S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (The online version contains supplementary material available at 10.1186/s12934-023-02024-2. Corydalis yanhusuo (named Yanhusuo) is a common traditional Chinese medicine that has been employed as an analgesic for thousands of years [C. yanhusuo is abundant in BIAs, including tetrahydroprotoberberines, quaternary protoberberines, aporphines, protopines, and benzophenanthridines [S)-stylopine, a protoberberine alkaloid, was identified as a promising compound due to its superior anti-inflammatory effect [Benzylisoquinoline alkaloids (BIAs) represent one of the most vital plant natural products in drug discovery, such as codeine, morphine, and berberine. The dry bulb of of years , 2. It hof years . C. yanhhridines . (S)-styy effect . SynthetN-terminal signal peptide, such that the crystallographic structural analysis of CYP450 remains challenging. Only a few studies have been reported on the crystal structures of plant CYP450s [Cytochrome P450s (CYP450s) are critical post-modification enzymes which are prevalent in plant secondary metabolic pathways –8 and co CYP450s , 12. Sem CYP450s . Commonl CYP450s . Semi-ra CYP450s , 16.S)-stylopine from (S)-scoulerine via (S)-cheilanthifoline [The CYP719 family is a member of the CYP71 clan , among whifoline . Althoughifoline –23.C. yanhusuo to explore their regioselectivity. In vitro enzyme assays demonstrated that CyCYP719As functionally catalyzed the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids -stylopine in yeast strain by synthetic biology.Currently, CYP719As from Papaveraceae and Ranunculaceae have been identified to catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids –24. HoweC. yanhusuo [C. yanhusuo and designated as CyCYP719A38, CyCYP719A39, CyCYP719A40, CyCYP719A41, CyCYP719A42  from A. thaliana, was utilized to heterogeneously express CyCYP719As [Five CyCYP719As have been functionally characterized to be involved in the methylenedioxy bridge-formation in BIAs biosynthesis. CYP719As . The expCYP719As , and difS)-scoulerine 1 to (S)-cheilanthifoline 2 -nandinine 6 (a compound containing an A ring methylenedioxy bridge structure) to (S)-stylopine 3, while CyCYP719A40 could not -scoulerine as substrate, while CyCYP719A40 only had a 2% conversion rate -cheilanthifoline 2 to (S)-stylopine 3, with a conversion rate of more than 90% -scoulerine 1 as a substrate to produce (S)-nandinine 6 -stylopine, the methylenedioxy bridge-formation on the D ring of protoberberine was prior to the methylenedioxy bridge-formation on the A ring. In addition, to determine the substrate regiospecificity of CyCYP719As, we screened 1-benzylisoquinoline and protoberberine alkaloids, including (S)-tetrahydrocolumbamine, (S)-coclaurine, (S)-norcoclaurine, (S)-N-methylcoclaurine, (S)-reticuline, (S)-3’-hydroxy-N-methylcoclaurine, jatrorrhizine, corytuberine, tetrahydropalmatine, corydaline, and columbamine as substrates -tetrahydrocolumbamine, none of the compounds could be employed as substrates for CyCYP719As. CyCYP719A41 could efficiently catalyze the formation of (S)-tetrahydrocolumbamine 4 to (S)-tetrahydroberberine 5, with a conversion rate of more than 40% -scoulerine to (S)-stylopine required the participation of two CYP719As with strict substrate regioselectivity, which catalyzed the methylenedioxy bridge-formation on the D and A rings of protoberberine alkaloids successively [In vitro enzyme assays and research reports revealed that the formation from -scoulerine, (S)-cheilanthifoline, and (S)-nandinine were selected as substrates for in vitro enzyme assays of CyCYP719As. The catalytic activity of CyCYP719As mutant was compared with that of wild-type CyCYP719As  resulted in an inability to catalyze the methylenedioxy bridge-formation on the A ring  catalyzed the methylenedioxy bridge-formation on the A ring, while lost its ability to catalyze the D ring  increased the catalytic conversion rate of methylenedioxy bridge-formation on the A ring more than two times  increased the conversion rate of methylenedioxy bridge-formation on the D ring by 20%  resulted in 90% reduction in catalytic activity for methylenedioxy bridge-formation on the A ring -scoulerine to (S)-stylopine via (S)-cheilanthifoline, although the two reactions could not occur simultaneously -scoulerine to (S)-stylopine in vitro, requiring stepwise catalysis) -scoulerine to (S)-nandinine -scoulerine. Docking analysis showed that P425 in CyCYP719A39 was closest to the active site heme and interacted with it. We speculated that the introduction of P425A mutation reduced the interaction between CyCYP719A39P425A and the substrate, leading to a shift of the D ring away from the heme and thus reducing its catalytic activity. However, the introduction of D289L mutation weakened the mutual exclusion between the double mutant CyCYP719A39P425A, D289L and the substrate A ring, which enabled the double mutant to catalyze the A and D rings of protoberberine alkaloids. Additional iterative mutation analysis based on the double mutant is needed to improve the efficiency of CyCYP719As in catalyzing the methylenedioxy bridge-formation on both the A and D rings of protoberberine.The methylenedioxy bridge-formation on the D ring is presumed to be prior to the formation on the A ring. We next focused on mutants which could catalyze the formation of methylenedioxy bridges on both the D and A rings of protoberberine alkaloids. Based on the previous analysis, it was indicated that D289 was essential for the methylenedioxy bridge-formation on the D ring of protoberberine catalyzed by CyCYP719A39, and L294 was crucial for methylenedioxy bridge-formation on the A ring catalyzed by CyCYP719A42. Conversion mutations at these residues resulted in the loss of original function for both CyCYP719A39 and CyCYP719A42. However, we found that although the mutant CyCYP719A39S)-stylopine has a low content in Papaveraceae such as C. yanhusuo and Chelidonium majus, while having positive anti-inflammatory and anti-fibrosis physiological activities [S)-stylopine makes chemical synthesis difficult. Therefore, we employed the synthetic biology method to construct an engineered yeast strain to achieve microbiological synthesis of (S)-stylopine. In previous work, we constructed a S. cerevisiae strain XJ0695, in which the (S)-scoulerine biosynthetic pathway was reconstructed and optimized by advanced metabolic engineering and synthetic biology strategies (Data unpublished). The construction of this strain started from the biosynthesis of the first BIAs molecule (S)-norcoclaurine by condensation of tyrosine-derived dopamine and 4-HPAA under norcoclaurine synthase (NCS). To improve the production of (S)-norcoclaurine, various optimization attempts have been conducted, including screening the optimal candidates (CYP76AD5 from sugar beet Beta vulgaris and CjNCS from Coptis japonica), enlarging the tyrosine pool  and ARO7* (G141S)), impairing the undesirable 4-HPAA scattering , increasing extra copies of rate-limiting enzymes (3 copies of CjNCS), introducing plant-derived tyrosine pathway, 4-HPAA biosynthesis pathway. Sequentially, we extended the biosynthetic pathway to produce the key intermediate (S)-scoulerine.