen) and digested with NcoI and HindIII . The gel purified insert  was cloned under control of the T7 promoter into the pT7-SC1 expression plasmid29 using sticky-end ligation  via NcoI (5\u2032) and HindIII (3\u2032) sites. Ligation mixture of 0.6\u2009\u00b5L was transformed into 50\u2009\u00b5L of E. cloni\u00ae 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37\u2009\u00b0C on ampicillin-containing (100\u2009\u00b5g/mL) LB agar plates. The identity of the clones was confirmed by sequencing.To allow cloning, a NcoI site (CCATGG) was introduced into the sequence of the wild-type GBP from MANKKVITLSAVMASMLFGAAAHAADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKKGSSHHHHHCCATGGCTAATAAGAAGGTGATAACCCTGTCTGCTGTGATGGCCAGCATGTTATTCGGTGCCGCTGCACACGCTGCTGATACTCGCATTGGTGTAACAATCTATAAGTACGACGATAACTTTATGTCTGTAGTGCGCAAGGCTATTGAGCAAGATGCGAAAGCCGCGCCAGATGTTCAGCTGCTGATGAATGATTCTCAGAATGACCAGTCCAAGCAGAACGATCAGATCGACGTATTGCTGGCGAAAGGGGTGAAGGCACTGGCAATCAACCTGGTTGACCCGGCAGCTGCGGGTACGGTGATTGAGAAAGCGCGTGGGCAAAACGTGCCGGTGGTTTTCTTCAACAAAGAACCGTCTCGTAAGGCGCTGGATAGCTACGACAAAGCCTACTACGTTGGCACTGACTCCAAAGAGTCCGGCATTATTCAAGGCGATTTGATTGCTAAACACTGGGCGGCGAATCAGGGTTGGGATCTGAACAAAGACGGTCAGATTCAGTTCGTACTGCTGAAAGGTGAACCGGGCCATCCGGATGCAGAAGCACGTACCACTTACGTGATTAAAGAATTGAACGATAAAGGCATCAAAACTGAACAGTTACAGTTAGATACCGCAATGTGGGACACCGCTCAGGCGAAAGATAAGATGGACGCCTGGCTGTCTGGCCCGAACGCCAACAAAATCGAAGTGGTTATCGCCAACAACGATGCGATGGCAATGGGCGCGGTTGAAGCGCTGAAAGCACACAACAAGTCCAGCATTCCGGTGTTTGGCGTCGATGCGCTGCCAGAAGCGCTGGCGCTGGTGAAATCCGGTGCACTGGCGGGCACCGTACTGAACGATGCTAACAACCAGGCGAAAGCGACCTTTGATCTGGCGAAAAACCTGGCCGATGGTAAAGGTGCGGCTGATGGCACCAACTGGAAAATCGACAACAAAGTGGTCCGCGTACCTTATGTTGGCGTAGATAAAGACAACCTGGCTGAATTCAGCAAGAAAGGCAGCAGCCATCACCATCACCATTAATGAAAGCTT30 on a circular plasmid template DNA (pT7-SC1 containing GBP gene) with Phire\u00ae Hot Start Polymerase (Finnzymes) by using two complementary primers. Six \u00b5M forward (GGTTATCGCCAACAACGATAGGATGGCA) and 6\u2009\u00b5M reverse primer (GCGCCCATTGCCATCCTATCGTTGTT) containing a codon encoding the mutation were used in 50\u2009\u00b5L final volume to amplify the gene . The PCR product was incubated with DpnI (1 FDU) for 1\u2009h at 37\u2009\u00b0C to digest the plasmid template. The remaining DpnI enzyme was deactivated at 55\u2009\u00b0C. The mixture was then incorporated into E. cloni\u00ae 10G cells (Lucigen) by electroporation. Transformants containing the plasmid were grown overnight at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009\u03bcg/mL ampicillin and 1% glucose. Single colonies were picked and inoculated into 10\u2009mL LB medium supplemented with 100\u2009mg/L of ampicillin for plasmid DNA preparation. The presence of the mutation was confirmed by sequencing of the plasmid.The GBP-A213R was constructed using the QuickChange protocol for site-directed mutagenesisE. coli strain MC1061 and purified as described previously.28 The cell lysate was mixed with 50\u2009mM potassium phosphate, pH 8.0, 200\u2009mM KCl, 20% glycerol (buffer A) supplied with 20\u2009mM imidazole and incubated with Ni2+-sepharose  resin for 1\u2009h at 4\u2009\u00b0C. Next, the resin was washed with 20 column volumes of buffer A supplied with 50\u2009mM imidazole, followed by protein elution with 500\u2009mM imidazole in buffer A. To prevent protein aggregation, 5\u2009mM EDTA was added immediately after elution. Afterwards the histidine-tag was cleaved off by adding 2.5% w/w His-TEV (S219V)31 and dialyzing overnight against 50\u2009mM Tris-Cl, 0.5\u2009mM DTT, 0.5\u2009mM EDTA, pH 8.0 to remove imidazole. The histidine-tagged tobacco etch virus (TEV) protease and residual uncut protein were removed by loading it on a small 0.5\u2009mL Ni2+-sepharose column collecting the flow-through. Next, proteins were further purified with size-exclusion chromatography on a Superdex-200 column  equilibrated with 50\u2009mM Tris-HCl, 200\u2009mM NaCl, pH 7.5. Peak fractions were collected in aliquots and were stored at \u221280\u2009\u00b0C after flash-freezing in liquid nitrogen.Substrate-binding domains carrying a histidine-tag were expressed in E. cloni\u00ae EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the ClyA-AS gene. ClyA-AS contains eight mutations relative to the S. Typhi ClyA-WT: C87A, L99Q, E103G, F166Y, I203V, C285S, K294R, and H307Y. Transformants were selected after overnight growth at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400\u2009mL 2xYT medium containing 100\u2009mg/L of ampicillin. The cells were grown at 37\u2009\u00b0C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of ClyA-AS was induced by addition of 0.5\u2009mM IPTG. The cell cultures were further grown overnight at 25\u2009\u00b0C. The next day, the bacteria were harvested by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 25\u2009min and the pellets were stored at \u221280\u2009\u00b0C. The pellets containing monomeric ClyA-AS were thawed and solubilized with 20\u2009mL lysis buffer  and incubated for 20\u2009min at 37\u2009\u00b0C. The bacteria were then lysed by sonication (Branson). The lysate was subsequently centrifuged at 6000\u00d7g at 4\u2009\u00b0C for 20\u2009min and the cellular debris discarded. The supernatant was mixed with 150\u2009\u03bcL of Ni-NTA resin (Qiagen) in wash buffer . After 1\u2009h, the resin was loaded into a column  and washed with ~5\u2009mL of the wash buffer. The protein was eluted with ~100\u2009\u03bcL elution buffer . Type I ClyA-AS oligomers were obtained by incubation of ClyA-AS monomers with 0.2% \u03b2-dodecylmaltoside (DDM) for 30\u2009min at 37\u2009\u00b0C. The oligomers were separated from monomers by blue native polyacrylamide gel electrophoresis  using 4\u201320% polyacrylamide gels20. The band corresponding to type I ClyA-AS were cut out from the gel and was extracted in 150\u2009mM NaCl, 15\u2009mM Tris-HCl, pH 7.5 supplemented with 0.2% DDM and 10\u2009mM EDTA.E. cloni\u00ae EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the His6-tagged GBP gene. Transformants were selected after overnight growth at 37\u2009\u00b0C on LB agar plates supplemented with 100\u2009mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400\u2009mL 2xYT medium containing 100\u2009mg/L of ampicillin. The cells were grown at 37\u2009\u00b0C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of GBP was induced by addition of 0.5\u2009mM IPTG. The cell cultures were further grown overnight at 25\u2009\u00b0C. The next day, the bacteria were harvested by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 25\u2009min. The protein was purified directly afterwards by osmotic shock. The pellets were thoroughly dissolved in 20% ice-cold sucrose solution and incubated, while shaking at 4\u2009\u00b0C for 10\u2009min. The cell suspension was centrifuged at 6000\u00d7g at 4\u2009\u00b0C for 10\u2009min and cells collected. The cells were re-suspended in 4\u00d7 volume of ice-cold MilliQ water supplemented with 5\u2009mM MgCl2 and incubated, while shaking, on ice for 20\u2009min. The cells collected again by centrifugation at 6000\u00d7g at 4\u2009\u00b0C for 20\u2009min. The supernatant was mixed with 150\u2009\u03bcL of Ni-NTA resin (Qiagen) in wash buffer . After 1\u2009h, the resin was loaded into a column  and washed with ~5\u2009mL of the wash buffer. The protein was eluted with ~500\u2009\u03bcL elution buffer . The protein (500\u2009\u03bcL) was then dialysed exhaustively overnight against dialysis buffer , whereby the dialysis buffer was replaced for fresh buffer at least three times. Protein concentration was measured by using the Nanodrop 2000C at 280\u2009nm (Thermo Fisher Scientific). Protein was stored with 10% glycerol in \u221280\u2009\u00b0C after flash-freezing until further use.sn-glycero-3-phosphocholine (DPhPC) in pentane (10\u2009mg/mL) was then added to the buffer  present in both compartments. After evaporation of the pentane, a lipid monolayer at the air\u2013water interface can self-assemble spontaneously by lowering and raising the buffer across the aperture.\u00a00.01\u20130.1 ng of purified ClyA-AS were added to the grounded cis compartment. The reconstitution of single nanopores was monitored electrically by applying a \u221235\u2009mV bias. ClyA-AS nanopores display a higher open pore current at positive than at negative applied potentials, which provides a useful tool to determine the orientation of the pore. After insertion of a single pore, GBP or SBD1 were added to the cis compartment. This was followed by addition of a glucose or asparagine sample or bodily fluid sample in the trans compartment.The setup consisted of two chambers separated by a 25\u2009\u03bcm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an aperture of approximately 100\u2009\u03bcm in diameter. The aperture was pre-treated with a solution of hexadecane [10% (v/v) in pentane] to leave a hydrophobic coating to support bilayer formation. 1,2-diphytanoyl-All traces were routinely recorded at \u221290\u2009mV or \u221260\u2009mV applied transmembrane potential by using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitised with a DigiData 1440\u2009A/D converter (Axon Instruments). The data was sampled by applying a 2\u2009kHz low-pass Bessel filter and at a frequency of 10\u2009kHz. Obtained traces were filtered digitally with a Gaussian low-pass filter with a 100\u2009Hz cutoff. Data were recorded by using the Clampex 10.4 software (Molecular Devices) and the subsequent analysis was carried out with the Clampfit software (Molecular Devices).\u03c4on) and ligand-independent opening (\u03c4off) of GBP were obtained from single exponential fits to histograms of the inter-event times and dwell times, respectively. The mean lifetime of the open state (\u03c4on) was converted to the on-rate constants by using kon\u2009=\u20091/\u03c4on[glucose], where [glucose] is the concentration of glucose added to the trans side. The \u03c4off was converted to the off-rate by using koff\u2009=\u20091/\u03c4off. The equilibrium binding constant was calculated by plotting the fraction of closed configuration against the concentration of substrate and the data fitted to a Hill function . Events shorter than 2\u2009ms were ignored. The mean dwell times for the ligand-induced closing  were used to determine the glucose in the bodily fluids. The solution of the glucose assay kit contains a hexokinase enzyme that uses adenosine triphosphate (ATP) as a co-factor to phosphorylate glucose. The glucose-6-phosphate (G6P) is then oxidised to 6-phospho-gluconate in the presence of oxidised nicotinamide adenine dinucleotide (NAD) in a reaction catalysed by glucose-6-phosphate dehydrogenase (G6PD). The generation of NADH was spectrophotometrically observed by measuring absorbance at 340\u2009nm. Under these conditions, the final concentration of produced NADH corresponds to the glucose concentration in the sample. In order to perform the experiment, a dilution of bodily fluid was added to the reaction mixture. The reaction was incubated for 15\u2009min at room temperature and then the absorbance was measured at 340\u2009nm (\u03b5\u2009=\u20096220\u2009M\u22121\u2009cm\u22121). For each sample, three different dilutions were measured. The commercial glucose metre Accu-Chek\u00ae uses a mutant variant of quinoprotein glucose dehydrogenase and has a measurement range between 0.6 and 33.3\u2009mmol/L32. The Accu-Chek\u00ae is considered to be a reliable device for monitoring glucose levels in blood from finger pricks33.A glucose assay kit (HK) from Sigma-Aldrich and a commercial glucose metre . The blood samples were collected shortly before the experiments to minimise ex vivo artefactual changes. Sweat, urine, and saliva samples were either freshly collected before, or stored at \u221220\u2009\u00b0C, before the start of experiments. The solutions were kept on ice for the duration of an experiment. The blood samples were diluted 500-folds and the urine sample 10-folds with buffer  before adding 5 and 2\u2009\u00b5L, respectively, to the kopening\u2009=\u20090.75\u2009\u00b1\u20090.12\u2009s\u22121, 500\u2009nM glucose), compared to the binding of asparagine to SBD1 , thus longer integration times are required for accurate reading. It follows that (GBP-A213R), which shows reduced affinity for glucose .All measurements were made by adding small volumes of biological samples to an electrophysiology chamber (500\u2009\u00b5L). We did not observe a particular instability of the bilayer . After mixing, a concentrator  was used to evaporate all liquids by vacuum evaporation at 60\u2009\u00b0C and 240\u00d7g, and the sample was reconstituted in an identical volume of water as the former fixed amount of supernatant. A final 5-min-long centrifugation at 20,000\u00d7g step was performed and the supernatant was stored at 4\u2009\u00b0C before analysis.Proteins in 150\u2009\u00b5L saliva and 60\u2009\u00b5L serum samples were precipitated by incubation with 8% trichloroaceticacid (CASnr 76\u201303\u20139) for 5\u2009min at 60\u2009\u00b0C and 450 r.p.m. using a thermomixer  followed by cooling on ice for 30\u2009min and centrifugation for 10\u2009min at 20,000\u00d7HPLC quantification of asparagine was performed on an Agilent 1100 HPLC binary system  equipped with an 1100 Fluorescence detector (FLD) and a Gemini C18 column  while a 40\u2009mM sodium phosphate buffer, pH 7.8 (solvent A) and a mixture of 45:45:10 acetonitrile/methanol/water (solvent B) were used. The fluorogenic labelling of amino acids was performed automatically by the auto sampler of the HPLC machine using a mixture of 10\u2009mg o-phtalaldehyde  and 10\u2009\u00b5L 3-mercaptapropionic acid  in water as reagent. The auto sampler mixed 0.5\u2009\u00b5L sample with 1.3\u2009\u00b5L 0.4\u2009N borate buffer, pH 10.2 followed by 0.5\u2009\u00b5L reagent and 7.6\u2009\u00b5L water right before each injection. After the sample injection, amino acids were separated by applying solvent B at 1.5%/min increase for 25\u2009min using a flow rate of 250\u2009\u00b5L/min . Thereafter, the column was cleaned for 5\u2009min at 66% solvent B followed by another 12\u2009min at 100% solvent B. Then the column was equilibrated back to 5% solvent B. Fluorescence was measured at an excitation wavelength of 340\u2009nm and an emission wavelength of 450\u2009nm.Quantifications were performed using the method of standard addition at three different concentrations of analyte . The concentration of the analyte was obtained by measuring the peak areas, while each sample was analysed in triplicate. Citrulline was added as internal standard at 200\u2009\u00b5M to correct for saliva and serum sample handling volume differences next to control run-to-run retention time shifts regarding all samples.Supplementary InformationPeer Review File"}
+{"text": "DNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase \u03b1-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol \u03b4 in connecting the nascent leading strand with the advancing replisome.  \u2022S.\u00a0cerevisiae DNA replication originsMapping of leading-strand start sites at two \u2022Leading-strand synthesis is established from \u201clagging-strand\u201d primers\u2022Pol \u03b4 likely plays a key role in establishing all nascent leading strands\u2022Replisomes remain interdependent until new leading strands are established Aria and Yeeles describe the mechanism by which leading-strand replication is established at eukaryotic DNA replication origins. \u201cLagging-strand\u201d primers, synthesized by replisomes on opposite sides of the origin, are elongated back across the origin by Pol \u03b4 until the 3\u02b9 ends are coupled to Pol \u03b5 at the advancing replication forks. Bidirectional DNA replication is initiated from specific regions of the genome, termed origins. In eukaryotes, assembly of the DNA replication machinery (replisome) begins in the G1 phase of the cell cycle when the ATP-dependent motor component of the replicative helicase, the hexameric Mcm2\u20137 complex (MCM), is loaded at origins by the origin recognition complex (ORC), Cdc6 and Cdt1 . The MCMin\u00a0vivo studies in budding yeast have reached conflicting conclusions regarding the location of leading-strand start sites relative to an origin. For example, one study concluded that the ARS1 origin contains a single leading-strand start site (Once sufficient single-stranded DNA (ssDNA) has been exposed at origins, synthesis of leading and lagging strands is initiated by the DNA polymerase \u03b1-primase complex (Pol\u00a0\u03b1). Lagging-strand synthesis requires repeated cycles of Pol \u03b1-dependent priming and subsequent primer extension by Pol \u03b4. Pol \u03b1 first synthesizes 7\u201312 nucleotide (nt) RNA primers before transferring them to the DNA polymerase domain, where further extension to about 20\u201325 nt takes place . Evidencart site . The sitart site , and theart site . Howeverart site . Consequin\u00a0vivo   C. Subseq (PIP is very similar to the 35% rate reduction that we observed previously when PCNA was omitted from reactions (PIP contributes significantly to leading-strand synthesis.At early time points (6 and 9\u00a0min), Pol \u03b5eriments E and S1Aeriments . In reacents  . Moreoveeactions . These fPIP in leading-strand replication, we created two additional Pol \u03b5 mutants: one in which catalysis was disrupted by a point mutation in the polymerase active site (Pol \u03b5Cat) (PIP/Cat). Replication with Pol \u03b5PIP/Cat proceeded at a similar rate to Pol \u03b5Cat   and a muPol \u03b5Cat F. In con. We reasoned that initiation via pathway 1 should be refractory to challenge by this mutant, whereas it should block initiation via pathway 2 , a potent block to replicative polymerases, 16 nt to the left of the ACS in the lagging-strand template A. The CPBased on our previous work , preventS.\u00a0cerevisiae origins. Synthesis is predominantly initiated outside the origin sequence; Left leading strands are started to the right, and Right leading strands are started to the left .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles  containing integrated expression constructs; or from Escherichia coli RosettaTM 2(DE3) cells (Novagen) (genotype: F\u2013 ompT hsdSB(rB\u2013 mB\u2013) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for protein overexpression. Plasmids details are reported in the Proteins were purified from The ARS306 templates used in this study were modified from the plasmid ZN3  using stTo generate linear replication templates all plasmids were purified with CsCl gradients, linearized at unique restriction sites and processed as previously indicated . ExperimCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGAGCATACGACTACCGCCGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTGCTGAGGACTTAAGTGCTGAGGGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACpol2 gene was modified in yJF1 (PIP) and D640A (Pol \u03b5Cat) were obtained by site directed mutagenesis, and subsequently sub-cloned into pRS304 (Pol2\u00a0+ Dpb4-CBP) (The endogenous  in yJF1  strain t in yJF1  as templpb4-CBP) . The Polpb4-CBP)  was mutaWith the exception of the Pol \u03b5 and Pol \u03b4 mutants (see details below), all proteins were purified as described previously . A list Pol \u03b5 mutants  were expressed and purified in S.