-stylopine, functionally characterized CyCYP719As and mutants were introduced to (S)-scoulerine producing yeast platform to synthesize (S)-stylopine. For comparison, both CyCYP719A39 and CyCYP719A39MS mutants were paired with CyCYP719A42 to construct recombinant plasmids, which were then introduced into the (S)-scoulerine yeast platform containing a copy of ATR1 from A. thaliana. It was induced with 20 g/L galactose after 72 h fermentation in shaker with 20 g/L glucose as carbon source. The results showed that the strain XJ0695 co-expression CyCYP719A39 and CyCYP719A42 could produce (S)-stylopine in vivo -scoulerine. The production of (S)-stylopine by the mutant strain CyCYP719A39MS-CyCYP719A42 increased approximately 10% to 32 mg/L -stylopine production and further validated the ability of the CyCYP719As mutant to increase the yield of the target product. The method provides a yeast strain for production of plant natural products by synthetic biology. Additional optimization strategies will be executed to improve the conversion of (S)-scoulerine to (S)-stylopine for high production of those compounds with methylenedioxy bridges.To construct the yeast strain for de novo synthesis of -scoulerine producing yeast platform for de novo synthesis of (S)-stylopine with a production of 32 mg/L. This yeast strain provided an alternative for producing (S)-stylopine.CYP450 is a crucial modification enzyme in plant natural products biosynthesis, providing groups for the methylation and glycosylation of active compounds . CYP719 D289L would increase after mutation to Leu containing multiple methyl groups compared with Asp with carboxyl groups, reducing its repulsive interaction with the substrate and making the methylenedioxy bridge produced on the A ring closer to the heme, while forcing the D ring away from the heme. Furthermore, functional characterization of CyCYP719A39D289L in the methylenedioxy bridge-formation on the A ring instead of the D ring proved this hypothesis. This function was enhanced after the introduction of M120S into CyCYP719A39D289L, which additionally reduced steric hindrance between the A ring and heme. While CyCYP719A42L294D lost its ability to catalyze the methylenedioxy bridge-formation on the A ring of protoberberine. In addition, we found that M120S mutation in CyCYP719A39 could improve the catalytic efficiency by reducing the hydrophobicity of proximal active pocket and narrowing the distance between the D ring and heme. We also obtained a double mutant CyCYP719A39P425A, D289L capable of continuous catalytic reactions, although with a relatively low conversion rate. Further iterative mutation analysis for CyCYP719A39 mutants was needed to identify the mechanism of continuous catalysis and to improve the catalytic efficiency to obtain an efficient mutant protein for continuously catalyzing both A and D rings formation. Afterwards, the output of (S)-stylopine product was fine-tuned by the introduction of various CyCYP719As and their mutants into the (S)-scoulerine-producing yeast.Although CYP450s shared different amino acid sequence identities, they contain a common tridimensional fold and catalytic center . This stS)-stylopine was constructed, which provided an alternative way for producing BIAs compounds. These results extend our knowledge of the structure–function relationship for CYP719As-mediated methylenedioxy bridge-formation and lay the foundation for the BIAs production by synthetic biology.In summary, we functionally characterized five CyCYP719As participated in the BIAs biosynthesis and identified key residues related to the methylenedioxy bridge-formation on the A ring (Leu294 in CyCYP719A42) and D ring (Asp289 in CyCYP719A39) by site-directed mutations and in vitro enzyme assays. Finally, an engineered yeast strain for production of -scoulerine, (S)-stylopine, (S)-cheilanthifoline, (S)-nandinine, (S)-tetrahydrocolumbamine, and (S)-tetrahydroberberine with purity ≥ 95% were obtained from Shanghai Yuanye Bio-Technology.The bulb of C. yanhusuo was reverse transcribed into cDNA using Superscript™ IV . According to the transcriptome data obtained in the laboratory, five CyCYP719As subfamily genes were screened, and specific primers were designed based on their open reading frames. Spe I restriction site was selected to design primers, as shown in Additional file ® HS DNA Polymerase  was utilized for amplification, and the target fragment was obtained by purifying the PCR product using the Quick Gel Extraction Kit . The target fragment was attached to the eukaryotic expression vector pESC-His by seamless stitching [E. coli Trans1 T1 and the positive clone was confirmed through PCR detection and DNA sequencing. Recombinant plasmid pESC-His-CyCYP719As was obtained from the strains using the Plasmid Miniprep Kit .Total RNA was extracted using a Total RNA Kit , and purity and integrity of the RNA were measured. Total RNA from the bulb of titching . PlasmidS. cerevisiae WAT11, which overexpressed CYP450 reductase (CPR) from A. thaliana [600 value of the bacterial solution reached 2–3. Then 20 g/L galactose was added to induce protein expression. Microsomal protein was extracted using the TESB method, whose detailed operation was referred to Ma et al. [Recombinant plasmid pESC-His-CyCYP719As was expressed in thaliana , and thea et al. . In addia et al. . In vitrLiu et al. [The catalytic products were tested by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The products were separated by Waters Atlantis T3 column . Gradient elution of the enzyme-induced products was performed with mobile phase solvent A composed of 0.1% formic acid (v/v) and 99.9% acetic acid (v/v) and solvent B composed of 0.1% formic acid water (v/v) at a flow rate of 0.4 mL/min. Elution procedure was referred to the paper by u et al. . The volS)-scoulerine and (S)-cheilanthifoline was simulated, and the docking results were displayed by PyMOL software. AutoDock4 software was used for docking analysis of protein models to search for key residues [The AlphaFold2 platform was employed for protein homology modeling of functional genes of CyCYP719A39 and CyCYP719A42, and to find the model with the highest similarity. Filter templates from the PDB database combining alternating sequence alignments and secondary structure predictions, and create multiple models based on these templates [residues . The pESS. cerevisiae strain XJ0695 that could produce (S)-scoulerine provided by Chen Yun's research group and a copy of ATR1 from A. thaliana was integrated into YORWΔ17CyCYP719A39 and CyCYP719A39MS mutant were introduced into the engineering yeast together with CyCYP719A42 for functional characterization in vivo and (S)-stylopine production. The recombinant pESC-Ura-CyCYP719As were constructed due to the lack of Ura tag in engineered yeast, and then cultured in the Ura-deficient liquid medium after transplanting into engineered yeast. Fermentation was performed in shaker with 20 g/L glucose as the sole carbon source until OD600 value reached 3, and 20 g/L galactose was added for induction. The reaction product was extracted with an equal volume of 30% acetonitrile, centrifuged and diluted with 15% acetonitrile, and tested by UPLC-QTOF-MS. The engineered yeast with pESC-Ura was utilized as negative control. All in vitro enzyme activity assays were repeated three times.According to the Additional file 1: S1. Amino acid sequences of CyCYP719As. Figure S1. Multiple sequence alignment of CYP719s. The conserved CYP719 regions, including helix-K, aromatic regions, and heme-binding regions, are highlighted. The conserved amino acid sequence in the I helix of CYP450 is represented by dashed line, leucine and serine are represented by “*”. All sequence information in the figure was shown in Table S2. Figure S2. UPLC-QTOF-MS analysis of the catalytic function of CyCYP719As. A: In vitro enzyme assays of CyCYP719As using (S)-scoulerine 1 as substrate. B: CyCYP719A41 and CyCYP719A42 catalyze (S)-tetrahydrocolumbamine 4 to produce (S)-tetrahydroberberine 5. C: CyCYP719A38, CyCYP719A39, and CyCYP719A40 catalyze (S)-nandinine 6 to produce (S)-stylopine 3. D: CyCYP719A41 and CyCYP719A42 catalyze (S)-cheilanthifoline 2 to produce (S)-stylopine 3. Figure S3. A: Mass spectrum of CyCYP719A41 product (2) compared with that of authentic (S)-cheilanthifoline. B: Mass spectrum of CyCYP719A42 product (6) compared with that of authentic (S)-nandinine. C: Mass spectrum of CyCYP719A41 product (5) compared with that of authentic (S)-tetrahydroberberine. D: Mass spectrum of CyCYP719A39 product (3) compared with that of authentic (S)-stylopine. Figure S4. Structure of the eleven compounds used for in vitro enzymatic assay of CyCYP719As. Figure S5. Relative yields of different products in vitro enzymatic reaction of CyCYP719A39 and CyCYP719A42 and their mutants with (S)-scoulerine, (S)-cheilanthifoline, and (S)-nandinine as substrates. Data reported are the means ±SD from triplicate analyses ** indicates P<0.01; nd, not detected. Data in vitro conversion of CyCYP719A mutants are shown in Table S4. Table S1. Primers used in this study. Table S2. Sequence information used in the phylogenetic analysis in Fig. 2. Table S3.In vitro conversion rate of CyCYP719A functional genes. Table S4.In vitro conversion rate of CyCYP719A mutants. Table S5. Concentration of (S)-Stylopine produced by fermentation engineered strains.


The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose–response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2. HeLa cells were maintained in minimum essential medium α supplemented with 2 mM GlutaMax and 10% fetal bovine serum.SARS-CoV-2 variants including the original Wuhan strain ), the strain containing the D614G mutation (EPI_ISL_940555), the alpha , the Beta , the Delta , and Omicron (EPI_ISL_7696645) variants were all propagated in Vero-E6 cells expressing TMPRSS2.A recombinant Sindbis virus (SINV) expressing HCV E1 glycoprotein was employed as previously described . Yellow in vitro. Mouse anti-GFP mAb was from Roche. Rabbit anti-calnexin antibody was from Abcam. Rabbit anti-ACE2 antibody was from Cell Signaling Technology. Mouse anti-transferrin receptor mAb was from SantaCruz. Cyanine 3-conjugated goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies were from Jackson ImmunoResearch.Mouse anti-dsRNA mAb (clone J2) was obtained from SCICONS. Polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody was from Novus. Mouse anti-HCV E1 mAb A4  was prodTo construct the fluorescent reporter, oligonucleotides 5′-AGCTTCACCAAAAAAAAAAAGAAAAGTAGGAGG-3′ and 5′-GATCCCTCCTACTTTTCTTTTTTTTTTTGGTGA-3′ containing the coding sequence of the nuclear localization signal of SV40 T antigen were phosphorylated, annealed and inserted by ligation between the HindIII and BamHI sites of the plasmid pEGFP-C1 (Promega), yielding pEGFP-NLS. The coding sequence of the cleavage site fused to that of cytochrome 5b C-terminus was amplified by PCR with primers 5′-TTTGGGATCCCCAGCGCTGTGCTGCAGAGCGGATTCCCTCCGGAAACTCTTATCACTAC-3′ and 5′-TTTGTCTAGATCAGTCCTCTGCCATGTAATCC-3′ and cDNA obtained by reverse transcription of Huh-7 cells total RNA as a template. The PCR product was purified and inserted between the BamHI and XbaI sites of pEGFP-NLS.A plasmid expressing a similar construct with no cleavage site was constructed by inserting a PCR product obtained with a sense primer without cleavage site coding sequence (5′-TTTGGGATCCCTCCGGAAACTCTTATCACTAC-3′) and the same antisense primer. Plasmids expressing mutants LR and QN were also constructed by PCR with sense primers 5′-TTTGGGATCCCCAGCGCTGTGCGGCAGAGCGGATTCCCT-3′ and 5′-TTTGGGATCCCCAGCGCTGTGCTGAATAGCGGATTCCCT-3′, respectively. All plasmids were verified by sequencing.The wild-type nsp5 sequence from GenBank accession MN908947.3 was codon optimized for mammalian cell expression and purchased from GeneCust. The nsp5 coding region was inserted in the pcDNA3.1(+) NheI/XhoI sites. An inactive version of nsp5 containing the mutations H41A and C145A was obtained by PCR overlap mutagenesis. The nsp5 coding sequence and flanking regions of these vectors were sequence verified.To construct the pTRIP-myc-ACE2 vector, the coding sequence of myc-ACE2 was amplified by PCR using the pCDNA3.1-ACE2 kindly provided by Gary Whittaker  as a template and the primers: 5′-CCGACTCTAGACCATGTCAAGCTCTT-3′ and 5′-ATATAGCTCGAGTTAAGCGGGCGCCACCTGGGA-3′. The PCR product was ligated into the pTRIP vector after restriction with XbaI and XhoI.Vero-81-derived cells were transfected using the TransIT-LT1 transfection agent (Mirus Bio). Transfected cells were selected with geneticin (2 mg/ml) for 2 weeks after transfection and GFP-expressing cells were enriched using a fluorescence-activated cell sorter. A cell line stably expressing the fluorescent reporter (F1G) was obtained by three successive limiting dilutions. Transient transfection of HeLa cells was obtained by using the same agent without subsequent selection.A plasmid encoding an mCherry-NLS protein was constructed similar to its GFP counterpart and the coding sequence of this construct was inserted in the pRRL.sin.cPPT.SFFV/IRES-puro.WPRE plasmid , kindly Cells were lysed and analyzed by immunoblotting as previously reported .Cells grown on glass coverslips were fixed and processed for immunofluorescence staining and imaged using an