\u00a0cerevisiae essentially as for wild-type Pol \u03b5 . Protein elution was carried out with 10 CV of Buffer T\u00a0+ 200\u00a0mM NaCl, supplemented with 2\u00a0mM EDTA and 2\u00a0mM EGTA. Eluted protein was diluted 1:2 in Buffer\u00a0T\u00a0lacking NaCl to reduce the salt concentration tot 100\u00a0mM, and then loaded on MonoQ column . The column was washed with Buffer T containing 100\u00a0mM NaCl and Pol \u03b4Cat was eluted with 20 CV of 100-600\u00a0mM NaCl. The peak fraction was dialysed against storage buffer .Pol \u03b4 protein , with so2; 50\u00a0mM potassium glutamate; 0.5\u00a0mM ATP; 60\u00a0\u03bcM dATP; 60\u00a0\u03bcM dGTP. Protein mix containing PCNA and Pol \u03b5 or Pol \u03b5PIP  was added to RPA-bound template. The two protein-DNA mixtures were split and increasing concentrations of RFC were added. The reactions were incubated at 30\u00b0C for 5\u00a0minutes and DNA synthesis started by adding nucleotide mix (60\u00a0\u03bcM of each dTTP and dCTP and 1\u00a0\u03bcCi [\u03b1-32P]-dCTP). After 12\u00a0minutes at 30\u00b0C, the reactions were stopped with stop solution  and separated on 0.7% alkaline agarose gels.Primer extension reactions were essentially performed as described previously but using M13-mp18 ssDNA (NEB), rather than \u03d5X174 . Singula2; 0.1\u00a0mg/ml BSA; 3\u00a0mM ATP; 5\u00a0nM DNA template; 75\u00a0nM Cdt1-Mcm2-7; 40\u00a0nM Cdc6; 20\u00a0nM ORC; 25\u00a0nM DDK. After a 10\u00a0min incubation S-CDK was added to 100\u00a0nM and incubation prolonged for a further 5\u00a0min. The loading reaction was 4-fold diluted into replication buffer containing the following components, reported in their final concentrations: 25\u00a0mM HEPES-KOH, pH 7.6; 250\u00a0mM potassium glutamate; 0.01% NP-40-S; 1\u00a0mM DTT; 10\u00a0mM Mg(OAc)2; 0.1\u00a0mg/ml BSA; 3\u00a0mM ATP; 200\u00a0\u03bcM CTP; 200\u00a0\u03bcM GTP; 200\u00a0\u03bcM UTP; 30\u00a0\u03bcM dATP; 30\u00a0\u03bcM dCTP; 30\u00a0\u03bcM dGTP; 30\u00a0\u03bcM dTTP; 1\u00a0\u03bcCi [\u03b1-32P]-dCTP. Samples were pre-warmed at 30\u00b0C for 1\u00a0minute and reactions were initiated by addition of replication proteins to the following concentrations: 30\u00a0nM Dpb11; 100\u00a0nM GINS; 30\u00a0nM Cdc45; 10\u00a0nM Mcm10; 20\u00a0nM Ctf4; 20\u00a0nM Csm3/Tof1; 20\u00a0nM RFC; 20\u00a0nM PCNA; 20\u00a0nM Pol \u03b5 (or\u00a0mutants); 10\u00a0nM Pol \u03b4 (or Pol \u03b4Cat); 100\u00a0nM RPA; 20\u00a0nM Pol \u03b1; 10\u00a0nM Topoisomerase I; 10\u00a0nM Mrc1; 12.5\u00a0nM Sld3/7; 30\u00a0nM Sld2. The samples were incubated at 30\u00b0C for the indicated times and were processed and imaged as described  through \u223c400\u00a0\u03bcL Sephacryl-S400 High Resolution  columns, previously equilibrated in 25\u00a0mM HEPES-KOH, pH 7.6; 100\u00a0mM potassium glutamate; 0.01% NP-40-S; 1\u00a0mM DTT; 10\u00a0mM Mg(OAc)2; 40\u00a0mM KCl. MCM loading and phosphorylation was performed by adding  3\u00a0mM ATP; 0.1\u00a0mg/ml BSA; 75\u00a0nM Cdt1-Mcm2-7; 40\u00a0nM Cdc6; 25\u00a0nM DDK. After 10\u00a0minutes at 24\u00b0C, S-CDK was added to 25\u00a0mM and incubation continued for 5\u00a0minutes. The nucleotide components were added  and the mix was pre-warmed at 30\u00b0C for 1\u00a0minute. Reactions were initiated by addition of replication proteins as described for standard replication reactions. Nhp6 and FACT were also added to final concentrations of 400\u00a0nM and 40\u00a0nM respectively. For the experiments on the ARS1 template the DDK concentration was 50\u00a0nM, the Sld3/7 concentration was 25\u00a0nM, the GINS concentration was 200\u00a0nM, the Mcm10 concentration was 5\u00a0nM and the RPA concentration was 60\u00a0nM. After quenching by addition of an equal volume of 50\u00a0mM EDTA, proteins were removed from the replication mix by proteinase K  \u2013 SDS (0.1%) treatment, for 15\u00a0minutes at 37\u00b0C, followed by phenol-chloroform-isoamyl alcohol extraction (Sigma-Aldrich). Unincorporated nucleotide was removed using Illustra G-50 columns . Samples were analyzed as described  in Cut Smart buffer, for 30\u00a0minutes at 37\u00b0C. Digests were stopped by adding EDTA to final concentration of 50\u00a0mM, followed by deprotinization with proteinase K - SDS treatment and phenol-chloroform extraction as described above. Sample aliquots were analyzed on 1% alkaline and 0.8% native agarose gels, where required. The remaining digested products were ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 10\u00a0mM Tris-HCl, pH 8; 1\u00a0mM EDTA. For the RNase HII experiments, digestion products were further treated with RNase HII enzyme (NEB) for 1 hour at 37\u00b0C. The reactions were stopped with 50\u00a0mM EDTA and processed as described above for the restriction digests. For polyacrylamide gel analysis an equal volume of 2x loading dye  was added to the samples. Samples were incubated for 3\u00a0minutes at 95\u00b0C, promptly transferred to ice, before being applied to a 40\u00a0cm x 20\u00a0cm denaturing 4% polyacrylamide (Bis-Acrylamide 19:1 \u2013 Fisher scientific), 7\u00a0M Urea, in 1x Tris-Borate-EDTA buffer (TBE) gel. Gels were run for 170\u00a0minutes at constant 40 Watt.Native agarose gels and acrylamide gels were dried directly onto 3MM chromatography paper . Alkaline agarose gels were fixed with two 15\u00a0min incubations at 4\u00b0C in 5% trichloroacetic acid solution before drying on 3MM chromatography paper . For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens  and imaged on a Typhoon phophorimager . Gels were also autoradiographed using Amersham Hyperfilm MP  for presentation.Quantification and data analysis were performed with ImageJ software and Prism 7. For pulse-chase experiments to determine maximum leading-strand synthesis rates  and S1 lFor the initiation site mapping experiments lanes profiles were generated in ImageJ. The migration positions of the replication products were converted to product lengths using standard curves generated from the molecular weight markers. To generate the standard curve the migration position of the marker bands were plot against the Log10 of their length and data were fit to a second order polynomial. Signal from the no enzyme lanes was subtracted from the profiles and data were normalized by dividing by the maximum peak value for a given profile. Initiation site positions relative to the 5\u0374 end of the ACS were then derived by subtracting the distance between the enzyme cleavage site and the 5\u0374 end of the ACS."}
+{"text": "CRISPR\u2013Cas9 enables recombination between homologous chromosome arms at predefined sites and also underscores the need for caution when applying CRISPR technologies in translational medicine. Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms\u2014often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.CRISPR\u2013Cas9\u2013based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in  CRISPR\u2013Cas9\u2013based genome editing has revolutionized genetic research, triggering the development of a plethora of technologies and applications that provide unprecedented control over genes in a growing list of model species , 6, 7, 8Drosophila. Although these findings expand the tool-box of CRISPR-based genome manipulation in research, they also raise concerns about the use of gene editing in therapeutic settings.Here, we set out to examine the occurrence and frequency of genetic exchanges between homologous chromosome arms initiated by Cas9-induced DSBs. We show that Cas9-triggered DSBs induce germline-transmitted recombination between homologous chromosome arms in up to 39% of the CRISPR events in Drosophila , is in-frame with the downstream ORF and resides within the unique 20-nt gRNA target, starting 4 nt upstream of the PAM sequence. Activation of CIGAR is achieved by Cas9-induced DNA cleavage within the STOPT codon. The induced DSBs will then be mended by NHEJ-mediated repair concomitantly severing or eliminating the STOPT codon. The resulting indel leads to repositioning of the upstream ATGs relative to the ORF, that is, causing one of the ATGs to be \u201cshifted in-frame\u201d with the ORF.NHEJ is a major repair mechanism triggered by CRISPR\u2013Cas9\u2013induced DSBs in osophila , 30. Levosophila . CIGAR cery cell , (ii) a ery cell , and /+), we found that in some of the F1 animals, the CRISPR target sequence of the CIGARmCherry reporter became located 5\u2032 of the eGFP ORF and vice versa. Sequence analysis of 84 animals from different crosses revealed a total of 26 animals in which the sequences on one side of the DSB had been exchanged ; molecular analysis of the target site. Deleted bp shown as: \u2013. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 84) for recombination on the X chromosome . Such individuals were crossed to yw animals, and their offspring were scored for recombination events ; molecular analysis of the target site. Deleted bp shown as: \u2013. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 156) for recombination on the fourth chromosome  differed. However, the recombination events could only be demonstrated by sequence analysis of the immediate vicinity of the CRISPR site; more distant phenotypic markers were not present on these chromosomes. Therefore, we could not rule out that, at least in some cases, other mechanisms, such as gene conversion, were responsible for the observed sequence exchange between the two CIGAR reporters in trans .w+-marked CIGARmCherry,102F, w+ and the recessive viable mutation svspa-pol close to the tip of the right arm on chromosome 4. We selected the Cas9 target site, targeted with sgRNA-3, in the 3\u2032UTR of the toy gene residing about 18 kb downstream of the CIGARmCherry,102F, w+ transgene insertion site. To induce recombination at the target site 1021,y+, svspa-pol embryos with active Cas9-sgRNA RNP complexes containing recombinant Cas9 and in vitro\u2013translated sgRNA-3 1021,y+, svspa-pol flies to visually detect recombination events in the offspring , the frequency of CRISPR-mediated targeted recombination in this experiment is estimated to be \u223c1.1%. This frequency is at least four orders of magnitude higher than that of the rare spontaneous recombination rate predicted for chromosome 4 , 41w+-mamosome 4 .Recombination between two visible markers.Table S4 mCherry 102F, w+/Dp(1021) y+, svspa-pol injected with Cas9.List of recombinants recovered from yw; CIGARTable S5 mini-white gene (+w), used as transgene reporter, is juxtaposed to heterochromatic regions via chromosomal rearrangements or translocations 1021,y+, svspa-pol animals exhibiting PEV were crossed to yw; ciD,svspa-pol/sv\u0394122 animals. Without exception, the tested PEV chromosomes were unable to complement the sv\u0394122 mutation, indicating that they must have lost distal parts of chromosome 4.The remaining 36 (out of 57) putative recombinants recovered from the above experiment were homozygous lethal and exhibited position effect variegation PEV;  in the aocations , 44, 45.d to PEV . We, theromosome . To assey+ marker on the short left arm of chromosome 4 and the w+-marked CIGARmCherry,102F transgene inserted at 102F of the right arm 1021, y+, svspa-pol were injected with RNPs containing Cas9 protein complexed with in vitro\u2013translated sgRNA-3. The RNPs induce DSBs 3\u2032 of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were crossed with yw animals to score recombination events between the y+ and the w+ marker  on the fourth chromosome , we repeated the experiment using the same experimental conditions, but assessed recombination between a + marker . We scre+ marker , TR wereIn summary, our experiments involving chromosome 4 revealed that germ-line\u2013transmitted recombinants can be recovered at frequencies ranging from about 1.1% (recombination between visible markers) to 26% . These percentages represent the number of recombinants recovered from the total number of animals analyzed. When only the detected Cas9-mediated events are taken into consideration, the frequency of recombination was even 39%.Drosophila, however, the first meiotic divisions occur at much later timepoints , likelysgRNAs are provided via transgenes (nanos (nos) promoter and the Cas9 transgene contains the nos 3\u2032UTR recapitulating germline-specific nos expression, transcript localization, and translational control -EcoRI fragment from pCaSpR3-Up2-RX polyA was subcloned into a pBluescript (pBS) vector between the EcoRI and XhoI sites using blunt-end ligation. The EcoRI-ubi-Acc65I fragment from the resulting plasmid was subcloned into pEPattB , a unique gRNA target sequence (referred to as sgRNA-1), the linker sequence, and the eGFP gene were designed (as shown below), synthesized by GenScript, and delivered ligated into the pUC57-Kan vector (pCIGAR-D0).To create the The pCIGAR-D0 insert:5\u2032-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCGGCGACAGCAGA ACGTAGCGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCATAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTC_EcoRI-3\u2032.KpnI and EcoRI and ligated into pre\u2013double-digested pKB342 vector in line with a tubulin 3\u2032 trailer, transformed, and purified, resulting in pKB342_CIGAR. To remove the CIGAR sequence including the tubulin trailer, pKB342_CIGAR was then digested with KpnI and XbaI, and the fragment was ligated into a KpnI- and XbaI-digested pUbiattB vector containing an ubiquitin-p63E promoter (see above). This resulted in the final product referred to as pUbiattB-CIGAReGFP.The synthetic sequence was excised with pUbiattB-CIGAReGFP were used for the construction of the pUbiattB-CIGARmCherry. The only difference was in the design of the target site; the gRNA (sgRNA-2) has a different unique target sequence and contains the mCherry gene as the fluorescent marker. The insert ligated into the pUC57-Kan vector was ordered from GenScript.The same sequential digestions and ligation as for the making of the CIGARmCherry insert:The 5\u2032-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCCCCGAGACAAGCACCTGACGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTTAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA_EcoRI-3\u2032.pCFD5 vector was a gift from Fillip Port   following the protocol described in the supplementary methods of pCFD5 cloning protocol.The Addgene) . pCFD5 ipCFD5 internal tRNA multi-gRNA Scaffold:5\u2032-GTCGGGGCTTTGAGTGTGTGTAGACATCAAGCATCGGTGGTTCAGTGGTAGAATGCTCGCCTGCCACGCGGGCGGCCCGGGTTCGATTCCCGGCCGATGCAGGGTCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3\u2032.sgRNA-1 and sgRNA-2 were inserted into the pCFD5 vector using primer (5\u2032-GCGGCCCGGGTTCGATTCCCGGCCGATGCACGGCGACAGCAGAACGTAGCGTTTTAGAGCTAGAAATAGCAAG-3\u2032) for sgRNA-1 and (5\u2032-ATTTTAACTTGCTATTTCTAGCTCTAAAACGTCAGGTGCTTGTCTCGGGGTGCACCAGCCGGGAATCGAACCC-3\u2032) for sgRNA-2.Through the process of Gibson cloning, both our sgRNAs were performed as described in reference IVT of sgRNA-3:IVT oligo used specific for CTGTTGATAAGCACGCAATCGTTTTAGAGCTAGAAATAGC-3\u20325\u2032-GAAATTAATACGACTCACTATAGG.sgRNA-R used for template PCR:IVT oligo used AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3\u2032.5\u2032-sgRNA primer and the sgRNA-R are shown in bold letters.Complementary sequences of the specific sgRNA . This corresponds roughly to a 1:2 ratio (SpCas9: sgRNA-3). SpCas9 has about 5\u00d7 the molecular weight of the sgRNA.The concentration of SpCas9 injected was about 800 ng/\u03bcl SpCas9  and about 300 ng/\u03bcl of sgRNA, ddH2O, and 10\u00d7 incubation buffer NEB and mix thoroughly. Gently add the SpCas9 and mix again thoroughly by pipetting up and down. It is mandatory to add SpCas9 as the last ingredient because low salt concentrations may cause SpCas9 to precipitate : add cipitate .sgRNA .X \u03bcl 1 \u03bcl 10\u00d7 NEB incubation buffer.2O (RNase free) to a total of 10 \u03bcl (X + Y = 6.5 \u03bcl).Y \u03bcl ddH2.5 \u03bcl SpCas9 .Mix gently and incubate the mix at 37\u00b0C for 2 min.Load the mix onto a column (Ultrafree-MC-HV 0.45 \u03bcm [Ref: UFC30HV00]).g.Spin for 1 min in a table-top centrifuge @14,000Reincubate the flow-through at 37\u00b0C for 2 min.Let the mix equilibrate at RT for \u223c30 min before injection. Never put the mix back on ice.Drosophila Stock Center (see the Acknowledgments section).Crosses were done at 25\u00b0C. Unless noted otherwise, fly lines were obtained from the Bloomington ZH-attP 5D [X chromosome] or ZH-attP 102F [fourth chromosome]) were generated by phiC31 integrase-mediated transgenesis ; CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG , respectively. PCR settings were as follows: 95\u00b0C, 5 min; 35 cycles of 95\u00b0C, 25 s; 60\u00b0C, 25 s; and 72\u00b0C, 30 s); final elongation of 72\u00b0C, 10 s. The same PCR setting was used to analyze the shifter sequence of the CIGAR reporters by single fly PCR.Transgenic CIGAR fly lines  was inserted into the attP 40 site .The The images were taken on Axio Zoom V16 (Zeiss) and were processed in Adobe Photoshop or Adobe Illustrator.CIGAReGFP or CIGARmCherry construct on the fourth chromosome at position 102F ). To test if CRISPR-Cas9\u2013mediated DSBs also result in recombination, the fourth chromosome, nos-Cas9/Y; U6:3-sgRNACIGAR/+; CIGAReGFP,102F, w+/CIGARmCherry,102F,w+ G0 males were crossed to yw females /+; CIGAReGFP,102F, w+/CIGARmCherry,102F, w+ G0 females to yw males). The offspring of such crosses were first scored at the larval stage to identify animals with either an activated CIGAReGFP,102F or CIGARmCherry,102F reporter. This preselection was made to ascertain that DSBs occurred in both constructs enhancing the likelihood of detecting recombination events on the fourth chromosome in case they occur. 17 vials  containing GFP and mCherry-positive larvae were selected for further analysis. A total of 172 y,w+ F1 animals harboring either a CIGAReGFP,102F or CIGARmCherry,102F reporter but lacking the sgRNA plasmid U6:pCFD5 (to avoid mosaic flies) from 13 of these crosses were randomly picked right after hatching. Single fly PCR of the target as well as part of the fluorophore region for the CIGAR reporters was performed for these 172 animals (Tables S2 and S3). PCR products and readable sequences were obtained in 156 cases.To determine if we could also induce CRISPR/Cas9\u2013mediated recombination on the fourth chromosome, we first generated flies harboring either a ion 102F . These aFor the recombination experiments using the CIGAR transgenes on chromosome 4, we estimate the recombination frequency to be 26%: 41 recombinants identified/156 flies analyzed \u00d7 100  located downstream of the w+ marker near the tip of chromosome 4  and is visible if the mutation is homozygous or over a null allele of sv such as sv\u0394122 .To confirm that indeed recombination between sister chromatids occurs after CRISPR\u2013Cas9\u2013induced DSBs, we tested if we could induce recombination between two visible markers that are separated by about 100 kb  and S3. mosome 4 . The Casion site . The rouyw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol embryos were injected with recombinant Cas9 protein complexed with in vitro\u2013translated sgRNA-3 targeting the 3\u2032UTR of the toy gene. From the eclosing G0 animals, a total of 135 crosses were set up. Either five G0 females (29 crosses) or single G0 males (106 crosses) were crossed with homozygous Dp1021,y+, svspa-pol flies. F1 offspring were either yw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol  or homozygous Dp1021,y+, svspa-pol animals  1021,y+, svspa-pol and would be scored as having rough, red eyes . The second recombination event (TR-B) that could occur would be yw; Dp1021,y+, sv+/Dp1021,y+, svspa-pol flies having white and smooth eyes . From a total of 8,604 scored F1 animals, 253 putative recombinant flies were recovered: 216 had rough red eyes  and 37 animals had white and smooth eyes (five independent G0 crosses). Notably, in one cross, 31 w\u2212, y+sv+ animals were present in one tube corresponding to about 25\u201330% of the offspring. 70 of the 253 putative recombinants  were backcrossed to homozygous Dp1021,y+, svspa-pol flies to establish stocks. 61 of the 70 putative recombinants were recovered from independent crosses . 13 of the 70 crosses remained without offspring. Most importantly, from 21 putative recombinants, we could generate homozygous viable lines. 17 of the 21 lines showed the y, w+, svspa-pol phenotype, whereas four lines were w, y+, sv+ marking all 21 lines as true recombinants. The remaining 36 putative recombinants were homozygous lethal .spa-pol) . Recombiy, w+, svspa-pol phenotype, the other was phenotypically w, y+, sv+, indicating that we may have recovered both chromosomes from the same recombination event. A third recombined animal from the same cross showed no CRISPR mark and thus can be counted as independent recombination event.We then investigated these animals by PCR specific for the CRISPR target site. Sequence analysis revealed that with the exception of two animals all CRISPR sites were without any indel . Interestingly, two recombinants from the same cross  showed the same CRISPR mark (6-bp deletion). However, they had the complementary phenotype: Whereas one fly showed the y+ marker on the short, left arm of chromosome 4 and the w+-marked CIGARmCherry,102F inserted at 102F 1021,y+, svspa-pol) were injected with Cas9 protein complexed with in vitro\u2013translated sgRNA-3. This again would induce DSBs 3\u2032 of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were this time crossed with yw animals to be able to score crossover events between the y+ and the w+ marker , we backcrossed such animals again against yw flies. Spontaneous recombination between y+ and w+ could be excluded as the above-mentioned backcross would have only revealed phenotypically y+w+ or yw flies. Instead, nondisjunction could be confirmed in all 11 cases because the backcross revealed phenotypically y+w+, yw, y+w, and yw+ flies. We determined the non-disjunction rate for the fourth chromosome to be 1 in 1,200, which is similar to the one observed for the X chromosome (To assess if CRISPR\u2013Cas9\u2013induced DSBs in general would enhance the frequency of recombination away from the Cas9 target site, we repeated the experiment and assessed the recombination frequency between a + marker . As experomosome ."}