EVOS M5000 imaging system, as reported previously . For col2 followed by image acquisition using an InCell-6500 automated confocal microscope (Cytiva) equipped with a 20x CFI Plan APO objective (NA: 0.75). Nine fields per well were analyzed using the 488 nm and 561 nm laser lines adjusted to 90% laser power for excitation of the GFP and mCherry channels, respectively, and applying a 4-band emission polychoric filter . Aperture values were set at 2.85 and technical triplicates were acquired for each experimental condition. In each experiment, identical sets of antiviral dilutions were applied to non-infected cells to control for the lack of impact of the compounds on the intracellular localization of GFP. Percentages of infection were measured by automated image analysis using the Columbus software  and an adapted script to distinguish between infected and non-infected cells  + MAD(negative control))/abs(median(positive control)-median(negative control)), MAD being the median absolute deviation.F1G-red cells were cultured in 30 μl of complete medium for 24 h in 384-well plates  and transferred to the BSL-3 facility. 30 μl of antiviral solutions in three-fold dilution series was added, after which the cells were inoculated with 10 μl of SARS-CoV-2 (MOI of 0.2) in a complete medium to obtain a final antiviral drug concentration ranging from ~0.1–45 μM. Cells were incubated for 16–18 h at 37°C and 5% COed cells . Data wePrevious experiments showed that the number of SARS-CoV-2 infected Vero-81 cells reached a plateau of about 70% at 24 hpi independent of the used MOI . As thisPicornaviridae), yellow fever virus , and Sindbis virus  was additionally assessed. Although some differences in infection levels were also observed between clones for these viruses, they were less pronounced than with SARS-CoV-2, and the susceptibility of the clones for these viruses did not correlate with the susceptibility to SARS-CoV-2 RNA viruses, namely coxsackievirus B4  with ACE2 expression . Plasmids expressing either the wild-type probe, the LR and QN mutants, or a construct with no cleavage site at all (CS-) were co-transfected in HeLa cells with a plasmid expressing SARS-CoV-2 nsp5 or an inactive version of the protease. These experiments confirmed that the probe was indeed cleaved in nsp5 active conditions . In contFinally, to find out if the quantification of SARS-CoV-2 infection with the fluorescent reporter would indeed generate reliable results, clone 6 cells expressing the fluorescent reporter were inoculated with different doses of SARS-CoV-2 and the number of infected cells was quantified at 16 hpi both by counting the number of cells with a green fluorescent nucleus and with a ds-RNA-positive signal. Both quantification methods showed a virus dose-dependent increase in infection. Again, the percentage of infection was however slightly lower when assessed with the fluorescent reporter than with the anti-dsRNA immunofluorescence . NonetheTo apply this reporter system to a high content format, a clonal cell line stably expressing the fluorescent probe was generated. A clone, named F1G, stably expressing the probe was obtained by successive limiting dilutions. Additionally, to allow visualization of the nucleus without the need for addition of any exogenous reagent (such as DAPI or Hoechst), a red fluorescent protein with a nuclear localization signal (mCherry-NLS) was stably expressed in the F1G cells, finally yielding a cell line named F1G-red, expressing the green fluorescent probe in the cytoplasm and a red fluorescent marker in the nucleus in non-infected conditions. As the green fluorescence shifts to the nucleus upon infection, both green and red signals co-localized in the nucleus in infected conditions . Next, w50 values of 2.8 μM for chloroquine, 0.75 μM for GC376, 6.1 μM for remdesivir, and 17 μM for molnupiravir . Again, these EC50 values might have been overestimated due the absence of a P-glycoprotein efflux inhibitor in these experiments.To assess the susceptibility to different SARS-CoV-2 variants, dose–response experiments were performed with GC376 using 6 SARS-CoV-2 variants, since it has been shown that Mto GC376 . As expeto GC376 , and the50 , the Z’-factor was tested as an assay quality indicator. Infections of F1G-red cells were performed at different MOI in the presence of DMSO or GC376 at a concentration of about 10xEC50 . A robus50 . This re50 .In December 2019, the world was introduced to a novel respiratory disease, COVID-19, caused by a newly emerged human coronavirus, later named SARS-CoV-2. After the emergence of SARS-CoV-1 in 2002 and MERS-CoV in 2012, SARS-CoV-2 was the third human coronavirus with high pathogenic potential. In sharp contrast to the 2 previous ones, this new virus surprised friend and foe with its rapid spread among the human population, resulting in a worldwide pandemic from March 2020 onward. Since then, massive amount of work has been done to identify anti-SARS-CoV-2 molecules that together with the available vaccines should help in controlling this pandemic, few of which have been approved for use in patients so far. However, the rapid need to screen massive amounts of molecules also highlighted the lack of a good cell-based model that allowed for a fast, effortless, and automated read-out of the SARS-CoV-2 infection, and that required minimal handlings to ensure maximum safety in the BSL-3 environment.The present paper reports the construction and characterization of a new reporter cell line for quantifying SARS-CoV-2 infection in living cells and shows that it can be used for automated dose–response calculations of antiviral compounds using a high content format. The main advantage of this reporter system is that it allows for a fast quantification of infection (before the onset of CPE) with minimally required handlings, as it does not require any additional reagent to measure infection. Indeed, there is no need to add any DNA probe, such as DAPI or Hoechst, to define the position of nuclei before recording images, and the detection of infected cells does not require adding any substrate or antibody, unlike reporter systems based on luciferase activity or immunostaining . This main situ detection of nsp5 proteolytic activity, which is highly specific to SARS-CoV(−2) infection, in contrast to other cell-based protocols relying on indirect and/or late impacts of infection, such as the measure of syncytia formation . The platform used in this work was supported by the European Union (ERC-STG INTRACELLTB grant 260901), the ANR (ANR-10-EQPX-04-01), the “Fonds Européen de Développement Régional” (Feder) (12001407 [D-AL] EquipEx ImagInEx BioMed), CPER-CTRL (Centre Transdisciplinaire de Recherche sur la Longévité), and the Région Nord-Pas-de-Calais (convention 12000080).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.


Escherichia coli expression system to obtain nine different SARS-CoV-2 RBD protein variants, including six single-point mutants, one double-point mutant, and two three-point mutants. Western blotting results show that nine mutants of the RBD protein had strong antigenic activity in vitro. The immunogenicity of all RBD proteins was detected in mice to screen for protein mutants with high immunogenicity. The results show that the mutants E484K, E484Q, K417T-E484K-N501Y, and K417N-E484K-N501Y, especially the former two, had better immunogenicity than the wild type. This suggests that site E484 has a significant impact on the function of the RBD protein. Our results demonstrate that recombinant RBD protein expressed in E. coli can be an effective tool for the development of antibody detection methods and vaccines.In the context of the COVID-19 pandemic, conducting antibody testing and vaccination is critical. In particular, the continued evolution of SARS-CoV-2 raises concerns about the effectiveness of vaccines currently in use and the activity of neutralizing antibodies. Here, we used the  The beta-coronavirus SARS-CoV-2 has become the seventh discrete coronavirus species that is capable of causing human disease . SARS-CoStudies have shown that SARS-CoV-2 spike (S) protein can serve as a suitable antigen with strong immunogenicity that can effectively stimulate the host immune system to induce the production of neutralizing antibodies ,6,7. TheEscherichia coli expression system, and their antigenicity and immunogenicity were compared and evaluated in mice in order to screen candidates for vaccine design and drug targets and to facilitate virus antibody detection kit development.Previous studies have shown that different SARS-CoV-2 mutants have different mutations in the RBD of the S protein, causing changes in viral pathogenicity and infectivity ,20. ThisE. coli XL1-Blue was used as a host to express the RBD protein. The bacterial strains in our experiment were cultured in LB medium at 37 °C. Ampicillin was added as needed. The fragment of the SARS-CoV-2 S protein gene (GenBank: NC_045512.2) (991–1749 bp) corresponding to amino acids 331−583 of the SARS-CoV-2 S protein (GenBank: YP_009724390.1) was synthesized. The expression vector pQE30 was digested with BamHI and HindIII restriction enzymes. The ClonExpress® Ultra One Step Cloning Kit  was used to ligate the target fragment into the vector. Primers used for cloning and mutant construction are shown in 2PO4, pH 8.0). Then, 0.5 mL lysozyme (50 mg/mL) was added and the lysate was placed on ice for 20 min. Ultrasonic disruption was performed with the following parameters: total time 16 min, working time 6 s, intermittent time 3 s, and power 300 W. After centrifugation for 20 min at 12,000 rpm and 4 °C, the supernatant was discarded, and the precipitate (inclusion body) was retained. Next, 20 mL of precipitation lysis buffer  was added to the inclusion body. After sufficient oscillation, ultrasonication was carried out. The supernatant (containing protein after inclusion body dissolution) was collected by centrifugation for 20 min at 12,000 rpm and 4 °C, and the precipitate was discarded. The RBD protein was purified under denaturing conditions with HisSep Ni-NTA Agarose Resin. The inactive RBD protein was added to refolding buffer  glycerol, pH 8.0) and incubated for 12 h at 4 °C. Finally, the refolded RBD protein was concentrated and desalted by a 10 kDa ultrafiltration tube.The recombinant strains were streaked on LB plates (containing 200 μg/mL ampicillin). Single colonies were inoculated in LB medium containing 100 μg/mL ampicillin and cultured overnight at 37 °C. Cultures were transferred to 1 L LB medium. When the OD600 value of the bacterial solution reached 0.5, IPTG was added at a final concentration of 0.5 mM and bacteria were cultured at 37 °C for 8–12 h. The cultured cells were harvested and resuspended in 15 mL lysis buffer . For Coomassie Bright Blue staining, CBB Fast Staining Solution  was used. For Western blot (WB) analysis, the proteins were transferred from the gel to a nitrocellulose membrane by semi-dry membrane transfer . The membranes were incubated in blocking buffer ) at room temperature for 2 h. After washing the membrane with TBST buffer three times, the membrane was incubated with mouse monoclonal anti-His tag  or human monoclonal anti-RBD  overnight at 4 °C. Following incubation, the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse  or HRP-conjugated goat anti-human for 1 h at room temperature. After washing, the membrane was immersed in a chromogenic solution and protein bands were imaged with a UVI gel imager .Ten RBD proteins were prepared and mixed with Freund’s adjuvants at a ratio of 1:1. In this experiment, three mice were immunized in each experimental group. Female BALB/c mice aged 6–8 weeks were subcutaneously immunized with 500 μL of RBD protein solution (30 μg of protein/mouse) or PBS and boosted on days 7 and 14. Blood was sampled on days 0 (prevaccination), 13, and 25. After coagulation at room temperature for 1 h, sera were collected by centrifugation  and stored at −80 °C until use.2SO4) was added to each well, and Infinite M200 fluorescent multifunctional enzyme marker  was used to read the absorbance at 450 nm.ELISA was used to analyze antibody responses in serum. Microplate wells were coated with RBD protein (100 ng/well) in PBS coating buffer and incubated overnight at 4 °C. Plates were washed three times using PBST containing 0.1% Tween 20 and blotted dry. The plates were sealed at room temperature with 5% BSA for 2 h. Continuously diluted serum with PBS buffer was added to the microplate (100 μL/well), which was incubated for 1 h at 37 °C. Then, the plate was washed. HRP-conjugated goat anti-mouse  was added to each well, followed by incubation for 1 h at room temperature. Next, the plate was washed six times and incubated with 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) solution for 15 min. To stop the reaction, 50 μL of stop solution  and GraphPad Prism software . Each experiment was independently replicated three times and statistical significance was defined as E. coli XL1-Blue cells and detected by SDS-PAGE. Although the recombinant RBD protein could be expressed in E. coli cells, the protein existed in the form of an inclusion body (The expression of the recombinant SARS-CoV-2 RBD protein was induced in ion body A. Therefion body B. When tion body C,D.In order to ensure that the purified wild-type RBD protein and mutant proteins have a biological function, their specific antigenicity was analyzed using WB. Mouse monoclonal anti-His tag A and humTo evaluate the in vivo immunogenicity of the mutated RBD proteins, mice were immunized with both wild-type RBD and the nine RBD protein mutants, and blood samples were taken on days 0, 13, and 25. The total antibody titer against the SARS-CoV-2 RBD protein was evaluated by ELISA using HRP-conjugated goat anti-mouse as the secondary antibody. Before prevaccination, no anti-RBD was detected in the serum of all mice A. On dayTo further evaluate the extensive antigenicity of mutants E484K, E484Q, K417T-E484K-N501Y, and K417N-E484K-N501Y, we coated microplate wells with E484K, E484Q, K417T-E484K-N501Y, and K417N-E484K-N501Y proteins, respectively, and after blocking, antibody titers in mouse serum collected on day 25 were determined. When RBD mutant proteins of E484K, E484Q, K417T-E484K-N501Y, and K417N-E484K-N501Y were coated separately as antigens, higher antibody titers were detected in the sera of mice immunized with E484Q B. These The cumulative multiple mutations of SARS-CoV-2 have formed highly infectious Delta and Omicron variants ,23, whicThe spike protein of SARS-CoV-2 consists of two major functional domains containing a total of 1273 amino acids. Located at the N-terminus of the S protein is the S1 functional region containing the NTD and RBD domains. The remaining part is the S2 functional region containing two trimeric structures mediating membrane fusion . TherefoE. coli, and after purification by affinity chromatography, BALB/c mice were immunized and antibodies against RBD were detected in serum (Several RBD mutants selected in this study were present in the above SARS-CoV-2 variants. The recombinant RBD protein was expressed by in serum B,C. The in serum D, indicain serum D. AlthouTo verify the above hypothesis, we used four RBD mutants, E484K, E484Q, K417T-E484K-N501Y, and K417T-E484K-N501Y, as antigens and detected the antibodies against RBD in the serum of all immunized mice. The results show that single mutants E484K and E484Q, when used as antigens, could not only detect high antibody titers in the sera of all immunized mice but also detect higher antibody titers in the sera of mice immunized with mutants E484K and E484Q as compared with mice immunized with wild-type and other mutant RBD proteins . It has In a pandemic where a virus frequently mutates, the broad-spectrum effectiveness of neutralizing antibodies and vaccines is crucial, so these studies are now the focus of many researchers. In the present study, different RBD mutants were selected as immunogens to investigate the differences in the levels of antibodies induced by them, and two significant mutants, E484K and E484Q, were found. Therefore, it can be inferred that the E484 amino acid residue on RBD not only significantly affects the binding affinity with the receptor ACE2, but also has a significant impact on the immunogenicity of the RBD protein, and it may even affect the transmissibility and pathogenicity of the mutant virus.