+{"text": "The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome\u2019s initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.  \u2022Reconstitution of collisions between a eukaryotic replisome and DNA damage\u2022Leading-strand damage specifically causes fork stalling and uncoupling\u2022The eukaryotic replisome can re-initiate leading-strands downstream of DNA damage\u2022Multiple mechanistic differences exist between leading- and lagging-strand priming To study the earliest events following DNA polymerase stalling during replication, Taylor and Yeeles reconstituted collisions between a yeast replisome assembled with purified proteins and template DNA damage. Surprisingly, re-priming of leading-strand synthesis beyond damage is inefficient but is promoted by RPA depletion, whereas lagging-strand priming occurs robustly after damage. Accurate and efficient DNA replication is vital to ensure faithful and timely transmission of genetic information. This task is accomplished by a complex and highly regulated molecular machine known as the replisome. Replisomes frequently encounter a wide variety of obstacles to their progression, including template DNA damage, DNA secondary structures, and the transcription machinery . ConsideEscherichia coli found that, following UV irradiation of nucleotide excision repair (NER)-defective cells, replication continued and nascent DNA was synthesized as small fragments interspersed with single-stranded DNA (ssDNA) gaps (E.\u00a0coli proteins (E.\u00a0coli replisome has the inherent capacity to \u201cskip\u201d over multiple leading-strand lesions via re-priming (Early studies in NA) gaps . Based oproteins . Howeverproteins . Mechani-priming .Experiments in eukaryotic cells also found that replication was discontinuous following UV irradiation . More rein\u00a0vivo following treatment of cells with DNA-damaging agents  by which it might operate, how efficiently it occurs, or how it might be regulated. In\u00a0this study, we sought to address these outstanding questions by analyzing the response of a reconstituted core eukaryotic replisome to site-specific DNA damage.More generally, how the in\u00a0vivo rate, DNA polymerase epsilon (Pol \u03b5) catalyzes the bulk of leading-strand synthesis in conjunction with PCNA, DNA polymerase delta (Pol \u03b4) is required for complete lagging-strand synthesis, and Mrc1 and Csm3/Tof1 are critical for maximum replication rates , which is one of the primary lesions generated by UV irradiation, at a specific location in plasmid DNA B and devLAG)  with a similar efficiency to those synthesized from an undamaged template  C. The retemplate D, nativents. LAG E, as pronts. LAG G illustrin\u00a0vitro studies  by linearizing plasmids with AhdI  required for the chase. Compared to an undamaged template, replication forks were almost universally delayed for several min by CPDLEAD  composed of stalled leading strands, Okazaki fragments, and a distinct population migrating at the expected position for leading-strand restart  were only visible after AvrII digestion in reactions lacking RPA but containing Pol \u03b1 in step 2  , \u223c60\u00a0bp ynthesis . It is ain\u00a0vivo occurs following treatment with DNA damaging agents, but it had not been possible to exclude other indirect effects of genotoxins or the involvement of DNA repair complexes and the transcription machinery. Because fork progression can be rapidly and efficiently rescued by artificially mimicking leading-strand \u201cre-priming\u201d with an oligonucleotide, we propose that the underlying basis for prolonged fork stalling and continued uncoupled synthesis may simply be a failure to re-prime the leading-strand template. If correct, then observations of stalled, slow-moving, and uncoupled replication forks in\u00a0vivo , isolated stalled forks should be rapidly rescued by forks from neighboring origins. We propose that this may be the principal mechanism by which stalled forks are rescued when DNA damage is infrequently encountered, which would generate daughter-strand gaps for processing by DDT pathways.In striking contrast to E.\u00a0coli , our resin\u00a0vivo. It is notable that some eukaryotes encode an additional primase, PrimPol   promote  PrimPol . PrimPol PrimPol .in\u00a0vitro raises the possibility that re-priming might fulfill a more significant role under conditions of catastrophic global fork arrest, which can have a dramatic effect on free RPA levels in\u00a0vivo .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles  modified to overexpress proteins of interest as detailed in the Key Resources table by transforming with linearized plasmids using standard genetic procedures; or Escherichia coli Rosetta\u2122 2(DE3) cells (Novagen) (genotype: F\u2013\u00a0ompT hsdSB(rB\u2013 mB\u2013) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for overexpression of proteins of interest as detailed in the Proteins were purified from Escherichia coli are listed in the ORC \u2013 ORC, carrying a CBP-TEV tag on the Orc1 subunit, was purified using affinity purification with Calmodulin Sepharose 4B , followed by gel filtration through a Superdex 200 column.Cdc6 \u2013 GST-tagged Cdc6 was bound to Glutathione Sepharose 4B  and released by cleavage with GST-tagged 3C protease. The eluted protein was further purified using a Bio-Gel HT hydroxyapatite column (Bio-Rad).Cdt1-Mcm2-7 \u2013 Cdt1-Mcm2-7 with a CBP-TEV tag on the N terminus of Mcm3 was purified using a Calmodulin Sepharose 4B column, followed by Superdex 200  gel filtration.DDK \u2013 DDK carrying a CBP tag on Dbf4 was bound to and eluted from Calmodulin Sepharose 4B resin. The eluted protein was dephosphorylated with lambda protein phosphatase and was then further purified by gel filtration (Superdex 200).S-CDK \u2013 The S-CDK complex carrying a N-terminal CBP-TEV tag on Clb5 was bound to Calmodulin Affinity Resin (Agilent). Protein was eluted by cleavage with Tobacco Etch Virus protease and was further purified by gel filtration (Superdex 200).Cdc45 \u2013 Cdc45 carrying a double internal FLAG tag was immunoprecipitated using Anti-FLAG M2 affinity gel (Sigma). The resulting eluate was further purified using a Bio-Gel HT hydroxyapatite column.Sld3/7 \u2013 Sld3/7 carrying a C-terminal TCP tag on Sld3 was first bound to IgG Sepharose 6 Fast Flow resin  and the protein eluted by cleavage with Tobacco Etch Virus protease. The His-tagged protease was removed by passing the eluate over Ni-NTA resin (QIAGEN) and the flow through was further purified through a Superdex 200 gel filtration column.Sld2 \u2013 3xFLAG-tagged Sld2  was precipitated from cell lysate with ammonium sulfate, resuspended in buffer, and then immunoprecipitated with Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide (Sigma) and the peptide was removed by binding and eluting the protein from a HiTrap SP HP column .Dpb11 \u2013 Dpb11-3xFLAG was purified using Anti-FLAG M2 affinity gel followed by MonoS  chromatography.Pol \u03b5 \u2013 Pol \u03b5, tagged with a C-terminal CBP tag on Dpb4, was purified sequentially over Calmodulin Sepharose 4B, HiTrap Heparin HP  and Superdex 200 columns.GINS \u2013 The GINS complex, modified with a 6xHis tag at the N terminus of Psf3, was bound to Ni-NTA and eluted with increasing imidazole concentration. Eluted protein was further purified over MonoQ  and Superdex 200 columns, followed a second Ni-NTA column.Pol \u03b1 \u2013 Pol alpha \u2013 primase, modified with an N-terminal CBP tag on Pri1 was purified sequentially over Calmodulin Sepharose 4B, MonoQ and Superdex 200 columns.Mcm10 \u2013 6xHis-Mcm10 was purified over Ni-NTA followed by two rounds of MonoS  chromatography.Ctf4 \u2013 CBP-TEV-Ctf4 was purified by Calmodulin Sepharose 4B chromatography, followed by MonoQ and Superdex 200 gel filtration columns.Mrc1 \u2013 Mrc1-2xFLAG was purified by FLAG immunoprecipitation followed by MonoQ chromatography.Topo I \u2013 CBP-TEV-Topo I was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column (Clontech) and the flow through was concentrated and separated through a Superdex 200 column.Csm3/Tof1 \u2013 Csm3/Tof1 carrying a N-terminal CBP-TEV tag on Csm3 was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column and the flow through was concentrated and separated through a Superdex 200 column.RFC \u2013 The RFC complex containing an N-terminal CBP tag on Rfc3 was purified by sequential chromatography over Calmodulin Sepharose 4B, MonoS and Superdex 200 columns.PCNA \u2013 Native PCNA was purified following overexpression in Escherichia coli. Nucleic acids were precipitated from the cell lysate with polymin P and then proteins were selectively precipitated with ammonium sulfate. Precipitated material was resuspended in buffer and then applied to HiTrap SP HP and HiTrap Heparin HP columns assembled in tandem. The flow through containing PCNA was further purified over HiTrap DEAE Fast Flow  and MonoQ columns.Pol \u03b4 \u2013 Pol \u03b4 with a C-terminal TEV-CBP tag on Pol32 was purified over Calmodulin Sepharose 4B, HiTrap Heparin HP and Superdex 200 columns.RPA \u2013 Native RPA was purified following overexpression in Escherichia coli. Protein was purified over HiTrap Blue HP , ssDNA cellulose (Sigma) and MonoQ columns.Isw1a \u2013 Isw1a carrying a C-terminal 3xFLAG tag on Ioc3 was immunoprecipitated from cell lysate using Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide and was further purified by MonoQ chromatography.Histones \u2013 Native histones were purified following overexpression in Escherichia coli by HiTrap Heparin HP and Superdex 200 chromatography.Nap1 \u2013 GST-Nap1 was bound to Glutathione Sepharose 4B and eluted from the resin with GST-3C protease. Eluted protein was further purified on a MonoQ column.FACT \u2013 His-tagged FACT was purified by TALON and MonoQ chromatography.Nhp6 \u2013 Native Nhp6 was purified following overexpression in Escherichia coli. Proteins were precipitated from the cell lysate with trichloroacetic acid. The precipitated protein was resuspended in buffer and further purified over a HiTrap SP HP column.Proteins were purified as described previously . All yeaThe DNA replication template was produced by direct synthesis of a modified version of the genomic DNA surrounding the early-firing origin ARS306 and subsequent cloning and modification in pBluescript II KS(\u2013). The complete sequence of the resulting two plasmids used for replication of damaged DNA introduced at two different sites is given below.Saccharomyces Genome Database. Modifications were made to (1) introduce restriction enzyme sites to facilitate cloning of three different fragments of this region into pBluescript II KS(\u2013) ; (2) introduce several unique diagnostic restriction enzyme sites in proximity to both sites of DNA damage cloning, used in a variety of different assays; (3) introduce two closely spaced restriction enzymes for cloning of cassettes containing BbvCI nicking endonuclease sites for the subsequent cloning of DNA damage at two different locations approximately 3 kb (PstI and BamHI) or 4.5 kb (SphI and SpeI) left of ARS306; (4) mutate any sequences outside of the origin with a strong ORC binding consensus, to facilitate tight origin specificity of replication initiation; (5) introduce other unique restriction enzyme sites for other purposes, including for asymmetric template linearization with respect the origin. The modified sequence was synthesized by Invitrogen GeneArt Gene Synthesis in three segments flanked by (1) KpnI and SalI; (2) SalI and EagI; (3) EagI and SacI. These segments were cloned using these restriction sites into the multiple cloning site of pBluescript II KS(\u2013). The resulting plasmids were then digested with either PstI and BamHI, or SphI and SpeI, for introduction of cassettes for the subsequent cloning of DNA damage at two different positions.A region of genomic DNA on chromosome III spanning approximately 7.5 kb left and 0.2 kb right of ARS306 was chosen from the \u02c6 indicates position of top strand nicking), was designed: CC\u02c6TCAGCACTTAAGTCC\u02c6TCAGC. This sequence and its reverse complement were incorporated into oligonucleotides flanked by overhangs compatible with the combination of either PstI and BamHI . To achieve this, the following cassette for cloning DNA damage, comprising an AflII restriction enzyme site (underlined) flanked by two Nt.BbvCI sites ) and ZraI (in the far left flank of the modified genomic DNA region) to excise approximately 1 kb DNA and the two blunt ends ligated to give a final plasmid size of approximately 9.7 kb. For the plasmid containing a cassette 4.5 kb from the origin, an additional two restriction enzyme sites, SacI and PsiI, were introduced approximately 1 kb and 1.6 kb respectively downstream of the cassette by site directed mutagenesis to facilitate characterization of the stalled fork. The final plasmids are hereafter referred to as ZN3 (plasmid with a cassette 3 kb from the origin) and ZN5SP1 (plasmid with a cassette 4.5 kb from the origin) and their sequences are given below:CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTATAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGCTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAAGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCTTTCAAAACAGAGTTGTATCTCTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACOligonucleotides for undamaged DNA  or DNA containing an abasic site  or CPD  were synthesized and purified  with 21\u00a0\u03bcL Nt.BbvCI  at 37\u00b0C for 2 h. A further 5\u00a0\u03bcL Nt.BbvCI (50\u00a0U) were then added per tube and digested for a further 1 h. Complete nicking to open circular DNA from covalently closed DNA was confirmed by agarose gel electrophoresis in the presence of ethidium bromide , contain2 with 110\u00a0U T4 DNA ligase (New England Biolabs M0202M) per \u03bcg plasmid at 16\u00b0C overnight in the dark  or AleI (for studying a lagging strand template CPD and undamaged controls), with the following exceptions. In Template linearization was performed in all cases by mixing 8-10\u00a0\u03bcg plasmid DNA in 50\u00a0\u03bcL final reaction volume containing 1X\u00a0CutSmart Buffer (New England Biolabs B7204), 2.5\u00a0\u03bcL each restriction enzyme  and incubating at\u00a037\u00b0C for 3 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected and DNA precipitated with 0.3\u00a0M NaCl\u00a0+ 2.8 volumes ice cold 100% ethanol in dry ice. The pellet\u00a0was harvested, washed with room temperature 70% ethanol, harvested, air-dried and resuspended in 15-20\u00a0\u03bcL TE for use in replication assays.2 with 233\u00a0U T4 DNA ligase (New England Biolabs M0202M) per \u03bcg plasmid at 16\u00b0C overnight in the dark. The following day, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37\u00b0C for 20\u00a0min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected. Ligated template DNA was isolated from oligonucleotides by passing the aqueous phase over Sephacryl S-400 High Resolution matrix  columns (prepared by applying 800\u00a0\u03bcL 50% slurry to 0.8\u00a0mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing 2\u00a0\u00d7 250\u00a0\u03bcL TE by centrifuging at 700 g for 1\u00a0min). 35-40\u00a0\u03bcL was applied per column and collected by centrifuging at 700 g for 2\u00a0min. DNA was pooled, frozen, lyophilized in a vacuum concentrator and resuspended to 250-500\u00a0ng/\u03bcl in TE.Undamaged (maxi prep) and CPD-containing plasmid DNA were linearized with SapI restriction enzyme (New England Biolabs R0569), which cuts approximately 0.5 kb right of ARS306. Biotinylated oligonucleotides  , 2\u00a0M NaCl, 1\u00a0mM EDTA) then mixed with 4\u00a0\u03bcg DNA\u00a0+ buffer A to a total volume of 80\u00a0\u03bcl, and incubated at 25\u00b0C for 30\u00a0min, shaking at\u00a0500\u00a0rpm. The supernatant was discarded and the beads washed with 2\u00a0\u00d7 100\u00a0\u03bcL buffer B (10\u00a0mM HEPES-KOH (pH 7.6), 1\u00a0M KCl, 1\u00a0mM EDTA), 2\u00a0\u00d7 100\u00a0\u03bcL buffer C (10\u00a0mM HEPES-KOH (pH 7.6), 1\u00a0mM EDTA), resuspended in 75\u00a0\u03bcL buffer C and stored at 4\u00b0C in the dark.32P]-ATP using 10\u00a0U T4 Polynucleotide Kinase (New England Biolabs M0201S) in total volume 20\u00a0\u03bcL at 37\u00b0C for 1 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10\u00a0mM Tris-HCl (pH 8.0), 1\u00a0mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . These markers were run as size standards in all gels and cropped from the final image for presentation.Molecular weight standards were prepared by first dephosphorylating 12.5\u00a0\u03bcg (25\u00a0\u03bcl) \u03bb DNA-HindIII Digest (New England Biolabs) with 10\u00a0U Antarctic Phosphatase (New England Biolabs M0289S) in total volume 30\u00a0\u03bcL at 37\u00b0C for 1 h. The DNA was then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 30\u00a0\u03bcL water. 1\u00a0\u03bcg of this DNA was labeled with \u03b3--dCTP, 12.5\u00a0nM Cdt1/Mcm2-7, 7.5\u00a0nM Cdc6, 3.3\u00a0nM ORC, 8.3\u00a0nM DDK, 20\u00a0nM S-CDK. In reactions omitting Pol \u03b4, 117\u00a0mM potassium glutamate was used in all experiments , 100\u00a0mM potassium glutamate, 0.01% NP-40-S, 1\u00a0mM DTT, 10\u00a0mM Mg(OAc)eriments , except eriments F or 4\u00a0mieriments F and S5Geriments  were addFor reactions not requiring post-quenching restriction enzyme digests or native gel analysis, unincorporated nucleotides were removed from the quenched reaction mix with illustra MicroSpin G-50 columns , which also exchanged the buffer to TE. Samples were supplemented with 20\u00a0mM EDTA, and 1/10 volume alkaline loading dye  added. For all other reactions, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37\u00b0C for 20\u00a0min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10\u00a0mM Tris-HCl (pH\u00a08.0), 1\u00a0mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . Except where indicated, samples for native gels were not digested with restriction enzymes, and mixed with native loading dye (20\u00a0mM Tris-HCl (pH 8.0), 50\u00a0mM EDTA, 10% Ficoll 400, 2% N-Lauroylsarcosine sodium salt solution) without additional processing. For restriction enzyme digests for the products of a standard 5-10\u00a0\u03bcL replication assay sample, 0.5-1\u00a0\u03bcL enzyme  was added in 1X CutSmart (New England Biolabs B7204). Enzyme (2-4\u00a0\u03bcl) and buffer volumes were scaled up for larger samples for 2D gels. Digests were performed at 37\u00b0C for 30\u00a0min, except SmaI which was incubated at 25\u00b0C. Digests were quenched with EDTA, samples split where appropriate, and added to native or alkaline loading dye.Denatured samples were analyzed in denaturing 0.6% agarose gels run at 24\u00a0V overnight in 30\u00a0mM NaOH, 2\u00a0mM EDTA. Native samples were analyzed in vertical 1% agarose gels run at 21-24\u00a0V overnight in modified 1X TAE (50\u00a0mM Tris-HCl (pH 7.9), 40\u00a0mM NaOAC, 1\u00a0mM EDTA (pH 8.0)), except for 2, 100\u00a0mM KOAc, 0.1% NP-40-S, 5% glycerol, 0.1\u00a0mg/ml BSA) on ice for 10\u00a0min. ATP (3\u00a0mM), creatine phosphate (40\u00a0mM) and creatine phosphate kinase (140\u00a0\u03bcg/ml) were added and chromatin assembly initiated by addition of DNA and transferring the reactions to 30\u00b0C for 1 h.Chromatin was assembled on 3\u00a0nM (20\u00a0ng/\u03bcl) AhdI- or AleI-linearized DNA in 40\u00a0\u03bcL total reaction volume. Nap1 (3\u00a0\u03bcM), histone octamers (370\u00a0nM) and ISW1 (30\u00a0nM) were pre-incubated in chromatin assembly buffer (25\u00a0mM HEPES-KOH (pH 7.6), 10\u00a0mM Mg(OAc)2, 40\u00a0mM KCl) and collecting by centrifuging at 700 g for 2\u00a0min. For helicase loading and phosphorylation, a volume of chromatin corresponding to the total reaction volume less the contribution from ATP, BSA, Cdt1/Mcm2-7, Cdc6, ORC and DDK was used. Chromatin replication was otherwise performed as for naked DNA templates except where noted in the figures and with the following exceptions: Sld2 was used at 30\u00a0nM; Pol \u03b4 was used at 10\u00a0nM in To prepare chromatin for replication assays, chromatin was exchanged to replication assay buffer by applying 40\u00a0\u03bcL chromatin assembly reaction to Sephacryl S-400 High Resolution matrix  columns (prepared by applying 800\u00a0\u03bcL 50% slurry to 0.8\u00a0mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing with 3\u00a0\u00d7 250\u00a0\u03bcL 25\u00a0mM HEPES-KOH (pH 7.6), 100\u00a0mM potassium glutamate, 0.01% NP-40-S, 1\u00a0mM DTT, 10\u00a0mM Mg(OAc)2 was added to 5\u00a0mM. 20\u00a0\u03bcL was collected and 1\u00a0\u03bcL 20-fold diluted MNase (100\u00a0U)  added and the mixture incubated at 37\u00b0C for 5\u00a0min. The reaction was stopped with 20\u00a0mM EGTA. 5 volumes of buffer PB  were added and the mixture applied to a QIAquick spin column, washed with buffer PE and eluted in 35\u00a0\u03bcL water. Products were analyzed by 1.5% agarose gel electrophoresis.After chromatin assembly CaCl32P]-dCTP. In 2, 0.1\u00a0mg/ml BSA, 3\u00a0mM ATP, 400\u00a0\u03bcM CTP, GTP, UTP, 30\u00a0\u03bcM dATP, dCTP, dGTP, dTTP, 66\u00a0nM \u03b1-[32P]-dCTP, 20\u00a0nM Pol \u03b5, 5\u00a0nM Mcm10, 20\u00a0nM Ctf4, 20\u00a0nM Csm3/Tof1, 20\u00a0nM Mrc1, 20\u00a0nM RFC, 20\u00a0nM PCNA, 10\u00a0nM TopoI, 20\u00a0nM Pol \u03b1. In Helicase loading and phosphorylation was performed by incubating equal volumes of DNA-bound Dynabead slurry in buffer C (prepared as described above) with a mixture containing reaction buffer components, proteins and ATP to give a final reaction mix as used for helicase loading and phosphorylation on a standard soluble template, but without KCl. After treatment with S-CDK as described above, an appropriate volume of the mixture was collected such that it would be diluted 5-fold in the final reaction mix. Replication was initiated as described above but in the absence of \u03b1--dCTP, 20\u00a0nM Pol \u03b1 and RPA (0-400\u00a0nM). RPA was first pre-bound to the template in the absence of Pol \u03b1 for 10\u00a0min. Reactions were initiated by addition of Pol \u03b1 and were incubated for 20\u00a0min. Samples were processed and run through alkaline agarose gels as described for soluble replication reactions.Primed template was prepared by annealing oligonucleotide JY180 (500\u00a0nM) (sequence: Denaturing gels were fixed with two changes of cold 5% trichloroacetic acid and dried onto chromatography paper (Whatman). Native gels were dried directly onto chromatography paper. Most gels were autoradiographed with Amersham Hyperfilm MP  for presentation. For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens  and screens were developed on a Typhoon phosphorimager .Replication product quantification was performed in ImageJ  after converting the .gel files to 16-Bit Tiff files using the Linearize GelData command. Lane profiles shown in In all cases, graphs of quantified data show the mean and errors (SEM) from n (stated in the figure legends) independent experimental repeats."}
+{"text": "DEDD), which is overexpressed in\u2009>\u200960% of\u00a0TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71\u2009kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with\u00a0TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with\u00a0CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with\u00a0TNBC.Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of\u00a0breast cancer. In this study, we reveal that\u00a0Death Effector Domain-containing DNA-binding protein ( The use of of CDK4/6 inhibitors to treat patients with\u00a0TNBC\u00a0is limited by loss of\u00a0Rb. Here, the authors show that a combination of CDK4/6 inhibitor and EGFR inhibitor is effective against DEDD-overexpressing\u00a0TNBC, independent of Rb status. Despite the heterogeneous nature of the tumor, targeted anti-cancer therapies have achieved clinical success through the exploitation of functionally essential genomic alternations, or tumor-specific vulnerabilities3. In the past decade, a compendium of the conceptual frameworks explaining mechanisms of tumor vulnerabilities has been proposed and experimentally validated, including oncogene/non-oncogene addiction4, synthetic lethality5, collateral lethality6, and synthetic essentiality7. A successful clinical translation of cancer genome studies  project)) for effective targeted anti-cancer regimens relies on two prerequisites: (1) tumor-specific genetic or epigenetic events that confer unique vulnerabilities (the Achilles\u2019 heel of cancer) to therapeutic targeting; (2) prognostic biomarkers coupled with an accessible clinical assay for selecting patients who will most likely respond to the targeted therapy.Cancer is a highly heterogeneous genetic disease8. Estrogen receptor (ER)-targeting agents  and human epidermal growth factor receptor 2 (HER2)-targeting therapeutics  have become first-line treatments for ER-positive breast cancer and HER2-positive breast cancer, respectively. Extensive pre-clinical and clinical studies have demonstrated that biomarker-based  patient selection for targeted therapies has led to a significant improvement in cancer treatment with reduced side effects compared to traditional chemotherapies9. Despite such clinical success, targeted therapy options for the most aggressive breast cancer subtype, triple-negative breast cancer (TNBC), remains limited. Because TNBC lack established therapeutic targets , non-specific chemotherapy remains the primary treatment option for TNBC patients. TNBC exhibits a higher level of genome instability and a distinct mutational landscape11. Despite the highly heterogeneous nature of TNBC genome landscapes, some TNBC-specific tumor vulnerabilities are emerging as clinically translatable targets for TNBC patients. For example, poly-ADP ribose polymerase (PARP) was identified to have a synthetic lethal dependency on BRCA1 mutation and has been targeted in TNBC clinical trials, ultimately resulting in recent Food and Drug Administration (FDA) approval of PARP inhibitors for TNBC12. Additionally, bromodomain and extra-terminal (BET) protein13 and the pyrimidine synthesis pathway14 have shown promise to be targetable TNBC vulnerabilities.Breast cancer targeted therapies are among the most successful examples of applying the concept of targeting tumor-specific vulnerabilitiesIn this study, we employed a pooled whole-genome RNA interference (RNAi) functional screen in TNBC cells to explore potential synthetic lethal pathways that synergize with epidermal growth factor receptor (EGFR)/HER2 inhibitor lapatinib (LAP).DEDD) as a potential vulnerability from our screen. Interestingly, DEDD is significantly upregulated in >60% of TNBC tumors. While DEDD has been known to function as a pro-apoptotic protein in the nucleus15, we found that DEDD is strongly expressed in the cytosol of tumor cells. Mechanistically, cytosolic DEDD promotes G1/S cell cycle transition through multiple mechanisms. First, DEDD interacts with heat-shock cognate 71\u2009kDa protein (HSC70) to enhance cyclin D1 expression. Second, overexpressed cytosolic DEDD promotes the proteasome-mediated degradation of retinoblastoma (Rb) family proteins to enable G1/S transition. Addicted to an accelerated G1/S cell cycle progression, tumor cells with DEDD overexpression exhibit an increased susceptibility to the combinatorial treatment of cyclin-dependent kinases 4/6 (CDK4/6) and EGFR inhibitors. Furthermore, a combinatorial regimen of CDK4/6 and EGFR inhibitors synergistically inhibited the progression of TNBC xenografts and patient-derived xenograft (PDX) in vivo. These pre-clinical results provide a strong rationale to extend recently FDA-approved CDK4/6 inhibitors to TNBC patients.Unexpectedly, we identified death effector domain-containing protein  were potentially critical for cell survival  are dysregulated (amplification and messenger RNA (mRNA) upregulation) in more than 40% of TNBC tumors  are\u00a0located close together on chromosome 1q23.3\u201342.1 -containing proteins. Without known enzymatic activity, DEDD executes its biological function primarily through protein\u2013protein interactions via its DED domain18. Previous studies suggested that DEDD may interact with cyclin B1, decrease Cdk1/cyclin B1 activity, and regulate cell size during pre-mitosis phases by facilitating the G1-phase rRNA synthesis19. However, most studies have focused on the capacity of DEDD to promote apoptosis through partnering with other DED-containing proteins20. Since DEDD is involved in pro-apoptotic processes, it is thought to have tumor suppressor activities21. Paradoxically, DEDD is aberrantly overexpressed in TNBC  than TNBC cells with moderate DEDD expression (HCC1806)  incorporation assay. We found that knocking down DEDD significantly decreased EdU incorporation in HCC38 and MDA-MB-468 TNBC cells  for 18\u2009h, and then released cell cycle inhibition by replacing nocodazole media with a fresh culture medium. We found that DEDD protein peaked in cells at the G1 phase, but not other cell cycle phases  Fig.\u00a0, suggestlls Fig.\u00a0, suggestion Fig.\u00a0, suggestDEDD nuclear localization leads to caspase activation, which induces apoptosis, and abolishing the nuclear translocation of DEDD decreases the apoptosis-promoting potential of the protein20. Interestingly, immunohistochemistry (IHC) staining for DEDD in TNBC tissue samples showed that DEDD primarily localizes to the cytoplasm of tumor cells that have high DEDD expression   DEDD or \u0394NLS-DEDD in DEDD-diploid normal cell lines including 293FT and normal human breast epithelial cells (HMECs) 22. Ectopic overexpression of WT DEDD promoted G1\u2013S transition in 293FT cells, which could be significantly diminished by preventing DEDD nuclear-cytosol shuttling using nuclear export inhibitor leptomycin (lepto)  was sufficient to override the G1/S checkpoint and increase the proportion of cells in S phase independent of mitogen activation (serum starvation) as determined by increased EdU incorporation  Fig.\u00a015. Then,to) Fig.\u00a0. This obion Fig.\u00a0. Additioion Fig.\u00a0. Cyclin wal Fig.\u00a0. InteresDEDD executes its functions\u00a0through protein-protein interactions23. To explore interaction partners of DEDD in the cytosol, we immunoprecipitated the \u0394NLS-DEDD protein complex and analyzed co-immunoprecipitated proteins by mass spectrometry. Interestingly, multiple peptides representing the HSC70 were identified  expression, which phenocopied the Rb protein expression pattern in Rb-WT TNBC cell lines  and RBL2 (p130), were reported to be CDK4/6 substratesnes Fig.\u00a0. MoreoveEDD Fig.\u00a0. We furtDEDD, we hypothesized that gain-of-function DEDD in TNBC promotes mitogen-independent G1/S transition and confers addiction to an accelerated cell cycle program, thereby creating a tumor-specific vulnerability for therapeutic targeting. CDK4/6 inhibitors show significant clinical efficacy in cancer treatment by targeting the central G1/S transition machinery29. Three CDK4/6 inhibitors have recently received FDA approval for ER\u2009+\u2009breast cancer, including albociclib (PALBO)\u00a0, ribociclib (RIBO)\u00a0, and abemaciclib (ABE) . Expression of WT Rb, the perceived CDK4/6 downstream target, is currently considered a prerequisite for the clinical anti-cancer efficacy of CDK4/6 inhibitors30.Based on the multifaceted cell cycle-promoting mechanisms of 31, TNBC patients are excluded from CDK4/6 inhibitor clinical trials. Interestingly, we found that targeting CDK4/6 in combination with LAP in DEDD-overexpressed TNBC significantly and synergistically inhibited cell proliferation in both Rb-WT  and Rb-deficient (MDA-MB-468) TNBC cells  rendered the KTB cells resistant to LAP mono treatment, confirming the proliferation-driving function of cytoplasmic DEDD. Interestingly, KTB cells with overexpressed DEDD were more responsive to CDK4/6 inhibitor treatment and Combo treatment  could further shift the sensitivity of TNBC cells to combo treatment. Overexpression of \u0394NLS-DEDD in MDA-MB-436 cells, which moderately express DEDD  is a transcription factor and has been shown to be frequently upregulated in multiple cancers and linked to sustained Akt signalingnes Fig.\u00a0. We nextent Fig.\u00a0. Ectopicent Fig.\u00a0, right. ent Fig.\u00a0. This fuEDD Fig.\u00a0, signifiEDD Fig.\u00a0, Left, slls Fig.\u00a0, right. lls Fig.\u00a0. To testlls Fig.\u00a0. Notablylls Fig.\u00a0.Fig. 6CyDEDD expression and localization as well as the sensitivity of TNBC cells to Combo treatment in vivo. First, we examined DEDD expression and localization in xenograft breast tumors derived from two TNBC cell lines  and two TNBC PDXs that were described previously35. IHC analysis of DEDD showed heterogeneous but strong DEDD expression that was localized predominately to the cytoplasm of epithelial cells within the tumors  transformation in soft-agar assays (data not shown). Likewise, DEDD did not influence overall survival and disease-free survival of TNBC patients who received traditional chemotherapies /AKT pathway through its lipid phosphatase activity, while a nuclear pool of PTEN maintains chromosomal stability41. DEDD has been well documented to be a nuclear-targeting protein through its three nuclear localization sequences18. DEDD functions as an apoptosis facilitator in the nucleus by interacting with caspase-8 and caspase-10, and stimulating caspase-dependent cell death42. Surprisingly, once overexpressed in TNBC cells, DEDD primarily resides in the cytoplasm  phosphorylates Rb on Ser567 in response to genotoxic stress. Hyperphosphorylation of Rb leads to its interaction with E3 ligases Hdm248. However, the functional relevance of the above in relation to observations to tumorigenesis in vivo has not been addressed. In this study, we discovered that aberrant overexpression of DEDD greatly accelerated ubiquitination and proteasomal degradation of Rb family proteins, including Rb protein and Rb family protein RBL1 in TNBC cells  is a transcription target of FoxM1, and FoxM1 promotes breast cancer tumorigenesis by transcriptionally activating PDGFA, which led to further Akt phosphorylation in breast cancer cells54. Supporting such findings, we observed that phosphorylation and total expression of FoxM1 were effectively blocked by CDK4/6 inhibitors  and palbociclib (P-7766) were purchased from LC Laboratories. Abemaciclib (HY-16297), ribociclib (HY-15777), and nocodazole (HY-13520) were purchased from MedchemExpress Inc. Leptomycin B (9676S) was purchased from Cell Signaling Technology. MG-132 (80053-196) was purchased from VWR. CHX (01810) was purchased from Sigma-Aldrich. The 293FT (R70007) cells were purchased from Thermo Fisher Scientific Company. BT-20, BT-474, HCC1806, HCC1937, HCC38, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were obtained from ATCC . The 293FT cell line was cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) high glucose supplemented with 10% FBS and 1% penicillin\u2013streptomycin (Pen-Strep). BT-20, BT-474, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were cultured in DMEM/Ham\u2019s F-12 (1:1) supplemented with 10% FBS and 1% Pen-Strep. HCC1806, HCC1937, and HCC38 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% Pen-Strep. The MCF-10A was originally purchased from ATCC and cultured in DMEM/F-12 medium supplemented with 5% horse serum (Invitrogen), 20\u2009ng/mL EGF, 10\u2009\u00b5g/mL insulin, 0.5\u2009\u00b5g/mL hydrocortisone, 100\u2009ng/mL cholera toxin, and 1% Pen-Strep. Normal human mammary epithelial lines from Komen Tissue Bank (KTB) were obtained from our collaborator Dr. Nakshatri and cultured in DMEM/F-12 supplemented with 10% FBS, 0.4\u2009\u03bcg/mL hydrocortisone, 5\u2009\u03bcg/mL insulin, 20\u2009ng/mL EGF, 24\u2009mg/L adenine, and 5\u2009\u03bcM ROCK  inhibitor (Y-27632). All cell lines were maintained at 37\u2009\u00b0C in a 5% CODEDD complementary DNA (cDNA) sequence  It was obtained from the UCSC Genome Browser.Human DEDD coding sequence:5\u2032-ATGGCGGGCCTAAAGCGGCGGGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGAGGAGACGGGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAAAACGCCGGAAGTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3\u2032.DEDD DEL-NLS coding sequence:5\u2032-ATGGCGGGCCTAGCGGCAGCAGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGCTGAGACTCGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAATCCTGCGGATCTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3\u2032.DEDD cDNA coding sequence was cloned into lentivirus mammalian expression vector pLOVE empty (Addgene #15948) or pLenti-CMV-puro-DEST (Addgene #17452) with an N-terminal 3\u00d7-FLAG tag. DEDD mutant entry vector was assembled through GeneArt Gene Synthesis (Thermo Fisher Scientific). For lentiviral production, a lentiviral expression vector was co-transfected with second-generation lentivirus packing vectors psPAX2 and pMD2.G (Addgene) into 293FT cells using CalFectin . Forty-eight hours after transfection, breast cancer cell lines were stably infected with viral particles. pLOVE-GFP served as packaging and infection efficiency control. Lentiviral-based pLKO.1 DEDD shRNA vectors were purchased from Sigma-Aldrich Co. . The control shRNA vector (pLKO.1 scrambled shRNA) was from Addgene Inc. .Human DEDD-targeting shRNA sequences used in the lentiviral constructs were: shDEDD\u2212493, 5\u2032-CCGGGAGGAGACATCAATTCGCTATCTCGAGATAGCGAATTGATGTCTCCTCTTTTTTG-3\u2032 (targeting CDs); shDEDD-867, 5\u2032-CCGGCATCATCTGTGACATCAAGTTCTCGAGAACTTGATGTCACAGATGATGTTTTTTG-3\u2032 (targeting CDs); shDEDD1056, 5\u2032-CCGGGAAACTCCTGAGGAACTTGATCTCGAGATCAAGTTCCTCAGGAGTTTCTTTTTTG-3\u2032.The Stable expression or knockdown clones were selected by culturing cells with puromycin (1\u20135\u2009\u03bcg/mL) for at least 48\u2009h. The uninfected cells were also treated with puromycin as a control. Infected cells have a multiplicity of infection (MOI) around 0.3\u20130.6.DEDD or 3\u00d7-FLAG-Del-NLS-DEDD:Primers for constructing 3\u00d7-FLAG-DEDD\u2014forward primer: melting temperature (TM): 72.4\u2009\u2103N-3\u00d7-FLAG-5\u2032-CACCATG GAC TAC AAA GAC CAC GAC GGC GAC TAC AAA GAC CAC GAC ATC GAC TAC AAA GAC GAT GAC GAC AAGATGGCGGGCCTA.DEDD\u2014reverse primer: TM: 72.1\u2009\u2103N-3\u00d7-FLAG-5\u2032-TCA GGG CAA TGC TTG CAG CAT CAA GTT CCT CAG GAG TTT CTG TCG GCC CAG CTC ATA GTC.Primers for detecting gene mRNA level:DEDD\u2014forward primer: TM: 56.6\u2009\u21035\u2032-ATGGACGTGACTTCTTATTG-3\u2032.DEDD\u2014reverse primer: TM: 56.4\u2009\u21035\u2032-ATACTTGTCTACAAGATCAGGG-3\u2032.RB1\u2014forward primer: TM: 57.2\u2009\u21035\u2032-CAGAAATGACTTCTACTCGAAC-3\u2032.RB1\u2014reverse primer: TM: 58.9\u2009\u21035\u2032-AATGTGGCCATAAACAGAAC-3\u2032.ABCB10\u2014forward primer: TM: 62.1\u2009\u21035\u2032-CCATATTTCAGGATTTCAGCC-3\u2032.ABCB10\u2014reverse primer: TM: 54.8\u2009\u21035\u2032-GACTAATAGTTCCAGAAGCAG-3\u2032.UAP1\u2014forward primer: TM: 56.7\u2009\u21035\u2032-GATAGTCAGAATGGGAAAGAC-3\u2032.UAP1\u2014reverse primer: TM: 56.3\u21035\u2032-GCATAGGAGATAAGAGGAGAG-3\u2032.17.In vitro pooled drop-out screening by infect HCC1806 cells at 0.3\u2009MOI with DECIPHER shRNA lentiviral library , which includes about 27,000 shRNA constructs targeting 5043 genes on human signaling pathways. After 8 days of puromycin (1\u2009\u00b5g/mL) selection, infected cells were treated with either dimethyl sulfoxide (DMSO) or LAP (2\u2009\u00b5M) for 14 days with replacing fresh medium containing drug every 48\u2009h. After 14 days of treatment, the cells are collected and sent for barcodes sequencing by Illumina sequencing. The row barcode counts were analyzed for the \u201cdrop-out\u201d hits using the MAGeCK software as described beforeN, N-dimethyl formamide) , 10% SDS (sodium dodecyl sulfate) ) overnight. The 96-well plate was read at 570\u2009nm absorbance by a microplate reader. We used the wells with cells untreated as 100% control and the wells with only medium as 0% control. Percentage of inhibition of cell proliferation was calculated as [100\u2009\u2212\u2009((OD treated\u2009 \u2212\u2009OD empty)/(OD untreated\u2009\u2013\u2009OD empty)\u2009\u00d7\u2009100]. For direct cell counting, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of the single treatment or combination treatment for designated times. Cell proliferation was determined by directly counting cell numbers in each well using hemacytometer and compared using the wells with untreated cells as 100% control and the wells with only medium as 0% control. For the clonogenic assay, cells were plated approximately 3000 cells/well in 24-well plates in 10% FBS. After 24\u2009h, cells were treated in triplicate with various concentrations of a single or combination treatment for approximately 2 weeks with media replacement and treatment every 48\u2009h. Cells were washed with phosphate-buffered saline (PBS), fixed, and stained with a solution containing 6% glutaraldehyde and 0.5% crystal violet. Plates were dried, and each well was photographed and quantified using ImageJ 1.43u .For MTT -2,5-diphenyltetrazolium bromide) assays, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of a single treatment or combination treatment for designated times. Cell proliferation was determined by incubating with MTT for 3\u2009h and then in lysing buffer (50% DMF (\u00ae Green Supermix (Bio-Rad). Relative expression levels were calculated using the 2\u2212\u0394\u0394CT method.Total cell RNA will be extracted using TriZol reagent (Invitrogen) following the manufacturer\u2019s instructions. One microgram of total RNA was subjected to reverse transcription to synthesize cDNA using the Verso cDNA Kit (Thermo Fisher Scientific). A 20\u2009\u03bcL volume reaction consisted of 2\u2009\u03bcL reverse transcription product and 20\u2009nM of both forward and reverse primer. The samples were incubated with 2\u00d7 iTaq\u2122 Universal SYBR\u00ae Plus EdU Alexa Fluor\u00ae 488 Flow Cytometry Assay Kit or by Click-iT\u2122 EdU Alexa Fluor\u2122 594 Imaging Assay. In the Click-iT\u00ae Plus EdU Alexa Fluor\u00ae 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) plus 2\u2009\u03bcg/mL propodium podide staining (Thermo Fisher Scientific), cells were rehydrated in 1% bovine serum albumin-PBS at a density of 1\u2009\u00d7\u2009106 cells/mL, transferred into flow cytometry analysis tubes, and stored overnight at 4\u2009\u00b0C prior to samples analysis by Beckman Coulter FC500 Flow Cytometer. EdU signal was detected in FL1 (515\u2013536\u2009nm), and PI signal was detected in FL3 (590\u2013615\u2009nm). Twenty thousand events will be recorded and analyzed using the FlowJo software. In the Click-iT\u2122 EdU Alexa Fluor\u2122 594 Imaging Assay, cells were mounted in ProLong\u2122 Gold Antifade Mountant for 24\u2009h before imaging using the Leica fluorescence microscope in HCRI Tissue Core Facility . All experiments were repeated three times, and quantitative results were analyzed by one-way analysis of variance (ANOVA) or t test, where P\u2009<\u20090.05 was considered statistically significant.Twenty-four hours before the experiment, the culture medium was replaced with medium without FBS. This would synchronize the cells in G0. After 24-h serum starvation, the cells were treated with 1\u2009ng/mL EGFs to induce cell cycle reentry. Cells were treated with 10\u2009\u00b5M of EdU for 1\u20136\u2009h. Cells were collected and processed following the protocol of either Click-iTDEDD plasmid. Forty-eight hours later, cells were treated with 1\u2009ng/mL of EGF and then with 1\u2009\u00b5M MG-132 for 6\u2009h before collection. Harvested cells were re-suspended in 500\u2009\u00b5L of M2 lysis buffer . After incubation for 1\u2009h on ice, cell lysates were incubated with 1\u2009\u00b5g of Rb (4H1) antibody overnight (18\u2009h). One percent of cell lysates were saved as input control. After that, the samples were incubated with 50\u2009\u00b5L of protein A/G agarose beads (Santa Cruz) for 2\u2009h at 4\u2009\u00b0C. Immunocomplexes were washed with M2 lysis buffer three times before immunoblotting. For immunoblotting, proteins were extracted using M2 lysis buffer and proteinase plus phosphatase inhibitor cocktail . Protein extracts were resolved using 4\u201320% gradient SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific #88018). Membranes were probed with primary antibodies overnight on a 4\u2009\u00b0C shaker and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific #34078).For immunoprecipitation, cells first were transfected with either empty or DEDD (Sc-271192) and HSC70 (sc-7298) antibodies were purchased from Santa Cruz Biotechnology. \u03b2-Actin (ab8226) was purchased from Abcam. FLAG M2 (F1804) was purchased from Sigma-Aldrich. Cyclin B1 (#4138), cyclin D1 (#2978), cyclin A2 (#4656), cyclin E1 (#4129), p-Erk (#4370), Erk (#4695), p-EGFR (#3777), EGFR (#4267), p-Akt (#4060), Akt (#4691), Rb (#9309), p-Rb (#9308), RBL1 (#89798), Ubi (#3933), p-FoxM1 (#14655), and FoxM1 (#5436) were purchased from Cell Signaling Technology. For immunoprecipitation assay, antibodies were diluted into 50\u2009ng/\u00b5L. For immunoblotting assay, antibodies were diluted into 2.5\u2009ng/\u00b5L. For immunofluorescence assay, antibodies were diluted into 25\u2009ng/\u00b5L. All immunoblotting original uncropped images were provided as Supplementary materials (Source Data (pptx)) with the manuscript.For immunofluorescence staining, cells were serum starved overnight before stimulation with 1\u2009ng/mL of EGF and treated with 1\u2009\u00b5M MG-132 for 6\u2009h before fixation. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilization in 0.2% Triton X-100 (PBS) and followed by anti-FLAG and Rb antibodies (dilution 1:50) incubation for 18\u2009h at 4\u2009\u00b0C. After incubation with tetramethylrhodamine-conjugated or fluorescein isothiocyanate-conjugated secondary antibody  for 1\u2009h at 25\u2009\u00b0C, images were captured with an Olympus FV1000 2-photon confocal microscope.DEDD plasmids for 72\u2009h before puromycin (1\u2009\u03bcg/mL) treatment for 48\u2009h. The cells were further cultured for two more doublings. The harvested cells were washed with 1\u00d7 PBS and re-suspended in 1:9 ratio of cell mass to extraction buffer (Dynabeads\u00ae Co-Immunoprecipitation (Co-IP) Kit, Thermo Scientific) with protease inhibitors. The cell lysates were incubated on ice for 30\u2009min and then centrifuged at 2600\u2009\u00d7\u2009g for 5\u2009min at 4\u2009\u00b0C to remove large cell debris and nuclei. BCA (bicinchoninic acid) assay was used to quantify the protein concentration in the supernatant. About 5000\u2009\u03bcg of protein was incubated with 1\u2009\u03bcg of anti-FLAG antibody -coupled Dynabeads (10\u2009\u03bcg of antibody) on a roller at 4\u2009\u00b0C for 1\u2009h. Another 2000\u2009\u03bcg of protein was incubated with 1\u2009mg of anti-mouse IgG1 -coupled Dynabeads (10\u2009\u03bcg of antibody) for non-specific control immunoprecipitation. One percent of cell lysates were saved as input control. The Dynabeads\u00ae Co-IP complexes were washed with extraction buffer for three times. The proteins bound to beads were released in 60\u2009\u03bcL of elution buffer provided by the kit. The samples were added five volumes of cold (\u221220\u2009\u00b0C) acetone overnight at \u221220\u2009\u00b0C.Cells were infected with lentiviruses containing pLenti-CMV-puro-3\u00d7-FLAG-\u0394NLS-41. When the tumor reached the volume of approximately 100\u2009mm3, the mice were randomly divided into groups with an even distribution of tumor sizes (10 mice/group). LAP was given every 48\u2009h at a dose of 100\u2009mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Abemaciclib was given every 48\u2009h at a dose of 25\u2009mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Tumor volume was calculated as: tumor volume\u2009=\u20091/2 (length\u2009\u00d7\u2009width2). As designated time point post treatment, tumors were collected and fixed in 4% PFA for 24\u2009h followed by 70% ethanol for another 24\u2009h before formalin-fixed paraffin-embedded embedding for histology analysis.All animal experiments were performed in strict accordance with IACUC guidelines. Rag1 knockout (Rag1\u2212/\u2212) and nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice were obtained from Taconic Farms . For TNBC xenograft experiments, 8-week-old female Rag1\u2212/\u2212 mice were transplanted with one million cancer cells suspended in 0.1\u2009ml of Matrigel mixture (1:1 mixture ratio of Matrigel and DMEM/F-12 medium without FBS) orthotopically into contralateral sides of the fourth mammary fat pads of the mice. For TNBC PDXs, 3.5-week-old female NOD SCID mice have received PDX tumor pieces as previously describedDEDD, Ki-67, or cleaved caspase-3 were detected by DEDD antibody (Santa Cruz Biotechnology #sc-271192), Ki-67 antibody (Ki-67 (D2H10), rabbit monoclonal antibody (IHC-specific) , or cleaved caspase-3 antibody (cleaved caspase-3 (Asp175) antibody, Cell Signaling Technology #9661) through IHC using the Polink-2 Plus HRP Rabbit with DAB Kit (GBI Labs) after 20\u2009min of antigen retrieval. The stained slides were scanned using the Aperio scanner in the HCRI Tissue Core Facility . Images were scored for the percentage of Ki-67 tumor cell nuclei per total tumor cell nuclei in each captured field using the ImageScope software. For DEDD and cleaved caspase-3, images were scored as the percentage of positive staining area per captured total area. All quantification was performed in a fashion that was blinded to the treatment group.PFA-fixed tumor samples were paraffin embedded and cut into 5\u2009\u03bcm thin slices and fixed on glass slides. The raw data for drug synergism analysis were generated by the MTT assay. The cells were evenly plated 24\u2009h before administration of drug dosages. Both single and combination treatments were completed in triplicate at each dosage (4\u20136 different dosages in a series dilution). Then, the absorbance reading of each well was calculated as FA by the treatment. The wells treated with DMSO served as non-affected controls. The wells with medium only served as full-affected control. The mean FAs at different concentrations were then analyzed by the CompuSyn software to generate simulation for the drug synergism analysis. The CI were plotted along with indicated FA for each cell line. CI\u2009>\u20091, antagonism; CI\u2009=\u20091, addition; CI\u2009<\u20091, synergism.http://www.cbioportal.org]. The oncoprint heatmap was generated by including mutations, putative copy-number changes, and mRNA expression z-scores (RNA-seq V2 RSEM with z threshold\u2009\u00b1\u20092). The survival data were extracted from the same dataset for Kaplan\u2013Meier plot in R using \u201cSurvival\u201d and \u201cSurvminer\u201d packages. The boxplot of the TCGA segmented copy-number changes without germline CNV (n\u2009=\u20091099) was generated using the University of California, Santa Cruz (UCSC) Cancer Browser. Gene expression data of commonly used cells lines were extracted from Broad Institute Cancer Cell Line Encyclopedia (CCLE). The heatmap of relative gene expression was generated in R using pHeatmap package. RNA-seq profiling of DEDD knockdown cells was conducted by standard Illumina TruSeq library preparation followed by Illumina sequencing using miSeq. The differential gene expression analysis was performed using DE-seq2 package. An adjusted P value of <0.05 was considered statistically significant. We performed GSEA analysis using GenePattern interface [http://www.broadinstitute.org/gsea/] using C6 v5.2 symbol (oncogenic signature) sub-datasets. Network analysis was conducted through the web-based bioinformatics package NetworkAnalyst [https://www.networkanalyst.ca/]-based on recommended protocol. All quantitative data were analyzed and plotted using the GraphPad Prism software. P values were generated using either two-sided t test or Fisher\u2019s exact test, or one-way ANOVA test.TCGA breast-invasive carcinoma provisional dataset (1105 samples) was sourced from cBioportal [Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data"}
+{"text": "Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and  Unlike the siRNA approach, CRISPR-Cas can permanently alter the function of genes by mutating them. CRISPR-Cas9 derived from Streptococcus pyogenes is the most widely used system because of its ease of use and effectiveness4. The system requires introducing two components: the nuclease Cas9 and a single-guide RNA (sgRNA) which targets the nuclease to a specific genomic locus based on base pairing at a 20-nt target sequence. The target sequence must be followed immediately by the motif 5\u2032-NGG-3\u2032, which is called PAM (protospacer adjacent motif)4. The sgRNA-guided Cas9 generates a double strand break (DSB) in the target site, which may modify the genome in two different ways. The first is random insertion/deletion (indel) mutagenesis which occurs when the DSB is repaired by the error-prone non-homologous end-joining (NHEJ) pathway, often resulting in a knockout or loss-of-function mutation4. The second is precise allele editing through the high-fidelity homology-directed repair (HDR) pathway, which may occur in a small percentage of the cells (<5%)6. More definitive than siRNA, more precise than older transgene insertion methods, and more efficient than older homologous recombination methods8, CRISPR has become one of the most widely used methods to alter a gene to interrogate its function or for medical applications10.The prokaryotic defense system CRISPR-Cas was discovered as an adaptive immunity against foreign DNA molecules and converted to a revolutionary genome editing tool16, but clonal selection and expansion would be still required unless the transduction efficiency and expression of the viral vectors are very high. Among the most widely used viral vectors, lentivirus and adeno-associated virus (AAV) are preferred for human gene therapy applications, but adenovirus (AV) has important advantages as a research tool18. AV has the highest transduction efficiency in diverse target cells20; unlike lentivirus, its genome does not integrate into the host genome, thus reducing off-target mutations; and AV has a substantially higher genome capacity than AAV21. We have used this capacity to co-package three CRISPR-Cas9 components along with their promoters: Cas9, sgRNA and a fluorescent protein to aid in sorting for transduced cells. A fluorescent protein such as GFP or mCherry is also used to monitor active viral production in packaging cell lines such as 293\u2009A and 91121. When active adenoviral particles are made and released from the cells, they infect neighboring cells forming comet-like plaques which can be monitored under fluorescent microscopy. Others have previously demonstrated the feasibility of using AV to deliver Cas9 and sgRNA into diverse target cells, including primary and immortalized cells in vitro, and in vivo18. Most have (like us) used a replication-incompetent AV5 vector lacking E1 and E3; one group used a new AV vector devoid of all viral genes14, but with the trade-off of a more cumbersome production process and less infectable AV. In this study, we demonstrate the efficacy and versatility of an all-in-one adenovirus CRISPR-Cas9 system to target a specific biological pathway, the circadian clock.However, CRISPR has limitations. Transfection of the Cas9 and sgRNA in plasmids typically only succeeds for a minority of cells, and in certain cell lines which do not proliferate or are not easily transfected with plasmids, clonal selection and expansion from the transfected heterogenic population may not be practical or possible. Various CRISPR viral vectors have been employed to try to overcome these problems24. Adverse consequences from having a faulty clock include compromised sleep quality, poor work performance, and increased risk for accidents in the short-term, as well as metabolic diseases and cancer in the long-term. Because the circadian clock is cell autonomous, and rhythms can be measured in cells in real-time using a luciferase reporter under control of a clock promoter, clock mechanisms can be studied in cell culture models such as mouse embryonic fibroblasts (MEFs) or human cell line U2OS27. Clock gene regulation, protein rhythms, and protein-protein interactions in cultured cells are comparable to those in in vivo tissues29. The molecular backbone of the circadian clock is a transcriptional negative feedback loop with interacting positive and negative elements31. CLOCK and BMAL1 are the positive elements, activating transcription of many downstream genes, including behavior-regulating genes and the main negative elements, Period  and Cryptochrome , whose products form an auto-inhibitory complex31. Unlike other core clock genes, Bmal1 has no redundant paralog. Disruption of Bmal1 results in complete arrhythmicity while disruption of a single Cry or Per family member produces subtle circadian phenotypes34.The circadian clock is a molecular circuit that controls daily rhythms in physiology and behavior, such as metabolic oscillations and wake-sleep cycles35, and adenovirus-mediated transduction is ~100% efficient and not cytotoxic, clonal selection and expansion may not be necessary to study the phenotype of knockouts if targeting is properly designed. We showed that a complete disruption of the circadian clock can be achieved in cell culture without clonal selection and expansion by targeting a splicing site in the essential clock gene Bmal1 using the all-in-one adenoviral CRISPR-Cas9 system. We believe interrogation of gene function can be performed in diverse cell lines in a rapid manner by using our all-in-one adenoviral CRISPR-Cas9 system since it does not involve conventional cloning, clonal selection and expansion.In the current study, we developed an all-in-one adenoviral vector where sgRNA can be easily cloned by the Gateway cloning system without labor-intensive conventional cloning and selection. Our vector achieves packaging of sgRNA, the SpCas9 transgene, and a fluorescent protein transgene into the same viral particle. Since indel efficiency of CRISPR-Cas9 is very high, up to ~100%4, we adopted the Gateway cloning technology. Gateway technology was developed by Invitrogen to transfer DNA fragments between plasmids using the bacteriophage lambda site-specific recombination system36. Briefly, a DNA fragment of interest is first cloned into an Entry plasmid where the DNA fragment becomes flanked by specific recombination sequences called attL1 and attL2. The DNA fragment in the Entry plasmid can then be transferred to any Destination vector by LR recombinase. Since only positive recombinants are selected, the subcloning procedure is highly efficient and rapid.To improve upon the conventional procedure of cloning sgRNA into plasmids using enzyme digestion and ligationCbh promoter (pShuttle-Cas9-DEST)  Fig.\u00a0. BrieflyST) Fig.\u00a0. Recombiase Fig.\u00a0. It has Bmal1 promoter27. This cell line is widely used as a model system to study clock mechanisms, and bioluminescence signal from Bmal1-dLuc (or a similar reporter) is widely used as a real-time clock reporter since it faithfully reports the activity of the endogenous clock mechanism.Before packaging the vector into adenovirus, we first tested the functionality of the all-in-one vector construct itself. We used it to edit essential clock genes\u2014whose null phenotypes were well characterized\u2014by transfecting it into a human cell line, U2OS, expressing a rapidly degraded luciferase (dLuc) under a Bmal1. In the mammalian system, Bmal1 is the only essential clock gene without redundancy32. Thus, if CRISPR-Cas9 generates frame-shifting mutations (via NHEJ) in early exons of both alleles of Bmal1, the C-terminal transcriptional activation domain of BMAL1 will be deleted, resulting in circadian arrhythmicity37. Aided by an in silico design tool (http://chopchop.cbu.uib.no), we identified sgRNA sequences targeting four exons of Bmal1  based on the 20 nt sequence immediately preceding SpCas9 PAM sequences in those exons  clones were detected as well as indels , and then the positive cells are expanded before subjected to functional assays. This approach can be applied to many cell lines, but it cannot be used in many primary cell lines that do not proliferate or cell lines that are very difficult to transfect. Moreover, clonal selection and expansion by FACS or drug selection may be labor-intensive and time-consuming, and demands a certain level of expertise.We thus confirmed the functionality of our all-in-one plasmid, but also confirmed the limitations of the typical paradigm of CRISPR-Cas9 mutagenesis21. Thus we hypothesized that high-titer infection of all-in-one CRISPR-Cas9 adenovirus into U2OS cells can achieve ~100% transduction efficiency and generate knockouts in the majority of cells, thus making the downstream clonal selection and expansion unnecessary. To test this possibility, we generated a new adenoviral shuttle vector expressing GFP and Cas9 from two different promoters (pAdTrack-Cas9-DEST)  Fig.\u00a0. The pShncy Fig.\u00a0. The BmaBmal1 exon 6 because our previous transfection experiments found that targeting exon 6 produced more arrhythmic clones (62%) than targeting other Bmal1 exons. Considering significant wt contamination (15\u201330%), targeting near this specific splicing site  machinery was revolutionary in editing the genome to produce precisely engineered knockout and knockin mice. The functional insights from these genetically engineered mouse models cannot be overstated. However, it is also important to recognize the mechanistic insights gained by studying mutant (and wt) cells in culture, as the clock mechanism is cell-autonomous and largely conserved across cell types42. Cell culture models will continue to be critically important for future advances in circadian biology, given their time- and cost-efficiencies. As our understanding of a biological pathway is most significantly advanced by identification of relevant new genes, a next frontier in circadian biology would be to identify new clock genes. We also have much to learn still from the hundreds of natural polymorphisms in known clock genes that lack a corresponding animal model. It has been already demonstrated that some of these polymorphisms are associated with circadian disorders such as Familial Advanced Sleep Phase Syndrome44. In the pre-CRISPR era, mutant cells were usually derived from mutant animals, as cell genomes could not be easily modified in culture. Now, however, cell culture models can be precisely edited using CRISPR-Cas9.As with most biological fields, our current understanding of the circadian clock has been shaped by genetic manipulations that have led to identification of a dozen essential clock genes to date19, but adenovirus can be easily concentrated by density gradient ultracentrifugation to achieve higher titers21. Adenoviral titer can be adjusted to maximize infection rate without cytotoxicity by visual inspection of fluorescence and cell morphology. We routinely measure robust bioluminescence rhythms from ~100% AV-infected MEFs and U2OS cells for several days, demonstrating that the physiology of these cells are not compromised45. Since AV transduction can be 100% efficient in delivering Cas9:sgRNA, whereas FACS sorting following transfection still includes non-transfected cells, AV is a more efficient way to achieve knockouts even when FACS sorting is feasible. A simple serial dilution without FACS sorting would be enough for clonal isolation and expansion for downstream assays , and not possible for all cell types. We show that CRISPR-Cas9 combined with AV can remove the need for clonal selection and expansion, thus addressing all of those issues in AV-infectible cells Fig.\u00a0. We and ays Fig.\u00a0.Bmal1-E7 and Per2-E15 in Fig.