Introduction: Abdominal aortic aneurysms (AAA) are among the most lethal non-cancerous diseases. A comprehensive analysis of the AAA-related disease model has yet to be conducted.Methods: Weighted correlation network analysis (WGCNA) was performed for the AAA-related genes. Machine learning random forest and LASSO regression analysis were performed to develop the AAA-related score. Immune characteristics and epigenetic characteristics of the AAA-related score were explored.Results: Our study developed a reliable AAA-related disease model for predicting immunity and m1A/m5C/m6A/m7G epigenetic regulation.Discussion: The pathogenic roles of four model genes, UBE2K, TMEM230, VAMP7, and PUM2, in AAA, need further validation by in vitro and in vivo experiments. The heart pumps arterial blood through the aorta, then into its branches to supply the body in the abdominal section called the abdominal aorta. An abdominal aortic aneurysm is diagnosed when the diameter of the abdominal aorta enlarges by more than 50% of its diameter for various reasons . It is cMost patients with abdominal aortic aneurysms are asymptomatic. Occasionally, patients find a “pulsing lump” on their stomach by accident or during a visit for another medical condition. If an abdominal aortic aneurysm suddenly causes severe abdominal pain, it is often a sign that it has ruptured or has ruptured. In addition, enlarged aneurysms can compress other organs in the abdominal cavity, such as intestinal compression, causing nausea, vomiting, abdominal distension, and discomfort. Compression of the ureter can cause hydronephrosis and so on.Abdominal aortic aneurysms (AAA) cause three main harms: 1) The enlarged abdominal aorta compresses the surrounding vital organs and tissues, affecting their physiological functions. 2) Thrombus is easy to form in the lumen, which blocks the lower limb blood vessels after the thrombus falls off, leading to acute ischemic necrosis of the limb, just like the sudden water cut or power cut in daily life. 3) Under the continuous impact of blood flow, the abdominal aorta gradually enlarges, and when it exceeds the maximum tolerance limit, the aneurysm will burst and cause sudden death . AlthougVascular inflammation is the first occurrence of AAA . In the As an important research direction of post-transcriptional gene regulation, N1 methyladenosine (m1A), 5-methylcytosine (m5C), N6-methyladenosine (m6A), and 7-methylguanosine (m7G) modified RNA is widely found in eukaryotic cells and plays a vital role in a variety of biological processes . They alIn this regard, we developed an AAA-related model and provided new mechanism insights for immunity and m1A/m5C/m6A/m7G regulation in AAA using large-scale bioinformatics analysis.GSE98278 (48 AAA samples) and GSE47472  from the Gene Expression Omnibus (GEO) database were collected and used for the follow-up studies. GSE98278 and GSE47472 were RMA normalized and conformed to a normal distribution.β = 16 was automatically generated by the pickSoftThreshold function, and a scale-free R2 = 0.86 automatically derived by the softConectivity function were used as soft-threshold parameters. Using the plotEigengeneNetworks function, the module dendrogram showing the link between the eigengenes and the macrophages was plotted after the module eigengenes had been recalculated. Enrichment analysis for Gene Ontology-Biological Process (GO-BP), Gene Ontology-Cell Component (GO-CC), Gene Ontology-Molecular Function (GO-MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the genes in the turquoise module was performed in GSE47472 by the R package clusterProfiler.Weighted correlation network analysis (WGCNA) was performed on the top 5,000 genes from GSE47472 using the R package WGCNA. Gene significance was used to quantify the relationship between specific genes and macrophage densities, while module membership was used to illustrate the relationship between module eigengenes and gene expression profiles. To guarantee a scale-free topology network and create a TOM matrix, a power of The R package limma identified the differentially expressed genes (DEGs) between AAA and normal samples. The R package pheatmap was used to visualize the DEGs between AAA and normal samples. The R package venn was used to visualize the intersected genes between the limma-based DEGs and the WGCNA-based genes in GSE47472. The R package Pi performed the GSEA for GO pathways related to the AAA-related genes in GSE47472.The R package ConsensusClusterPlus was used to identify the AAA-related clusters based on the AAA-related genes in GSE98278 using the kmdist method. The R package pheatmap was used to visualize the AAA-related genes in the AAA-related clusters in GSE98278.The R package limma identified the DEGs between the AAA-related clusters in GSE98278. Random forest (machine learning) was performed for dimension reduction of the limma-based DEGs between the AAA-related clusters in GSE98278. LASSO regression analysis (machine learning) was performed to develop the AAA-related score, which lambda.min = 0.0063. The AAA-related score was calculated as the sum of gene*coefficient. The R package pROC was used to generate the ROC curves of four model genes in predicting the AAA-related clusters in GSE98278.The R package pheatmap was used to visualize the association between AAA-related score and CIBERSORT-based immune cells in GSE98278. The R package Pi performed the GSEA for GO pathways related to AAA-related score in GSE98278. m1A-related genes, m5C-related genes, m6A-related genes, and m7G-related genes were collected from the previous study .Alzet osmotic minipumps  were implanted into APOE−/− mice (C57BL/6J) at 8 weeks of age. Pumps were filled either with saline vehicle or solutions of Ang II  that delivered (subcutaneously) 1,000 ng/min/kg of Ang II for 28 days. Pumps were placed into the subcutaneous space of mice through a small incision in the back of the neck. All procedures involving animals were approved by the Animal Care and Use Committee at Dalian Medical University.https://www.ncbi.nlm.nih.gov/gene/60; F ACCCTGAAGTACCCCATCGAG R AGCACAGCCTGGATAGCAAC; Product length 224bp). H-UBE2K . H-TMEM230  H-VAMP7 . H-PUM2 .Six AAA and six normal samples were collected from our mice model. Total RNA was extracted by Trizol. The absorbance was measured at 260 and 280 nm, and the concentration and purity were calculated. The RT-qPCR assay was performed with SYBR method. All the primers were designed with primer 5.0. H-actin , plasma cells, activated dendritic cells, naïve B cells, activated CD4 memory T cells, and CD8 T cells. Double-strand break repair gulation . In addigulation .In our study, AAA-related scores positively correlated with most m1A/m5C/m6A/m7G-related genes. The AAA-related score was significantly associated with m5C-related genes DNMT1, NSUN2, NSUN5, and YBX1. The AAA-related score was significantly associated with m6A-related genes FTO, HNRNPC, METTL3, and RBM15. The AAA-related score was significantly associated with m7G-related genes CYFIP1, EIF3D, EIF4E3, NSUN2, and NUDT11. M1A, m5C, m6A, and m7G are new types of RNA methylation. M1A, m5C, m6A, and m7G are widely involved in regulating cardiovascular diseases, including heart failure, cardiac hypertrophy, aneurysm, and vascular calcification. Studies have also shown the expression pattern and functional significance of m6A-related genes in AAA . m6A-relin vitro and in vivo experiments.Our study developed a reliable AAA-related disease model for predicting immunity and m1A/m5C/m6A/m7G epigenetic regulation. The pathogenic roles of four model genes, UBE2K (hazardous), TMEM230 (favorable), VAMP7 (favorable), and PUM2 (favorable), in AAA, need further validation by