\u00a0Bmal1-E6. As a future refinement, we propose that the efficiency can be further increased by using two different adenoviruses targeting two different early exons. We have shown that MEFs could be doubly transduced by AV26. Although AV has been tested to deliver Cas9 and/or sgRNA into cells and showed knockdown of target proteins up to 90% in some cases15, to our knowledge, our present study is the first one to demonstrate a functional knockout without clonal selection by an all-in-one AV. All AV viral genes can be removed in the AV vector to increase packaging capacity and reduce immune response in vivo14. However, when the AV vector devoid of all viral genes is used, packaging efficiency suffers, resulting in vector titers ~two orders of magnitude lower, even after multiple rounds of amplification, compared to ours14. AV can also be employed to deliver catalytically-inactive Cas9 as a synthetic transcription factor. It has been shown that a catalytically-inactive Cas9 can be targeted to promoters to activate or inhibit transcription of their downstream genes10. AV is superior to lentivirus for that purpose because expression levels of transgenes are much higher by AV than lentivirus.Even if transduction of Cas9-sgRNA is near 100% and if Cas9-sgRNA can induce indels in ~100% of cells, one may expect frame-shifting mutations to occur only in 2/3 of cases. However, based on our results, the NHEJ repair process is not random, but rather locus-specific  was PCR-amplified with primers with attL1 and attL2 sequence and subcloned into pEAR A1A .pEntry-sgRNA was generated by Mutagenex  as follows. U6-sgRNA fragment from pU6-(BbsI)_Cbh-Cas9-T2A-mCherry plasmid (Addgene #6432421). Then, R1-CmR-ccdB-R2 fragment from the Gateway pDEST  was subcloned into the intermediate vector _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324) was subcloned into XbaI and NotI sites of pShuttle , respectively _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324), and R1-Cmet al.4 The final PCR products were purified and mixed with either pShuttle-Cas9-DEST or pAdTrack-Cas9-DEST along with LR clonase (Thermo Fisher Scientific #11791020) to generate final all-in-one vectors. Bacterial colonies transformed with the reaction mixture were screened by colony PCR that amplifies DNA region flanked by R1 and R2 (negative clones), or B1 and B2 (positive recombinants). Two different sets of primers were used for pShuttle and pAdTrack as follows.The first round of PCR was performed using two sets of primers as described in Table\u00a0pShuttle-Cas9-DEST:attR1 Up Fwd: 5\u2032-GAGCCCACTGCTTACTGGCTTATC-3\u2032Cbh Rev: 5\u2032-CGTACTTGGCATATGATACACTTGA-3\u2032Size of PCR amplicons: 1,966\u2009bp negative clone and 691\u2009bp for positive recombinant.pAdTrack-Cas9-DEST:KpnIattR1F: 5\u2032-ATAGGTACCCCCACTGCTTACTGGCTTATCGAAATTAATAC-3\u2032KpnIattR2R: 5\u2032-CCGTAAGTTATGTAACGGGTACCTCTAGATCAACCAC-3\u2032Size of PCR amplicons: 1820 bp for negative clone and 545\u2009bp for positive recombinant.Bmal1 promoter was described previously. For transfection with the all-in-one plasmids, cells were plated into 6\u2009cm dishes to be approximately 60% confluent on the day of transfection. Cells were transfected with Polyfect  according to the manufacturer\u2019s protocol and incubated for 2 days before they were subjected to trypsinization and FACS sorting using BD FACSAria SORP equipped with an Automated Cell Deposition Unit (ACDU) for sorting into 96 well plates. Cells were trypsinized with Trypsin (0.25%)-EDTA (2.21\u2009mM) for 3\u2009min and were filtered through mesh with 50um pores. FACS-sorted cells were collected into three groups: negative, intermediate and bright mCherry, and plated into 35\u2009mm dishes and grown for 2 days before harvest and genomic DNA extraction. Before the cells were harvested, they were inspected for mCherry expression. Even the bright mCherry group included 10\u201330% of non-mCherry cells. Harvested cell pellets were homogenized in 250 ul solution A containing 0.1\u2009M Tris-HCl pH\u2009=\u20099.0, 0.1\u2009M EDTA, and 1% SDS, and incubated at 70\u2009\u00b0C for 30\u2009minutes. 35 ul 8\u2009M potassium acetate was added and incubated at room temperature for 5\u2009minutes. The samples were centrifuged at 13,000\u2009rpm for 15\u2009minutes and genomic DNA was purified by subjecting the supernatant to phenol-chloroform extraction and ethanol precipitation. The extracted genomic DNA pellet was dissolved in 100 ul water and used as a template to PCR amplify the target genomic locus with a set of primers . Digestion products were resolved on 8% acrylamide/bis TBE gel and visualized with EtBr.A U2OS cell line expressing luciferase under a Bmal1 exon 7 and Per2 exon 15. Wt contamination was confirmed: 4 out of 16 were wt for Bmal1 exon 6 and 2 out of 15 were wt for Per2.PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 6, 8 and 9, and Per2 exon 5 were set up for bioluminescence monitoring as described previously19. For immunoblotting for BMAL1, a previously described anti-BMAL1 antibody (GP3) was used47. For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1\u2013200 peptide by Cocalico Biologicals, Inc . hP2-GP49 was used in the current study. The antibody was validated by oscillations of human PER2 in U2OS cells and a novel monoclonal antibody against human PER2 (hP2-C6A3). The monoclonal antibody was selected from clones of antibodies raised against aa 1\u2013200 of human PER2 by Boreda Biotech . The monoclonal antibody was also able to detect oscillations of PERs in U2OS cells similarly . When compared with 293 cells, expression forming units (efu) of the adenovirus was 5\u201310 fold lower for U2OS cells. However, U2OS cells could be infected with ~100% efficiency without further purification, unlike infection for MEFs19. To ensure 100% infection in U2OS cells with the all-in-one adenovirus, the cells were infected at MOI of 50 twice, two days apart.Adenoviruses expressing mutant BMAL1 lacking the DNA-binding domain or wt BMAL1 were described previouslyCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATATGCCATGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCNNN: MCST7-F 5\u2032-TAATACGACTCACTATAGGGT3-F 5\u2032-ATTAACCCTCACTAAAGGGA1 Seq-F 5\u2032-GATTAAGTTGGGTAACGCCAGGGA1 Seq-R 5\u2032-CTTGAGCGTCGATTTTTGTGATGCA1 Flk-F 5\u2032-GTAATACGACTCACTATAGGGC.Supplementary figures"}
+{"text": "Chronic exposure of pancreatic \u03b2-cells to excess free fatty acids is thought to contribute to type 2 diabetes pathogenesis in obesity by impairing \u03b2-cell function and even leading to apoptosis. In \u03b2-cells, lipid droplet-associated protein perilipin 5 (PLIN5) has been shown to enhance insulin secretion by regulating intracellular lipid metabolism; the roles of PLIN5 in response to lipotoxicity remain poorly understood.INS-1 \u03b2-cells were transfected with PLIN5-overexpression adenovirus (Ad-PLIN5) and treated with palmitate. C57BL/6\u2009J male mice were fed with high fat diet and tail intravenous injected with adeno-associated virus overexpressing PLIN5 (AAV-PLIN5) in \u03b2-cells.Our data showed that palmitate and PPAR agonists including WY14643 (PPAR\u03b1), GW501516 (PPAR\u03b2/\u03b4), rosiglitazone (PPAR\u03b3) in vitro all induced PLIN5 expression in INS-1 cells. Under palmitate overload, although upregulating PLIN5 promoted lipid droplet storage, it alleviated lipotoxicity in INS-1 \u03b2-cells with improved cell viability, cell apoptosis and \u03b2-cell function. The protection role of PLIN5 in \u03b2-cell function observed in cell experiments were further verified in in vivo study indicated by mitigated glucose intolerance in high fat diet fed mice with \u03b2-cell-specific overexpression of PLIN5. Mechanistic experiments revealed that enhanced FAO induced by elevation of PLIN5, followed by decreased ER stress may be a major mechanism responsible for alleviation of lipotoxicity observed in the present study.Our finding substantiated the important role of PLIN5 in protection against lipotoxicity in \u03b2-cells.The online version of this article (10.1186/s12986-019-0375-2) contains supplementary material, which is available to authorized users. The prevalence of type 2 diabetes (T2DM), a chronic metabolic disorder, has been increasing steadily, partially due to the rising obesity rates. Excess adiposity and dyslipidemia commonly seen in obesity not only induce insulin resistance but also directly damage \u03b2-cell function, a fundamental defect in diabetes . Indeed,Excessive FFA are mainly stored in the form of triglyceride (TG) in lipid droplet (LD) in most of mammalian cells. LD consist of a score of neutral lipids  surrounded by a phospholipid monolayer in which are embedded proteins named LD-associated proteins . In mammThe perilipin family proteins (PLINs) were the major LD-associated proteins that encompass five members, from PLIN1 to PLIN5. In contrast to PLIN1, which is mostly highly expressed in white adipose tissue, PLIN5  is mainly expressed in tissues with high oxidative capacity such as red muscle, heart, liver and brown adipose . As a LD12 viral genome/ml. After 5-month dietary manipulation, AAV-PLIN5 or AAV-MIP was delivered to HFD mice by tail vein injection at 5\u2009\u00d7\u20091011 viral genome per mouse. CHOW mice were injected with saline as the control group. 1\u2009week after injection, fasting (12\u2009h) and fed glucose (2\u2009h) levels were determined from tail vein blood with a glucometer . 10\u201314\u2009days after injection, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted. For GTT, mice were fasted for overnight (16\u2009h) and then injected i.p. with D-glucose at 1.5\u2009g/kg of body weight. For ITT, mice were performed after 6\u2009h fasting and injected i.p. with human regular insulin  at 0.75\u2009IU/kg of body weight. Blood glucose was measured from tail vein blood at the indicated time interval after the injection using a glucometer (FreeStyle). The areas under the curves for blood glucose in GTT (AUCg) and ITT (AUCitt) were calculated. Serum insulin at 0, 15, 30\u2009min after GTT  and fasting serum FFA  were measured according to manufacturers\u2019 instructions. At the end of the study, mice pancreatic islets were obtained as described previously  reacting with mitochondrial dehydrogenase to produce an orange water soluble formazan dye. Prior to the analysis, the INS-1 cells were plated in 24-well cell culture plates at a density of 5\u2009\u00d7\u200910Cell apoptosis was measured by flow cytometric analysis. Briefly, after treatments, the cells were trypsinized, washed with PBS, and suspended in 195\u2009\u03bcl binding buffer. Then, cells were incubated with 5\u2009\u03bcl Annexin V-FITC for 15\u2009min and 5\u2009\u03bcl propidium iodide  for 5\u2009min at 4\u2009\u00b0C in the dark. The cells were then analyzed on a FAC Scan flow cytometer  to quantify apoptosis.INS-1 cells were treated with adenovirus for 24\u2009h in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), after which 0.5\u2009mM PA was added to the medium for another 12\u2009h or 48\u2009h. Cells were starved for 2\u2009h in glucose-free RPMI 1640 and then were washed twice with glucose-free Krebs-Ringer bicarbonate (KRB) buffer (pH\u20097.4). GSIS was investigated as described previously, with minor modifications . BrieflyS6K, Bcl-2, phospho-mTOR (p-mTOR), total mTOR which were all purchased from Cell Signaling and PLIN5 from Progen Biotechnik, ACO from Abcam, CHOP from Immunoway, CPT-1 and BiP from Santa Cruz. All the secondary antibodies and internal reference GAPDH were all purchased from Santa Cruz. Immunoreactive bands were visualized with an ECL reagent kit (Millipore). Optical densities of each band were calculated and analyzed by using Image J analysis software.Proteins were extracted and immunoblotted as described previously . The folCyclin D1, Cyclin D2, spliced XBP-1 (XBP-1(s)), sterol-regulatory element binding protein-1c (SREBP-1c), CHOP, BiP, and PLIN5 were detected using primers indicated in Table\u00a0Total RNA from INS-1 cell line was isolated using TRIzol reagent . First-strand cDNA was synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase and random hexamer primers . Gene expressions were analyzed using the SYBR Green PCR system, following the manufacturer\u2019s recommendations . t-tests. To compare data sets of more than two groups, we used one-way ANOVA followed by Bonferroni\u2019s multiple comparison tests. Two-way ANOVA determined significance for GTT and ITT. P\u2009<\u20090.05 (two-tailed) was considered significant.All quantitative data were presented as means \u00b1 SEM and cell experiments were performed at least three times. Differences of numeric parameters between two groups were assessed using Student\u2019s PLIN5. Hence, the upregulation of PLIN5 by PA may dependent on PPAR\u03b1 activation in INS-1 cells. And the induction role of PLIN5 expression by PPAR\u03b2/\u03b4 and/or PPAR\u03b3 activation is indirect which needs to be investigated in the future study.Recently, PLIN5 was confirmed to be an LD protein in both human and murine islets and altering expression level of PLIN5 can impact insulin secretion by regulating lipolysis . However. In fasted islets, elevated PLIN5 expression and high TG content was observed recently [SREBP-1c . However, upregulating PLIN5 compromised this induction effect of PA which suggesting the mitigation of ER stress induced by PA overload . Furthermore, overexpressing CPT-1A in PLIN5 deficient cells alleviated the induction of BiP, CHOP by PA incubation  and GFP, with sites for ECORI, BamHI, NdeI, SacI and ApaI. The size of AAV2/8 vector, MIP and PLIN5 was 4,492\u2009bp, 874\u2009bp and 1,392\u2009bp respectively. Left: map of AAV-PLIN5; Right: map of AAV-MIP. Sequences of MIP: 5\u2032-TTGTAGCTGGAATAGAGCATGCACTAACAGATGGAGACAGCTGGCTTTGAGCTCTGAAGCAAGTATTACATATGGAGACTTGCTGGCCTTCAGGTGCTTATCTTGTTATTGGATACTGCAGGAGGATGTACCACAGGGCTTCAGCTCAGCTGACCCCCAAGTGGGATATGGAAAGAGAGATAGAGGAGGAGGGACCATTAAGTGCCTTGCTGCCTGAATTCTGCTTTCCTTCTACCTCTGAGAGAGAGCTGGGGACTCGGCTGAGTTAAGAACCCAGCTATCAATTGGAACTGTGAAACAGTCCAAGGGACAAAGATACTAGGTCCCCAACTGCAACTTCCTGGGGAATGATGTGGAAAAATGCTCAGCCAAGGACAAAGAAAGCATCACCCACTCTGGAACAATGTCCCCTGCTGTGAACTGGTTCATCAGGCCATCAGGGCCCCTTGTTAAGACTCTAATTACCCTAGGACTAAGTAGAGGTGTTGACGTCCAATGAGCGCTTTCTGCAGACCTAGCACCAGGGAAGTGTTTGGAAACTGCAGCTTCAGCCCCTCTGGCCATCTGCTGACCTACCCCACCTGGAGCCCTTAATGGGTCAAACAGCAAAGTCCAGGGGGCAGAGAGGAGGTGCTTTGGTCTATAAAGGTAGTGGGGACCCAGTAACCACCAGCCCTAAGTGATCCGCTACAATCAAAAACCATCAGCAAGCAGGAAGGTACTCTTCTCAGTGGGCCTGGCTCCCCAGCTAAGACCTCAGGGACTTGAGGTAGGATATAGCCTCCTCTCTTACGTGAAACTTTTGCTATCCTCAACCCAGCCTATCTTCCAGGTTATTGTTTCAACA-3\u2032 Sequences of PLIN5: 5\u2032-ATGTCTGAAGAAGAGGCGGCTCAGATCCCCAGATCCAGTGTGTGGGAGCAGGACCAGCAGAACGTGGTGCAGCGTGTGGTGGCTCTGCCCCTGGTCAGGGCCACGTGCACCGCGGTCTGCGATGTTTACAGTGCAGCCAAGGACAGGCACCCGCTGCTGGGCTCCGCCTGCCGCCTGGCTGAGAACTGCGTGTGCGGCCTGACCACCCGTGCCCTGGACCACGCCCAGCCGCTGCTCGAGCACCTGCAGCCCCAGCTGGCCACTATGAACAGCCTCGCCTGCAGGGGCCTGGACAAGCTGGAAGAGAAGCTTCCCTTTCTCCAGCAACCTTCGGAGACGGTGGTGACCTCAGCCAAGGACGTGGTGGCCAGCAGTGTCACGGGTGTGGTGGACCTGGCCCGGAGGGGCCGGCGCTGGAGCGTGGAGCTGAAGCGCTCCGTGAGCCATGCTGTGGATGTTGTACTGGAAAAATCAGAGGAGCTGGTGGATCACTTCCTGCCCATGACGGAGGAAGAGCTCGCGGCACTGGCGGCTGAGGCTGAAGGCCCTGAAGTGGGTTCGGTGGAGGATCAGAGGAGACAGCAGGGCTACTTTGTGCGCCTCGGCTCCCTGTCAGCACGGATCCGCCACCTGGCCTACGAGCACTCTGTGGGGAAACTGAGGCAGAGCAAACACCGTGCCCAGGACACCCTGGCCCAGCTGCAGGAGACGCTGGAGCTGATAGACCACATGCAGTGTGGGGTGACCCCCACCGCCCCGGCCCGCCCTGGGAAGGTGCACGAGCTGTGGGGGGAATGGGGCCAGCGCCCTCCGGAGAGCCGCCGCCGGAGCCAGGCAGAGCTGGAGACGCTGGTGCTGTCCCGCAGCCTGACCCAGGAGCTGCAGGGCACGGTAGAGGCTCTGGAGTCCAGCGTGTGGGGCCTGCCCGCCGGCGCCCAGGAGAAGGTGGCTGAGGTGCGGCGCAGTGTGGATGCCCTGCAGACCGCCTTCGCTGATGCCCGCTGCTTCAGGGACGTGCCAGCGGCCGCGCTGGCCGAGGGCCGGGGTCGCGTGGCCCACGCGCACGCCTGCGTGGACGAGCTGCTGGAGCTGGTGGTGCAGGCCGTGCCGCTGCCCTGGCTGGTGGGACCCTTCGCGCCCATCCTTGTGGAGCGACCCGAGCCCCTGCCCGACCTGGCGGACCTGGTGGACGAGGTCATCGGGGGCCCTGACCCCCGCTGGGCGCACCTGGACTGGCCGGCCCAGCAGAGAGCCTGGGAGGCAGAGCACAGGGACGGGAGTGGGAATGGGGATGGGGACAGGATGGGTGTTGCCGGGGACATCTGCGAGCAGGAACCCGAGACCCCCAGCTGCCCGGTCAAGCACACCCTGATGCCCGAGCTGGACTTCTGA-3\u2032 (EPS 980 kb)"}
+{"text": "Clerodendron cyrtophyllum, was isolated from root of Clerodendron cyrtophyllum Turcz, which was able to produce the highest activity of \u03b2-glucosidase up to 33.72 U/mL at 144 h during fermentation on Potato Dextrose Broth (PDB). The obtained fungus was grown on isoflavones-rich soybean extract to produce genistein and daidzein, achieving the conversion rate of 98.7%. Genistein and daidzein were isolated and purified by column chromatography using hexane/acetone (29:1/1:1), reaching purities of over 90% of total isoflavones, as identified and determined by TLC, LC-MS/MS, and 1H and 13C NMR spectroscopy. These results imply that the isolated P. citrinum is a potential fungal strain for industrial-scale production of genistein and daidzein from isoflavones-containing soybean extracts. These products may serve as potential raw materials for manufacture of functional foods that are based on aglycones.Soybeans offer an abundant source of isoflavones, which confer useful bioactivities when existing in aglycone forms. The conversion of isoflavones into aglycones via fermentation of soybean products is often realized by \u03b2-glucosidase, an enzyme produced by fungi. In this study, a filamentous fungus,  Isoflavones are polyphenolics that exert estrogen-like effects and have been widely utilized in manufacture of foods and cosmetics . IsoflavGlycine max) ; ;  with a flow rate of 1.0 mL/min. Mass spectra of Electron Spray Ionisation (ESI) were recorded on the Agilent 1100 Series mass spectrometer connected with Varian 320-MS . 1H-NMR spectra were recorded by 500 MHz  with DMSO-d6 or acetone-d6 as solvents and tetramethylsilane as an internal standard. 13C-NMR spectra were recorded at 125 MHz (Bruker XL-500) with DMSO-d6 or acetone-d6 as solvents and as internal standard.To perform TLC (thin layer chromatography) analysis, silica gel 60 Fp < 0.05 via Student\u2019s t-test. Statistical analysis was performed in the JMP Pro 13.2 software.Experiments were carried out in triplicate for the accuracy of data. Statistical significant differences were realized at Clerodendron cyrtophyllum Turcz sp. were used as microbial sources to isolate fungi with \u03b2-glucosidase activity on PDA medium. At 10\u22125 dilution, colonies were isolated separately. They were then subcultured on sterilized PDA plates several times to obtain pure culture for identification and \u03b2-glucosidase enzyme assay. After several subcultures at 30 \u00b0C for 72 h, 10 fungi were isolated with different morphological characteristics of colony. Five fungi were isolated from fresh samples (designated as BC) of C. cyrtophyllum Turcz\u2019s while five other fungi were isolated from dried plant samples (designated as BSD).Roots from For enzymatic screening and fungal selection, the ten fungal isolates were tested for their capability of enzyme production. The \u03b2-glucosidase activity of these fungi is shown in the Aspergillus species.On SA medium, colonies of BC2 grew fast and displayed a compact green or yellow basal felt enclosed by a layer of white of erect conidiophores. The diameter of colonies was approximately measured as 2.4 cm. Microscopically, conidiophore stipes are long and smooth-walled and the color of hyaline turned dark towards the vesicle. Conidial heads were biseriate, large, globose, and showed tendency to radiate, splitting into several loose columns with age. Additionally, phialides, usually in the form of septate metulae, were also observed. These morphological characteristics affirm that this fungus belongs to the Penicillium species.The shade of colonies of BSD5 growing on SA medium was green in color and a dense felt of conidiophores was observed. The average diameter of colonies after three days of incubation was 1.2 cm. Observing under a microscope, conidiophores were hyaline and smooth-walled. In addition, terminal verticils, carrying 3\u20135 metulae each, were observed on conidiophores. In each metula, around 3\u20137 phialides were recognized. Regarding conidia, they are smooth-walled and their shape appeared to be globose or subglobose. In addition, the production of conidia was basipetal from the phialides. Therefore, the described fungus belonged to the Penicillium sp. was identified to ensure that this fungus belonged to Penicillium sp. Penicillium sp. was sequenced as:After identification by morphological characteristics, genomic DNA of the \u201cCATGCTCCGGCCGCCATGGCGGCCGCGGGAATTCGATTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGCCCCTCGGGGCCCAACCTCCCACCCGTGTTGCCCGAACCTATGTTGCCTCGGCGGGCCCCGCGCCCGCCGACGGCCCCCCTGAACGCTGTCTGAAGTTGCAGTCTGAGACCTATAACGAAATTAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCCCCCGCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGCTCTAGTAGGCCCGGCCGGCGCCAGCCGACCCCCAACCTTTAATTATCTCAGGTTGACCT\u201d.Penicillium citrinum existing in the database, as evidenced by the excellent bootstrap results. Therefore, the examined fungus presumably belonged to the Penicillium genus and firmly aligned with the P. citrinum species, as demonstrated by the the strong sequence similarities with the said species.To gain insights into the evolutionary relationship, two methods for creation of the phylogenetic trees, namely Neighbor Joining (NJ) and Maximum Parsimony (MP), were employed, resulting in almost identical topologies. The aligned dataset consisted of 42 taxa and 100 characters. Our sequence (marked as unknown) achieved 100% matching with those of The optimum incubation time is the time interval at which the highest \u03b2-glusosidase activity was attained. After incubation for 24, 48, 72, 96, 120, 144, and 168 h, \u03b2-glusosidase activity was 0.23, 2.56, 8.16, 21.25, 23.55, 33.37, and 23.23 U/mL , respectP. citrinum was tested to grow in fermentation medium containing various carbon sources under the temperature of 30 \u00b0C, agitation speed of 200 rpm, and for six days. Carbon source is a critical factor affecting the production of enzymes. Therefore, v/v) volumetric ratio of P. citrinum inoculum to CDN substrate, the transformation into aglycones from glucoside forms complete took place during 72 h of fermentation (v/v) of P. citrinum enzyme to CDN with fermentation time of 72 h at 30 \u00b0C.entation . This in15H10O4, daidzein) with molecular weight of 254.23 (15H10O4). Data indicates that daidzein obtained after extraction and purification from fermentation broth is of high purity. Similarly, genistein ion fragments appeared at m/z 269.0 ) appeared as yellow-brown with 5% FeCl3 and dark green with Ce(SO4)2 (data not shown). 