STMN2, in the cerebellum of 95 FTLD-TDP cases and 25 non-neurological disease controls. Soluble TDP-43 was decreased in the cerebellum of FTLD-TDP cases but a concomitant increase in insoluble TDP-43 was not seen. Truncated STMN2 transcripts, an indicator of TDP-43 dysfunction, were elevated in the cerebellum of FTLD-TDP cases and inversely associated with TDP-43 levels. Additionally, lower cerebellar TDP-43 associated with a younger age at disease onset. We provide evidence of TDP-43 loss of function in the cerebellum in FTLD-TDP, supporting further investigation into this understudied brain region.Frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) is a neurodegenerative disease primarily affecting the frontal and/or temporal cortices. However, a growing body of evidence suggests that the cerebellum contributes to biochemical, cognitive, and behavioral changes in FTLD-TDP. To evaluate cerebellar TDP-43 expression and function in FTLD-TDP, we analyzed TDP-43 protein levels and the splicing of a TDP-43 target, The online version contains supplementary material available at 10.1186/s40478-022-01408-6. Frontotemporal dementia (FTD) is an umbrella term for syndromes presenting with deficits in behavior, executive function, and language , 2. ApprC9orf72) gene as the most common genetic cause of FTD and amyotrophic lateral sclerosis (ALS)—often referred to as c9FTD/ALS—stoked interest in the cerebellum. The cerebellum of c9FTD/ALS cases is marked by robust dipeptide-repeat protein pathology and foci of repeat RNA transcripts [C9orf72 repeat expansion [C9orf72-associated pathology, leaving the causes of cerebellar dysfunction in FTLD-TDP unanswered.The cerebellum has historically been underappreciated in FTLD-TDP given the absence of TDP-43 inclusions and significant neurodegeneration in this neuroanatomical region. However, functional imaging studies show cerebellar involvement in cognitive abilities such as working memory, emotion, language, and attention processing , 7. In anscripts –14. Reponscripts , 16, andnscripts , 18, andxpansion –24. In aSTMN2 [STMN2 (tSTMN2), generated by the aberrant inclusion of a stop-codon containing cryptic exon, is detected in the central nervous system of ALS and FTLD cases with TDP-43 proteinopathy [tSTMN2 was also detected in the cerebellum of some ALS and ALS/FTLD cases [Previous studies discovered that TDP-43 suppresses the inclusion of cryptic exons in numerous transcripts , includiSTMN2 , 27. We inopathy . IntriguLD cases , suggestGiven the mounting evidence implicating cerebellar anomalies in FTLD-TDP, we evaluated TDP-43 splicing function and expression in the cerebellum of a cohort of well-characterized FTLD-TDP cases.Post-mortem brain tissue from individuals with neuropathologically confirmed FTLD-TDP and those without neuropathological features were provided by the Mayo Clinic Florida Brain Bank. All participants or their family members gave written informed consent, and all protocols were approved by the Mayo Clinic Institution Review Board and Ethics Committee. Sample size was determined based on the availability of tissue in our brain bank. A description of patient characteristics is provided Table tSTMN2 transcript were determined using 250 ng of RNA using the NanoString PlexSet platform. Data was analyzed using transcript nSolver4.0 software (NanoString Technologies) and normalized to hypoxanthine phosphoribosyltransferase (HPRT1), a housekeeping gene we have previously used to normalized transcript levels [tSTMN2 5’-AGAAGACCTTCGAGAGAAAGGTAGAAAATAAGAATTTGGCTCTCTGTGTGAGCATGTGTGCGTGTGTGCGAGAGAGAGAGACAGACAGCCTGC-3’, and HPRT1 (NM_000194.3), 5’- CTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGG-3’.RNA was extracted from postmortem frozen cerebellum tissue using the RNAeasy Plus Mini Kit (Qiagen) per the manufacturer’s instructions. RIN was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies), and only samples with an RNA integrity number  were used. Levels of the t levels . Probe sRadioimmunoprecipitation assay buffer (RIPA)-soluble and urea-soluble protein fractions were obtained from postmortem cerebellar tissue, as previously described . ApproxiAll statistical analyses were done using GraphPad Prism 9 (GraphPad Software). For each figure the type of analysis used, and the number of subjects is indicated in the figure and/or legend.tSTMN2 RNA and TDP-43 protein in the cerebellum between controls and FTLD-TDP cases, all FTLD-TDP cases combined and the three genotypes separately, were analyzed with single-variable (unadjusted) and multivariable linear regression models (adjusted). Multivariable models were adjusted for age at death, sex and RIN for tSTMN2 RNA, and for age at death and sex for TDP-43 protein. Both tSTMN2 RNA and TDP-43 protein were analyzed on the base 2 logarithmic scale due to their skewed distributions. The regression coefficients (β) and 95% confidence intervals (CIs) were estimated and interpreted as the difference in the means, bases on the 2 logarithmic scale, between all FTLD-TDP cases combined or the individual genotypes and the controls (reference group). P values less than 0.0125 were considered statistically significant after adjusting for the four different statistical test that were performed for all FTLD-TDP and the separate FTLD-TDP groups  vs. controls.To compare tSTMN2 RNA, age at disease onset, or disease duration after onset were evaluated in FTLD-TDP cases using single-variable and multivariable linear regression models. Both TDP-43 protein levels and tSTMN2 RNA were analyzed using the base 2 logarithmic scale. The multivariable model examining TDP-43 protein levels and tSTMN2 RNA was adjusted for age, sex and genotype. The model evaluating TDP-43 and age at disease onset was adjusted for sex and genotype, and the model assessing associations between TDP-43 protein and disease duration was adjusted for sex, age at onset and genotype. P values less than 0.0167 were considered statistically significant after adjusting for multiple comparisons.For associations between TDP-43 protein levels and To compare levels of soluble (RIPA-soluble) or insoluble (urea-soluble) TDP-43 protein a Mann–Whitney test was used.STMN2 missplicing, we used the NanoString PlexSet platform to measure tSTMN2 RNA in 95 FTLD-TDP cases ([C9orf72 repeat expansion and 33 with a mutation in progranulin (GRN)) and in 25 cognitively normal controls pathologically-confirmed to have no TDP-43 pathology  fraction, we measured cerebellar TDP-43 in soluble and insoluble fractions for a subset of FTLD-TDP cases and controls. More specifically, we selected the FTLD-TDP cases showing the most drastic depletion of soluble TDP-43 Fig. D as theytSTMN2 RNA production, we assessed whether detergent soluble cerebellar TDP-43 or tSTMN2 RNA levels associate with age of disease onset or disease duration in FTLD-TDP cases. Lower cerebellar TDP-43 associated with a younger age at disease onset in both unadjusted analysis  showed a significant accumulation of tSTMN2 RNA arguing that even a minor change in TDP-43 levels results in aberrant STMN2 splicing. These data suggest that tSTMN2 and other cryptic exon-containing transcripts normally suppressed by TDP-43 are sensitive markers of TDP-43 loss of function, thus providing a rationale to study their possible utility as biomarkers for distinguishing patients with FTD caused by TDP-43 proteinopathy vs. tauopathy—an endeavor of great importance to the FTD field.We noted that loss of soluble TDP-43 protein was more pronounced in the cerebellum of mutation carriers with the decrease in non-mutation carriers failing to reach statistical significance. However, each FTLD-TDP group (i.e., non-mutation carriers, tSTMN2 RNA associating with lower age at disease onset, in agreement with our earlier study finding a significant association between tSTMN2 in the frontal cortex and disease onset in a larger FTLD-TDP cohort [We additionally observed that lower levels of cerebellar TDP-43 associated with an earlier age at disease onset, suggesting that maintaining cerebellar TDP-43 levels may be protective in FTLD-TDP. We also noted a trend of higher P cohort . In combP cohort , 18, 31,P cohort –24, thesOverall, we conclude that TDP-43 loss of function may be more pervasive than initially appreciated, and that further study of the role of the cerebellum in the pathogenesis of FTLD-TDP is warranted.Additional file 1: Table S1. Associations of cerebellar tSTMN2 RNA with age at onset, and disease duration in FTLD-TDP cases.