1H-NMR : \u03b4 (ppm) 6.28 , 6.41 , 6.90 , 7.46 , 8.163 , 13.03 . 13C-NMR : \u03b4 (ppm) 94.15 , 99.52 , 105.87 , 115.65 , 122.75 , 123.76 , 130.86 , 153.98 , 163.64 , 158.76 , 158.13 , 164.70 , 181.35 .For genistein , light yellow needle crystals, Rf = 0.30 ) appeared as dark-green with Ce(SO4)2 and colorless with 5% FeCl3 (data not shown). 1H-NMR : \u03b4 (ppm) 6.89 , 6.98 , 7.47 , 8.06 , 8.14 . 13C-NMR : \u03b4 (ppm) 102.04 , 115.07 , 116.58 , 123.43 , 127.23 , 129.99 , 125.74 , 157.11 , 157.37 , 162.45 , 174.63 .For daidzein ; white needle crystals, RPenicillium funiculosum was 30\u201336 U/mL, achieved on 3% rice bran or defatted oil cakes after 288 h fermentation. By stark contrast, the peak activity was found at a much lower level of 2.8 U/mL with Penicillium miczynskii cultured on 3% pineapple peel within 216 h of fermentation  to their aglycones by ectively , on soy hila J18 . MoreoveIsoflavone aglycones can be derived from soybean waste by extraction with ethanol/water solvent followed by acid hydrolysis and purification , reachinP. citrinum fungus strain that produces the highest \u03b2-glucosidase activity of 33.72 U/mL, thus placing it amongst the most active fungi in this regard, has been successfully isolated from roots of Clerodendron cyrtophyllum Turcz. The fungus demonstrated a catalytic capacity to hydrolyze isoflavones-rich soybean extract into aglycones  with a hydrolysis yield of 98.7% after 72 h of fermentation at 30 \u00b0C. Purification of the hydrolyzed mixture (rich in genistein and daidzein) by column chromatography using hexane/acetone (29:1/1:1) resulted in aglycone products with purity of over 90%. These results imply that soybean extract is a promising raw material for manufacture of functional foods derived from aglycones. Our study demonstrated a potential pathway for the production of aglycones from residual soybean extract via fermentation with an isolated fungi as a biocatalyst, with applciations in the pharmaceutical and functional food industries.The"}
+{"text": "The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology enables purification of specific genomic regions interacting with their associated molecules. In enChIP, the locus to be purified is first tagged with engineered DNA-binding molecules. An example of such engineered DNA-binding molecules to tag the locus of interest is the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically-inactive form of Cas9 (dCas9) and guide RNA (gRNA). Subsequently, the tagged locus is subjected to affinity purification for identification of interacting molecules. In our previous studies, we developed enChIP systems for analysis of mammalian genome functions. Here, we developed an enChIP system to analyze bacterial genome functions.Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) in bacteria. Inducible expression of 3xFLAG-dCas9 in Escherichia coli was confirmed by immunoblot analysis. We were able to purify specific genomic regions of E. coli preserving their molecular interactions. The system is potentially useful for analysis of interactions between specific genomic regions and their associated molecules in bacterial cells to understand genome functions such as transcription, DNA repair, and DNA recombination.We generated a plasmid inducibly expressing  To understand the regulatory mechanisms underlying genome functions such as transcription, it is essential to identify the molecules associated with a genomic region of interest in vivo. The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology we developed recently, enables specific isolation of genomic regions of interest interacting with their associated molecules , 2. ExamStreptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) and gRNA in bacteria. The developed enChIP system isolated target genomic regions from Escherichia coli. The system might enable identification of molecules associated with a specific genomic region in bacteria, and thus help to elucidate their genome functions.Previously, we developed enChIP systems for analysis of mammalian genome functions. Here, we report development of an in-cell enChIP system for bacterial cells Fig.\u00a0a. The syBglII and BstZ17I and treated with bacterial alkaline phosphatase (E. coli C75) . The double-strand DNA (dsDNA) containing the coding sequence of the 3xFLAG-tag and the N-terminal portion of dCas9 (agatctaaagaggagaaaggatctATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTCATGGATAAGAAATACTCAATAGGCTTAGCTATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATAC)  was synthesized (Invitrogen) and digested with BglII and BstZ17I. The cleaved pdCas9-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated.To construct the doxycycline (Dox)-inducible 3xFLAG-dCas9 expression plasmid 3xFLAG-dCas9/p-bacteria (Addgene #64325), pdCas9-bacteria (Addgene #44249)  was digeSpeI and HindIII and treated with bacterial alkaline phosphatase (E. coli C75). The dsDNA for targeting the lacZ gene (ACTAGTGCTCTGCTACCTGCGCCAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTT) (underlined: the target sequences) was synthesized (Invitrogen) and digested with SpeI and HindIII. The cleaved pgRNA-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated. For construction of gRNAs targeting the promoter region of the rpoH gene, oligodeoxyribonucleotides   was digeDH5\u03b1  was transformed with 3xFLAG-dCas9/p-bacteria alone or together with gRNA expression plasmids, and transformed bacteria were selected with chloramphenicol (Cam) (25\u00a0\u00b5g/ml) for 3xFLAG-dCas9/p-bacteria alone or a combination of Cam (25\u00a0\u00b5g/ml) and ampicillin (Amp) (50\u00a0\u00b5g/ml) for 3xFLAG-dCas9/p-bacteria plus gRNA expression plasmid.DH5\u03b1 transformed with 3xFLAG-dCas9/p-bacteria was cultured in 2\u00a0ml of LB media containing Cam (25\u00a0\u00b5g/ml) at 37\u00a0\u00b0C overnight with shaking. One hundred microliters of the culture liquid was mixed with 900\u00a0\u00b5l of LB media containing Cam (25\u00a0\u00b5g/ml) and incubated for 1\u00a0h with shaking. Subsequently, Dox (2\u00a0\u00b5M) was added to the culture media for induction of expression of 3xFLAG-dCas9. After incubation with shaking for 4\u00a0h, 400\u00a0\u00b5l of the culture liquid was centrifuged at 5000\u00a0rpm for 1\u00a0min, and the bacterial pellets were suspended in 100\u00a0\u00b5l of 4\u00d7\u2009SDS buffer. After boiling at 100\u00a0\u00b0C for 5\u00a0min, 10\u00a0\u00b5l of the sample was subjected to SDS-PAGE with a 5\u201320% gradient gel followed by immunoblot analysis with anti-FLAG M2 antibody (Ab) .600 of the culture media reached 0.5, Dox (2\u00a0\u00b5M) was added for induction of 3xFLAG-dCas9 expression. After incubation at 37\u00a0\u00b0C for 4.5\u00a0h with shaking, the bacterial cells were fixed with 1% formaldehyde at 37\u00a0\u00b0C for 5\u00a0min and neutralized with glycine at room temperature for 10\u00a0min. After centrifugation, the cell pellets were suspended in 800\u00a0\u00b5l of modified lysis buffer 3 , and DNA was fragmented by sonication using Ultrasonic disruptor UD-201 (TOMY SEIKO) with conditions: Output, 3; Duty, 100% (continuous); Time, Free; 6 cycles of sonication for 10\u00a0s and cooling on ice for 20\u00a0s (the average length of chromatin fragments was 1 kbp). The sonicated chromatin was subjected to enChIP-real-time PCR analysis as described previously . First, we designed two gRNAs targeting the promoter region of the rpoH gene  was markedly lower than that of the rpoH promoter region  factor . This gee sigma 3 factor [e sigma 3 factor [e sigma 3 factor [E. coli genome while preserving their chromatin structures, and potentially contributes to the understanding of bacterial genome functions such as transcription and DNA repair.In this study, we developed an enChIP system for analysis of bacterial genomes. This system enables efficient isolation of specific genomic regions from the Further studies might be necessary to assess the utility of this system combined with MS and NGS to identify molecules associated with the target genomic regions in bacteria."}
+{"text": "The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain\u2013containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)\u2013brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator. In mammals, circadian rhythms are generated by a molecular oscillator in which the circadian locomotor output cycles protein kaput (CLOCK)\u2013brain and muscle ARNT-like protein 1 (BMAL1) transcription factors drive expression of the genes coding for their own repressors, the CRYPTOCHROME (CRY) and PERIOD (PER) proteins. A key feature of the oscillator is that the protein stability of its components is highly regulated. Previous studies had implicated the JmjC domain\u2013containing protein 5 (JMJD5) in regulation of the circadian clock in plants and flies. Here, we show that cells and livers that lack JMJD5 exhibit dysregulation of circadian gene expression. Mechanistically, JMJD5 is required for CRY1 degradation, including its destabilization by AMP-regulated protein kinase (AMPK), by facilitating its interaction with the proteasome. We found that JMJD5 is needed for normal CRY1-mediated transcriptional repression, thereby uncovering an inverse relationship between CRY1 stability and circadian repression. Finally, we showed that JMJD5 impinges on non-clock roles of F-box/leucine-rich repeat protein 3 (FBXL3) and CRY1. Altogether, our studies demonstrate that JMJD5 is a novel link between the oscillator and other physiological processes. Bmal1 gene transcription, which contributes to robust amplitude in circadian rhythms. However, the function of the circadian oscillator involves a much larger repertoire of factors that include other transcription regulators, kinases, phosphatases, ubiquitin ligases and peptidases, and chromatin regulators. Together, this large cohort of molecules acts in concert to generate circadian rhythms, coordinate the clock with other physiological processes, and enable environmental information to be integrated into its function.Circadian rhythms are endogenous, approximately 24-hour oscillations in behavior and physiology that evolved as an adaptation to the day\u2013night cycle. These rhythms are generated by a cell-autonomous timekeeping mechanism known as the molecular circadian oscillator. At its most basic, the oscillator is a transcription\u2013translation circuit formed by two interlocked delayed negative feedback loops . In one \u03b2-TRCP1/2 ubiquitin ligase complex  and 300 mM NaCl) and incubated on ice for 45\u201360 minutes. This was then centrifuged at 3,000 rpm for 10 minutes at 4 \u00b0C. The resulting supernatant was the nuclear extract and was used in subsequent applications. Nuclear extracts from the MEFs were prepared using this protocol, and confluent 35-mm dishes of cells were used.Whole-cell lysates from cells and livers were prepared using lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.5% TX-100, 0.5% NP-40, 0.25% Sodium Deoxycholate 0.025% SDS along with EDTA-free protease inhibitor cocktail and phosphatase inhibitors . Briefly, cells or crushed tissue was incubated with lysis buffer for 30 minutes on ice and then spun at 10,000 rpm for 10 minutes at 4 \u00b0C. To prepare the nuclear lysates, liver tissue was homogenized in a hypotonic buffer , cells were lysed using a lysis buffer containing 200 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 1% NP-40 supplemented with phosphatase and protease inhibitors. HEK293T cells were seeded out in 6-well plates and transfected the next day with a total of 2.5 \u03bcg of DNA . Forty-eight hours post transfection, the cells were lysed, incubated on ice for 30\u201345 minutes, and spun at 10,000 rpm for 10 minutes at 4 \u00b0C. The supernatant was incubated with the M2 beads overnight at 4 \u00b0C while tumbling. Subsequently, the beads were washed 3 times with chilled 1X TBS for 5 minutes each. The protein was eluted from the beads with equal volume of 3X flag peptide (Sigma Cat# F4799-4MG). The eluate was boiled in NuPAGE LDS Sample Buffer and reducing buffer and subjected to SDS-PAGE-immunoblot analysis.HEK293T cells were transfected with 1.5 \u03bcg V5-FBXL3, 400 ng HA-CRY1K:R, and 600 ng of V5-JMJD5. Forty-eight hours post transfection, the cells were lysed using the lysis buffer described above and immunoprecipitated with HA antibody bound to protein G beads for 1 hour at 4 \u00b0C. The beads were washed 3 times with chilled 1X TBS for 5 minutes with mild tumbling. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis.Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs were transfected with HA-RPN1 and 48 hours later were lysed with buffer containing 400 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 0.25% Sodium Deoxycholate, supplemented with phosphatase and protease inhibitors. The lysates were incubated on ice for 30\u201345 minutes and spun at 10,000 rpm for 10 minutes at 4 \u00b0C. Protein G beads were prebound with anti-HA tag antibody. The lysates were incubated with the antibody\u2013bead complexes for 1 hour at 4 \u00b0C and washed 5 times with 1X TBS containing 0.5% Triton X-100. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis to detect endogenous CRY1 levels bound to RPN1.FLAG M2 (Sigma), Anti-HA (12CA5), V5 (Abcam), and Anti-CRY1 were the antibodies used in this study. Anti-CRY1 (687) antibody was a kind gift from Satchin Panda.Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs were seeded out in 6-well plates at 350,000 cells per well and shocked 48 hours later with 100 nM of Dexamethasone for 2 hours before supplementing with fresh medium. Thirty-six hours post shock, the cells were harvested using 1 ml TRIzol (Fisher Scientific Cat# 15596026) and stored at \u221280 \u00b0C every 4 hours. Total RNA was then prepared using the manufacturer\u2019s instructions. For real-time qPCR, 1 \u03bcg of RNA was reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences Cat# 95048\u2013025). FastStart Universal SYBR Green Master (Rox) (Roche Cat# 4913850001) was used to perform the qPCR reaction in a BIO-RAD CFX384 Touch Real-Time PCR System.mDbp-F: GAG CCT TCT GCA GGG AAA CAmDbp-R: GCC TTG CGC TCC TTT TCCmCLOCK-F: AGAACTTGGCATTGAAGAGTCTCmCLOCK-R: GTCAGACCCAGAATCTTGGCTBmal1-F: GCC CCA CCG ACC TAC TCTmBmal1-R: TGT CTG TGT CCA TAC TTT CTT GGmCry1-F: ATC GTG CGC ATT TCA CAT ACmCry1-R: TCC GCC ATT GAG TTC TAT GATmCry2-F: GCA GAG CCT GGT TCA AGCmCry2-R: GCC ACT GGA TAG TGC TCT GGmPer1-F: GCT TCG TGG ACT TGA CAC CTmPer1-R: TGC TTT AGA TCG GCA GTG GTmPer2-F: TCC GAG TAT ATC GTG AAG AAC GmPer2-R: CAG GAT CTT CCC AGA AAC CAmNr1d1-F: GGA GCT GGG CCT ATT CAC CGCmNr1d1-R: GCT GCT CCA CCG AAG CGG AA.mJmjd5-F: CGCAGTCCTCCAGACACACCmJmjd5-R: CAAGATCACAGGCCTCCCAGmmMrpl46-F: GGTCCGGTCATTTTTTTTGTCAmMrpl46-R: GGGAGCAGGCATTCCTACAGmRORA-F: GTGGAGACAAATCGTCAGGAATmRORA-R: TGGTCCGATCAATCAAACAGTTCmABCG5-F: AGG GCC TCA CAT CAA CAG AGmABCG5-R: GCT GAC GCT GTA GGA CAC ATmGSTM3-F: CCC CAA CTT TGA CCG AAG CmGSTM3-R: GGT GTC CAT AAC TTG GTT CTC CAmLPL-F: GGG AGT TTG GCT CCA GAG TTTmLPL-R: TGT GTC TTC AGG GGT CCT TAGmLIPG-F: ATG CGA AAC ACG GTT TTC CTGmLIPG -R: GTA GCT GGT ACT CCA GTG GGmPCK1-F: CTG CAT AAC GGT CTG GAC TTCmPCK1-R: CAG CAA CTG CCC GTA CTC CmANGPTL4-F: CAT CCT GGG ACG AGA TGA ACTmANGPTL4-R: TGA CAA GCG TTA CCA CAG GChJMJD5-F: GGC CCG TGA TCC TGA AAG GhJMJD5-R: GGC TCA TTC ACG ATG TAT TTG ChCRY1-F: ACA GGT GGC GAT TTT TGC TTChCRY1-R: TCC AAA GGG CTC AGA ATC ATA CThFBXL3-F: GCA GCT TGT GAT ATA CTA TCG CAhFBXL3-R: TGG TCG AGC AGT TGA AAT AAG TChHPRT1-F: CCT GGC GTC GTG ATT AGT GAThHPRT1-R: AGA CGT TCA GTC CTG TCC ATA AAll the shRNA and siRNA knockdowns were performed using transient cotransfection of the constructs along with overexpression constructs of FLAG-CRY1, HA-JMJD5, and V5-FBXL3 in HEK293T cells in 6-well plates.shRNA hFBXL3 Sigma MISSION shRNA TRCN0000369031 (5\u2032-CCGGCTGATCAGTGTCACGGCTTAACTCGAGTTAAGCCGTGACACTGATCAGTTTTTG-3\u2032), shRNA hCRY1 Sigma MISSION shRNA TRCN0000231065 (5\u2032-CCGGGGAACGAGACGCAGCTATTAACTCGAGTTAATAGCTGCGTCTCGTTCCTTTTTG-3\u2032), siRNA hJMJD5 Sigma MISSION siRNA EHU149061, siRNA universal negative control Sigma MISSION siRNA SIC001.Jmjd5LKO mice between 6 and 9 weeks old were entrained in a light tight chamber to a 12-hour light:12-hour dark cycle for 10 days and released into constant darkness for 24 hours. Mice were then killed every 4 hours for a 24-hour period.Control or C57BL/6NTac-Jmjd5tm1a(EUCOMM)Wtsi frozen embryos were obtained from the European Conditional Mouse Mutagenesis Program (EM:04155), and embryo reconstitution was performed at Charles River. Born heterozygotes were crossed with the FLP deleter strain B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ (Jackson Laboratory 009086) to excise the lacZ-neo cassette. Thereby, conditional Jmjd5flox knockouts were created, which were bred for several generations onto a C57BL/6 background. To generate liver-specific JMJD5-null animals, Jmjd5flox/flox mice were bred with a transgenic mouse line that specifically expresses CRE in the liver at high levels /Cre+ littermates were used as controls.The following gBLOCKS were ordered from IDT and cloned into pGL3 BASIC (Promega).Wild-type E-boxes: AGTGCTAGCCATCACCCACTCACCCCTTAACGACACGTGGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCACGTGCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.E-boxes mutated: AGTGCTAGCCATCACCCACTCACCCCTTAACGACAgGTcGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCAgGTcCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.S1 Fign = 3) and mice livers (mean \u00b1 SEM n = 4) were determined by qPCR analysis. JMJD5, JmjC domain\u2013containing protein 5; MEF, mouse embryo fibroblast; qPCR, quantitative PCR.mRNA levels of JMJD5+/+ and JMJD5\u2212/\u2212 MEFs (mean \u00b1 SEM (TIF)Click here for additional data file.S2 FigPer2-Luc promoter in Jmjd5+/+ and Jmjd5\u2212/\u2212 MEFs . JMJD5, JmjC domain\u2013containing protein 5; MEF, mouse embryo fibroblast.Real-time bioluminescence measurement from overexpressed (TIF)Click here for additional data file.S3 FigJmjd5LKO, JmjC domain\u2013containing protein 5 liver knockout; loxP, locus of X-over P1.Schema of targeting vector and resulting floxed allele. Exons shown as solid boxes; Frt and loxP sites are respectively shown as green and purple triangles. Frt, flippase recognition target; (TIF)Click here for additional data file.S4 FigH321A (MUT) in the real-time luciferase assay in (A) Relative protein levels of FLAG-JMJD5 and FLAG-JMJD5(TIF)Click here for additional data file.S5 Fig(A) FLAG-CLOCK and BMAL, (B) FLAG-PER1, and (C) FLAG-PER2. BMAL, brain and muscle ARNT-like protein; CLOCK, circadian locomotor output cycles protein kaput; JMJD5, JmjC domain\u2013containing protein 5; PER, PERIOD.(TIF)Click here for additional data file.S6 FigJmjd5LKO, JmjC domain\u2013containing protein 5 liver knockout; WLE, whole liver extract.Total protein stain was used as loading control. CRY1, CRYPTOCHROME 1; (TIF)Click here for additional data file.S7 FigShown are the inputs for the coimmunoprecipitations of FLAG-tagged CRY1 and mutant CRY1s with (A) V5-FBXL3 and (B) HA-JMJD5. CRY1, CRYPTOCHROME 1; FBXL3, F-box/leucine-rich repeat protein 3; JMJD5, JmjC domain\u2013containing protein 5.(TIF)Click here for additional data file.S8 FigHEK293T, human embryonic kidney 293T; HPRT1, hypoxanthine phosphoribosyltransferase 1 gene; qPCR, quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA.(TIF)Click here for additional data file.S9 FigImmunoprecipitation of FLAG-CRY2 in HEK293T cells shows no interaction with HA-JMJD5 both in the absence and presence of V5 FBXL3. FLAG-CRY1 interaction with HA-JMJD5 is shown as a positive control. * denotes an unknown band observed only when FLAG-CRY2 is transfected. CRY, CRYPTOCHROME; FBXL3, F-box/leucine-rich repeat protein 3; HEK293T, human embryonic kidney 293T; JMJD5, JmjC domain\u2013containing protein 5.(TIF)Click here for additional data file.S10 Fig(A and B) Shown are the areas of the blot used to quantify the accumulation of CRY1. (C) Quantification of the main band (non-ubiquitylated CRY1) reveals a similar pattern of accumulation as quantifying all the forms of CRY1 as shown in (TIF)Click here for additional data file.S11 Fig71A/280A; CRY1, CRYPTOCHROME 1; JMJD5, JmjC domain\u2013containing protein 5.Real-time luciferase assays from (TIF)Click here for additional data file.S12 FigCRY1, CRYPTOCHROME 1; MEF, mouse embryo fibroblast.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."}
+{"text": "Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties. Published 1, December 2015In the Materials and Methods subsection \"In situ hybridization\" the sequences of radiolabeled oligodeoxyribonucleotide probes used for CKAMP52 and CKAMP59 were mixed up. These are the correct sequences:Ckamp52ins1 = 5\u2019AATGTCAGCCAGAGCCCTGTGGATGTTCATCTCTCGCGGACkamp59ins1 = 5\u2019GCGGCATAGCACGCCAGTCGAGGTTGGAGGGCTTCATGGTGTTWe apologize for the mistake.The article has been corrected accordingly."}
+{"text": "Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5\u2019 deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3\u2019 deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers,  brinker (brk) and short-gastrulation (sog) from the genome of D. melanogaster fruit fly using CRISPR-Cas9 genome editing. Surprisingly, opposite phenotypes relating to some target genes are associated with the enhancer deletions. Deletion of one enhancer generally exhibits phenotypes in early embryo patterning similar to respective gene mutants; whereas, in contrast, deletion of the other presents unique phenotypes including change in cell number for a particular tissue in the embryo, the amnioserosa. In summary, this study shows that coacting enhancers driving similar expression outputs can support distinct, and sometimes complementary, functions to differentially impact the development of embryos and that the individual mutation of these enhancers can provide insight into new gene functions.Genes expressed during development are often regulated by multiple cis-regulatory sequences, non-coding DNA, also known as enhancer sequences. Many instances have been found where two or more distinct enhancer sequences support similar spatiotemporal outputs relating to a single gene. These enhancers have generally been considered redundantly acting, or overlapping in activity, though few have been studied functionally. We created deletions of coacting enhancer pairs associated with the genes  Drosophila embryos, brinker (brk) and short gastrulation (sog), which are each associated with two enhancers that control their expression in the early embryo  when both enhancers should be active (yw), brk\u03945\u2019, brk\u03943\u2019, and brk\u03945\u2019\u03943\u2019. First, as proof of principle, total counts of brk mRNA were measured and, as expected, the brk\u03945\u2019 data is similar to wildtype (as the brk3\u2019 enhancer is predominantly acting at late stage 5), whereas counts are decreased for the brk\u03943\u2019 and brk\u03945\u2019\u03943\u2019 datasets  and level (NanoString) of expression allowed us to more fully investigate the changes in gene regulation in these mutant embryos  or antagonistic effects (i.e. opposite phenotypes) on target gene expression outputs. NanoString allows us to detect mRNA abundance, which is complementary to the spatial data provided by stainings. For example, dization , and levdization . During s mutant . The brkgenotype . However embryos .brk encodes a transcription factor that acts as a transcriptional repressor to limit BMP signaling and support patterning in the early embryo and at other stages of development including in the wing disc .All flies were reared at 23\u00b0C on standard fly media. ftz-lacZ , sogY506ftz-lacZ , brk 3\u2019-vep-lacZ , sog.intvep-lacZ , sog.disvep-lacZ , and Hishttp://flycrispr.molbio.wisc.edu/tools) was used to identify the PAM sequences with no predicted off target hits. The gRNA plasmids were then injected into either y2 cho2 v1 P{nos-phiC31\\int.NLS}X; attP2 (III) (NIG-Fly #TBX-0003) or y1 v1 P{nos-phiC31\\int.NLS}X; attP40 (II) (NIG-Fly #TBX-0002). Stable gRNA transgenic lines were created and then crossed to a Cas9 expressing line . Individuals from the next generation were screened by PCR for the deletions. Alternatively, for the sog\u0394int and sog\u0394dist, a homologous recombination cassette (HRC) was created by adding 1kb homology arms to the plasmid pDsRed-attP . Lines were screened for RFP in the eyes and the RFP cassette was subsequently removed in the same way as for the single mutants. All lines represent isogenic backgrounds differing only in the gRNA line used to create the specific deletion.For CRISPR-Cas9 deletions within the genome, gRNA constructs were created by modifying the pCFD4 plasmid  to targsog, aos, zen, sna, and rho were transcribed from cDNA subcloned into pGEM-T vector or full-length cDNA as for brk . For fluorescent in situ hybridization (FISH), probes were detected using Sheep anti-digoxigenin (Life Technologies PA185378), Mouse anti-Bio (Invitrogen 03\u20133700), and Rabbit anti-FITC (Invitrogen A889). Fluorescently labelled secondary antibodies were all from ThermoFisher (used at 1:400). Enzymatic detection was performed using digoxigenin labelled probes followed by detection with Anti-Digoxigenin-AP antibody  and standard AP staining using NBT/BCIP. Other antibodies used in this study were Hnt , pSMAD1/5 , and dpErk .Embryos were fixed and stained following standard protocols. Antisense RNA probes labeled with digoxigenin, biotin, or FITC-UTP were used to detect reporter or in vivo gene expression as described previously . Probes brk\u03945\u2019 and the brk\u03945\u2019\u03943\u2019), there were no obvious defects in cellularization seen in any of the mutants. Using nuclear shape and size combined with percent cellularization to characterize developmental stage was judged to be an adequate method to synchronously stage embryos that is independent of the genotype.Specific staging of embryo images is indicated in the associated figure legend. For early and late stage 5, the shape and length of the nucleus as well as the membrane front of the cellularizing cells was used to define either early or late stage 5. Early stage 5 (nuclear cycle (nc)14 A and B) included all embryos in nc14  that arThe following primers were used to generate riboprobes:dppi-f ccagaactagaaaaccggaagcdppi-t7-r gaaatTAATACGACTCACTATAgggCGCCTGTGCTAAAGACCCTGstumps-f TGGCCCAGAACATCGTCAGTTTstumps-r-t7 gaaatTAATACGACTCACTATAgggATGAGACTTCACCTGCTCCTGGATtld B-f ATGTGGATGAGTGTTCAATtld B-r-T7 gaaatTAATACGACTCACTATAgggTCCCTTCGCTGGACCTCTCATRace-f ATGAGACTGTTTCTGCTAGCCCTGCRace-T7-r GAAAATTAATACGACTCACTATAGGGACGCAAGCAGAAGGCACAGATAnetA\u2013f ATGATCCGTGGAATCTTGCTCCTGCnetA-T7-r AATTTAATACGACTCACTATAGGGCTTTGCACTCATTGGCTTCCTTGGCpnr-f ATCTCAAACCCTCGCTCAGCpnr-T7-r aagtaatacgactcactatagggagaCGAGGTGGCCATCAGTTTGGsog ex1-f TCAGGTTCAGTCGCTCTTGAsog ex1-T7-r AATTTAATACGACTCACTATAGGGGTGTCGGACTCCTCGAACATdpp and rho was measured using ImageJ. First, an ellipse was fit to the outside of the embryo image, and the perimeter of this ellipse was measured; then, an arc was manually drawn along this ellipse matching the extent of fluorescent signal for each gene measured. The length of the arc was measured, and a ratio of arc length to full circumference was determined. The width of zen at stage 6 was measured using chopped sections that were co-stained with DAPI to mark each of the nuclei. The cells expressing zen were counted manually. The amnioserosa cell number was counted using stage 10\u201313 stage embryos stained with Hnt antibody. Nuclei on the lateral side of the embryo only were counted manually in ImageJ by planing through the z stacks and using the Cell Counter plug-in. Box plots were created using BoxPlotR (http://shiny.chemgrid.org/boxplotr/).Embryos were sectioned along the anterior-posterior axis manually using a razor blade, and cylindrical mid-embryo sections were mounted on the cut side allowing for imaging of the axial plane. Percentage of embryo circumference for \u03c72 [The width of the pMad gradient was measured as described  with the\u03c72 , we used\u03c72 . The scastumps were made in ImageJ. Z-stack projections were created using the sum slices function and then the heat map was applied using the LUT function.Heatmap representation of images for pMad and Grids were drawn on standard 10 cm apple juice collection plates, dividing each into 160 quadrants. One embryo was transferred from a two hour collection into each of the quadrants of the grid. The plates were aged at 25\u00b0C in a humidified chamber for 30 hours, and the number of hatched and unhatched embryos was manually counted. This was repeated for a minimum of three times on different days for each genotype.brk mutant lines and nuclear morphology was observed on a Zeiss LSM800 microscope. Embryos were collected at nc14C for each genotype. This specific stage was determined to be when the nuclei were elongated but not yet disorganized, and the membrane front  fully encapsulates the nuclei [WT, brk\u03945\u2019\u03943\u2019) or 5  individual embryos were analyzed for each genotype.NanoString was run as described . H2A-RFPe nuclei . Once exbrk\u03945\u2019 and brk\u03943\u2019 were verified by crossing deletions made with different gRNA lines in trans to check for offsite effects. For brk\u03945\u2019\u03943\u2019, two independent mutant lines made using the same gRNA pair was crossed in the same manner. No differences were seen between the single mutant embryos and the trans-heterozygous embryos in the expression of brk, dpp, Race, or zen so all analyses were done using a single mutant line for each deletion.Individual deletion lines for brk or sog gene expression, specifically. For brk analysis, males of the genotype YW, brk\u03945\u2019, brk\u03943\u2019, or brk\u03945\u2019\u03943\u2019 were crossed to females of genotype brkM68/FM7 ftz-lacZ. To identify trans-heterozygous embryos  and assay for changes in amnioserosa, embryos were stained with a combination of antibodies: anti-\u03b2Gal to detect the balancer, anti-Sxl to identify female embryos, and anti-Hnt to detect amnioserosa. Similar trends were seen as for homozygous enhancer mutants :3\u2019grna-f attttaacttgctatttctagctctaaaacCGCCTCGGCCGGCGTCGCTGCgacgttaaattgaaaataggtc3\u2019grna-r attttaacttgctatttctagctctaaaacCGGGGTGGAAAAGCGACCCGCgacgttaaattgaaaataggtc5\u2019grna-f tatataggaaagatatccgggtgaacttcGTACCTGTTCGATCCTTCATgttttagagctagaaatagcaag5\u2019grna-r attttaacttgctatttctagctctaaaacTTGGGCTTTCGTTGCACAACgacgttaaattgaaaataggtcSog.int grna-f tatataggaaagatatccgggtgaacttcGTCAAAATCTTTAGTTAAAGgttttagagctagaaatagcaagSog.int grna-r: attttaacttgctatttctagctctaaaacTAGATCCCGGGATTTGTGCCgacgttaaattgaaaataggtcSog.distgrna-f:tatataggaaagatatccgggtgaacttcGATGATAGGTGGACTCTTGATgttttagagctagaaatagcaagSog.distgrna-r:attttaacttgctatttctagctctaaaacAGCGAATACGTGGAATTCCTCgacgttaaattgaaaataggtcSet 2 (second independent generation of deletions used to test for 2nd site mutations):3\u2019grna2-f tatataggaaagatatccgggtgaacttcGTTCCAAAACTTTAATCTGTTgttttagagctagaaatagcaag3\u2019grna2-r attttaacttgctatttctagctctaaaac GCAATCATTCTTCAATTCATCgacgttaaattgaaaataggtc5\u2019grna2-f tatataggaaagatatccgggtgaacttcGACCCGATGAAGGATCGAACgttttagagctagaaatagcaag5\u2019grna2-r attttaacttgctatttctagctctaaaacGCTTTCGTTGCACAACTTTAgacgttaaattgaaaataggtcHDR templates:Sog.int LA-DSR-f gtacgtgaattccgaaactcgcgtgtgttatctaSog.int LA-DSR-r ctagcggcggccgctaactaaagattttgactagtSog.int RA-DSR-f gtacgtggcgcgccatcccgggatttgtgccSog.int RA-DSR-r ctagcgctgcaggcggcagacagttgaataaaSog.dist LA-DSR-f gtacgtcccgggtccatccccacaccatttatSog.dist LA-DSR-r ctagcggcggccgcgaatacgtggaattcctttcgSog.dist RA-DSR-f gtacgtactagtaagagtccacctatcatcccagtSog.dist RA-DSR-r ctagcgggcgcgccagatgcgccagaagtacgCRISPR-Cas9 Deletions:Deleted base pairs capitalized. Indel/added sequence shown in bold italics.brk\u03945\u2019:tggtattaaaactgaaaatcaatctaaaaatcaaccattgataacattttattgaatcaaaccaaaagccaaattgattcctgaatccaaaagacccgatgaaggatcgaacaggtactacgatgatattggtcgGAAAATACCTGCGCATCCTGGTGGTTTATGGTGCGGCCGTAAATGCAAGCCAAGTTCTTTACGGCTTCTCTGGCACAAACCCTAAATGTGGATTACGCTAATATTGCCCCCCCTAATAAAAACGGTCGTTGTCCAGGGCCGAATATTGCGTCTGATTGGTTTTTCCCACGATTACAATTAGCCGGACGGACACAAACTGACCTGAGCTGACCCGCAAAAAGACACGGTTGTCCGGCAGTCGGAACTGAAGGAAACTAAAGGAAACTGAGGGCAGGTCAGCGCTATGGATTGTGCACTAAGTTGCTTAATCCGACGGGAAATCCAAAACACAACCCGAGCCCGATCCTTCGCTCCTTCGATTTAAGCCAAAGTTAGAGGCACAGGCACACATGTGTGTTTGGTTTGAACGGGAAAGCCCCATTTTAAAGCTGGCCAACCAACGGCAACACATGTTCATGTTAGGACCGATACAGGTTGACATTCCCTGGAAGGATGCACCTCTGGGAGATTCCCACAACCGGCAGCAGGTCATGTCCAACCGATCGTTGCGGGAGCCACTTGTCCCGAAAAAATCCAAAGAAACTATCAAGTGGCGTTTAGGGAAACTCAAAACTTTCCAACCACACCATATCTTTCTAACGCCACACAATAAACTGGTATGATCACTGTTAAGATCAAAATGGGCTAAATAAAGCGATCATGAATCATTTACTATAAACTGAAGAGTTTTCTATTCTTTAATATAAAAGAAAAGATGAGTAACACCCTACAAAAATTTTAGGACTACAGATCTACCCACCGATGATGATCAACCCTTCCCAAAAAAAAGTCACAGCTTGTATTTCGTAAAGATTTCAGATTTCTTAAGACACGATCACCAACTGATCTATGGACTTCTTAAACCATTGGGCTTTCGTTGCACAACTTTAGGGATTTGTTTTTTTTTTTTTTTTTTTGATTCCACATCCGATTTCTTCTTCGAAGCACTTCGATCTTTTCTCacatcagtttgatcgacaatcgacgatcgatggctgtgtgaaaacaagtttcgtaactatgtaaattaacctccabrk\u03943\u2019:GGAGAGCAGAGAGCAAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCgggggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtatttgtatttgtacgagacgggcagaaagaacgagaggaatggagatagagggbrk3\u2019 in brk\u03945\u2019\u03943\u2019:gaagcgaatgccaatgcacaaggagctaaacgagtgcgagcgagacgggcagaaagaacgagaggaatggagatagagggAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtasog\u0394int:GCGGCCGCGGACATATGCACACCTGCGATCATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTAAAGATCTCCATGCATAAGGCGCGCCAAGGGGTATTAACTGTTGCCTGTTGCTTTCGACATTTCTACCTCCGCTGCGATTGCATAAGTTGCCAATGCCATTGCGCATACGCCGTGTCGTCTATATGGCTATATGGCTATATGGCTGTATGGTGCGGGGAAATCCCCGTAATCGCAGGTAGAATTCCAGCCGGTGCCGAGGCGGGACCTGCTCGCACCTCTAATCCCGCCAGGGTTTTCGGGACATGGGATATTCCCGACGGCACAGCATAGCACTCCGTTTTCTTTTTTTTTTTTTATTATTATTGTGTCCAGTTTTAATCCGGAAAGCGGGAATTCCCTTCCGCTCGCTGCCTGCACTGCGCTGCGCAGACGCATCGGCGTCCGTAAGCCGCTTACCAAAAAGATACGGGTATACCCAAATGGATGCCTGCCCATGTATATAGACCATTGGGTGGTATGGACCATGGACCATAAAGCGGCACCCAATTGCAATTTGTTGCAACTCACACGCTTGATTAGCTGCATTTCCTGTTTGCAAACCCCCCTCCCAATACTCCCTACACTCATATATTATATATGCATATAAATGTATGTGTATATTGCATACTATTATGTTCCCGAGTTGCAGTTTTTTTTTCCTTTTTTTTCCACTATTTCTTTCTTGGTTCGCTGCCGTTGTTGCATTTTCAATTAAAAACAGATTGCGGTTTACGCTGCAGCGACTTTGAAACGTTACTCGGCTTACACACAAAATGCAGCGCGATAGTAATGATGATTAACAGGTTATGTTAGGGCTTAGTTGATACGCCGTAGatcccgggatttgtgccggaaccggaacgaatatcgaatcaatcgcatatccttaaagttcgaactagtcaaaatctttagttasog\u0394dist:GGCCGCGGACATATGCACACCTGCGATCGTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAGATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTGGGTAGTCCACATTCCATTCCATCGAAAGGAATTCCACGTATTCGCTGGGCGTTTCGGGGGCGGACTGGGCATATGGGCGTGGTGGGAGGGGGTGGGCGTCACGGGGCGTGGCCACACGTGAAACGGCTAATTTGTTGATGCAAATGCGGGTGTGGCCTGTTCTCGGATCGGAGTCCCCAGCACGCGCCCACAAATAAATAAATGGCTGGAGATGGAAATGGAAACCGATGACGGGGAATAGGGAATGACGGCTGGGTAGCGGGGGAGACGGGGATGGGAACTGGGGGGCTGGGGTCTTGGACCGAGATCGCAGCCAATGGCGACAATTGGCATTCACCGTCGCAATTAATCATCATCACCGTCCGATTGACAATCGGGCCACAAATAAATCAAAATCAAAGGCATAAGAAAGCGAATGCAGCGTCGGCATACAACTGCACTGAAAGAAAAATATGTATGAAGCTTCTGGTCAAGCAAACCGCAAGAAGCGCATTTAAGCTCAAGTATGCTCAAGTATTTGTTGTGTAACTTTAGATGATTCAAAATCGCTTTCTTATGTCTTTTTTGACTGCACTTGCCCTGAATTTCGTCGAGTGTAGTTCCCAATCATTGCAACAATTCATCAAGTATGCAAACACACGCGGCCGAATCAATCAATGGCGAGTAGTAGGAGTGCATGGAGGATGAGAAGGAGGAGAAGTTGGTTGAGAGGTCATCGTTGCGATTCTGCGATTCAGCAGTTCCACAGAAGGTGTCGTAATCCTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTCCTGTGCCACCAGATGACGCACGATGCGGCCGGAAAAACCCAAAATCAAAAACCGAAAACCGAAAACCTGGTCAGAGTTTCCGAAAACCAAAGAGCCAACATCGAATGCGGCACAATAACCCGATTGTCTGCGAATACCCACGATGATCTAGAATCGCACGGAGAGCACTCTCACGCATCCGTGGCCATATGGGTGCGGCCAAATCGGAAATTCCCAGGACAGGTAGAATGCATTGGATATACGGGTATACGGATTGGAATTGGGATTGGGATTGGGACTAGCACCAGGTTGCAACGCCCGCCAAGAAGCCAATTTAAATAAGCAGCATAAACAAAAGCGACAGCGTTTTATGATCCCCGCTCCTTATCCTTGCACAAGGATATCGCCATGGCCACGCAGGTAGGAATAGCAGATATGGCGGCAATGATGCGCCAACCGCACTGCTTCGTCCTGGTCCTGGTCGGATGGGCTTTTCCCACGCAACCGCGACCTTATCTGCGCCCCTTTTATGAGGCTGCATCTGTTTTCGCACCTCGATGCCGTTGGCATTATAGCCACATGTGTATGGTGGGAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAACCCGGGAATCTGTCCATCCTGAGCTTCCACACACACACACACACACACACAGGTCAGTCGGCATCAATTGGCTGCCATAAACATATAACAATCAATATTGAATCCTTTATCGTAGAATTTGTTGTATATGCCCATTGCAGTCCTTCGATTAAATGATGGCTAAAATGAATAAAATGAGTTGCTAGGTTCGAAGTAGGTGAAAACTGGTCAATAAGCCATATCCTTTATGATTAGATATTCTAGTAATCTGGCATGAAATACACAAATATAATAGCGAGTGATTAGTTTTATATAAGTATTTCTATTAAACCTCTAATAATTACCGCTTTACATATAGATTTTTTTAGTTGGATGCGGGCTTTAGTACCATATAGCTTTACCTGAATCTTATCGTTTGCTTTGGCTGATCCCCCAGGATCTGCGACATAATGCCAAACACAAGCAACTTGTTCGCCTGATCTTCGCAATCCCAGCAGGTTAACTACCCGATTTTATCGAGCAACAGCAAACTCTTTGAACTGATTTGCTATATTTCATTTATGGCCGATTCCAAGTCCATGTCCATTTCCCATTTCCATGTTCGTGTCCAAGTCCGTTGTTCGTTGTCCATTTCGATGTCAGTCATTCATTTTTGTGCTCGACTGCTAATGCCTTAAAGCCAAAATCCATTTCGTCGTTTGGCCATATAATCCAACATTATACATTAAATATGGCACTTGAAAGCACCGCGCGTCTGCATGCCAATTTCAAGGCCCCTTTTCCTCCACTTCACCACCCACAGCATACCATCCCATATCCCACCGGAATTTTCCGATTTTCCCCATTTTCCCCCAACAACACCACCACCTTGCTGCGATCGCACTTGTCGCTCGCGCCGGCGCCTTCAGGGTCGTCAATTTTTTGAGCGACATTTCGAAACGGTGGCTAAAATCACAAAAGCCAACAGAAAAAAATAAAAATCAACTATGGCGAAAAAGTAATACAAATGAAATCCGAAAACAAATAAGCGATTTTGTTTGTGGGGAGAAATGATGATGATCGGCGCCAGACGGACATAAAAACGCCAAAAGAAACGGAAAGGAAGCCTGGGAAAGGCGCATTTCCACTTTGTTTATTCGGAGCTCTGAAGCTCTTTTTAATGGTTTCTTTTTTTTTTTTTTTTTGGCCAAATGGCCAATTAATGCGATTTACTTGATACGCTTTTGCATTTTGTTGTTGCTTTACGACGGCGGATATTAATTAATTGGCCCGTCTCTGGGGAGCAATTCCCATTGTGCCCATCaagagtccacctatcatcccagttcgcagtccaaacctgacaatcgattaaaattacctctccagttgcacagatcaacggggtagtccacattccattccatcgaaaggaattccacgtattcgcS1 Fig(A) Dark field images of lateral view of cuticle preps from first instar larvae. (B) Bright field images taken with 40X objective of dentical bands in the A2 abdominal segment. Ventral view with anterior to the left.(TIF)Click here for additional data file.S2 Figbrk\u03945\u2019\u03943\u2019) or 5  individual embryos are shown by black bars. WT shown in black, brk\u03945\u2019 in gold, brk\u03943\u2019 in blue and brk\u03945\u2019\u03943\u2019 in grey. Genes broken into groups based on expression level for ease of display.Results for 70 genes assayed by NanoString in nc14C (late stage 5) embryos see . The res(TIFF)Click here for additional data file.S3 FignetA and (B) stumps gene loci. Brk binding is significant  only in late stage 5. Flybase defined protein coding regions for each gene shown in blue under Brk ChIP-seq tracks.(A-B) Screen shots from database of Brk ChIP-seq data  showing (TIF)Click here for additional data file.S4 Figtsg and (B) vn, or immunostained with an antibody to (C) dpErk. (D) Cross section of stage 6 embryos showing zen expression (green). Magnified image shows only dorsal one-third of embryo. Representative images for each genotype, further quantified in E. (E) Box plot of width, in number of cells, expressing zen. P-values determined by Welsh\u2019s t-test comparing brk\u03945\u2019, brk\u03943\u2019, and brk\u03945\u2019\u03943\u2019 to WT for zen were P = 0.4, P = 5.5x10-5, P = 0.06, respectively. Significance indicated in graph by *P<0.05, ***P<0.0001. (F) Percentage of embryos showing normal (blue) vs disrupted (orange) expression of Race in early stage 6 embryos. Number of embryos counted for each graph in this figure indicated under genotype.(A-C) Dorsal view of stage 6 embryos hybridized with riboprobes to (A) (TIF)Click here for additional data file.S5 Figbrk and egr. egr expression is diminished or lost in the brk\u03945\u2019 and brk\u03945\u2019\u03943\u2019 embryos. (B) In situ hybridization of late stage 5 embryos, dorsal views, with riboprobes to egr. egr expression remains low in the brk\u03945\u2019 but is expanded in the brk\u03943\u2019 embryos. (C) FISH staining of late stage 5 embryos, lateral views, with riboprobes to Cv-2. White arrows indicate expanded Cv-2 expression in the brk\u03945\u2019 and brk\u03945\u2019 \u03943\u2019 embryos. (D) Model of canalization loop acting to regulate amnioserosa cell number, reproduced from [egr, (F) Cv-2, and (G) zen loci.(A) FISH staining of early stage 5 embryos, lateral views, with riboprobes to ced from . (E-G) Sced from  showing (TIF)Click here for additional data file.S6 Figtld (green), ind and sna (both purple). All embryos are trans-heterozygous females of the genotypes indicated. Consistent with the patterns seen in the homozygous brk CRISPR mutants, tld is expanded ventrally, beyond the domain of ind expression in the trans-heterozygous embryos with brk\u03945\u2019 and brk\u03945\u2019 \u03943\u2019 but not significantly in brk\u03943\u2019. (F) Comparison of number of amnioserosa cells in homozygous brk enhancer mutants to trans-heterozygous combinations with brk- gene mutant. Homozygous mutant data is reproduced from (A-E) FISH staining of late stage 5 embryos, lateral views, with riboprobes to (TIF)Click here for additional data file.S1 DatasetExcel file containing raw counts for all graphically represented data depicted in Figs (XLSX)Click here for additional data file."}