Drosophila pupal wing development. We present evidence that these pathways generate planar polarity via distinct mechanisms. Core pathway function is consistent with strong feedback capable of self-organizing cell polarity, which can then be aligned with the tissue axis via weak or transient global cues. Conversely, generation of cell polarity by the Ft-Ds pathway depends on strong global cues in the form of graded patterns of gene expression, which can then be amplified by weak feedback mechanisms.Planar polarity describes the coordinated polarization of cells within a tissue plane, and in animals can be determined by the “core” or Fat-Dachsous pathways. Current models for planar polarity establishment involve two components: tissue-level “global” cues that determine the overall axis of polarity and cell-level feedback-mediated cellular polarity amplification. Here, we investigate the contributions of global cues versus cellular feedback amplification in the core and Fat-Dachsous pathways during   •The core and Fat-Dachsous planar polarity pathways function via distinct mechanisms•The core can self-organize planar polarity and be oriented by weak upstream cues•Fat-Dachsous are oriented by strong gradient cues but show poor self-organization Brittle et al. investigate how two different pathways generate planar polarity. Behavior of the Frizzled-dependent “core” pathway suggests the ability to self-organize planar polarity that can be oriented by weak tissue-level direction cues. Conversely, the Fat-Dachsous pathway shows only weak self-organizing properties and dependence on strong tissue-level gradient cues. Planar polarity is the polarization of cells within a tissue plane and is needed for the correct formation and function of many tissues . In animDrosophila wing. In these “puncta” core protein complexes are highly aligned in the same orientation . Unlike classical cadherins, Ft and Ds do not bind homophilically in rodimers B′  and Ft and Ds tagged with EGFP at their endogenous loci . To enabMeasurement of mean Fz-EGFP intensity on whole junctions on twin-clone boundaries confirmed enrichment on distal cell junctions, with intermediate levels on lateral junctions D and 2E In the pupal wing, Ft-Ds cellular asymmetry radiates out toward the wing margin in a broadly anteroposterior orientation, following the gradients of Ds and Fj expression . We agaiNotably, asymmetry of Ds-EGFP and Ft-EGFP within puncta is higher compared with non-puncta and whole junctions A–S1F, alWhen examining Ds-EGFP on clone boundaries, in addition to bright puncta on posterior cell edges, we also see weaker but distinct Ds-EGFP puncta on anterior edges  and mApple (in the apposed twin-clone) on a scatter graph. As expected, if the Ds signal is always higher on posterior cell edges, the points clustered into two populations: puncta on posterior Ds-EGFP clone edges (green dots) with higher Ds-EGFP/lower Ds-mApple, and puncta on anterior Ds-EGFP clone edges (magenta dots) with higher Ds-mApple/lower Ds-EGFP E. If theWe carried out a similar experiment to examine core pathway puncta. As we do not have Fz tagged with a red fluorescent protein, we instead generated EGFP-(Strabismus) Stbm/mApple-Stbm-expressing twin-clones D for theIn conclusion, for both Ft-Ds and the core pathway, individual puncta have a common orientation. However, Ft-Ds puncta contain detectable populations of Ds on both anterior and posterior cell edges that FRAP reveals to be in stable complexes E. This sde novo after ∼16 h APF  promoter in a ds background. We first confirmed that Ds expressed under the arm promoter throughout development polarizes normally on the anteroposterior axis . Control puncta are expected to have on average a 1:1 ratio of normalized EGFP to mCherry signal. However, imaging noise and stochastic variation in the amounts of EGFP and mCherry in puncta is expected to produce some variation in ratios in control puncta  compared with that in control puncta not on twin-clone boundaries inside To quantify these differences, for each condition a line was fitted to the heterozygous puncta distribution and the difference of each individual punctum from this line was calculated see . A valueTo explore further, we considered just puncta where the EGFP signal was on either anterior or posterior twin-clone boundaries , and plotted the relative EGFP/mCherry intensities G and 6H.It has been suggested that Ft-Ds complex sorting could be a result of reciprocal action of Fbxl7 on Ft and Ds, with Fbxl7 binding to Ft stabilizing Ft localization and destabilizing Ds by mediating its ubiquitination and removal . HoweverWe examined the effects of loss of Fbxl7 on Ft and Ds localization. This revealed a small decrease in Ft (and possibly Ds) levels at junctions, but no significant decrease in polarity A–7D. We These results support Fbxl7 playing a role in stabilizing both Ft and Ds at cell junctions, most likely via its binding to Ft. The failure to observe a reduction in cell polarity upon loss of Fbxl7, and the failure of Fbxl7 overexpression to destabilize Ds, both argue against a role in sorting of Ft-Ds heterodimers.In this work we compare the mechanisms used to generate planar polarity by the core and Ft-Ds pathways, and assess the relative contributions of upstream global signals and feedback amplification.In the core pathway, there is good evidence for feedback amplification e.g., . The corThe presence of punctate Ft and Ds in developing wings  led us tFrom these results we make two proposals regarding mechanisms of planar polarization by the Ft-Ds pathway in the pupal wing: (1) upstream global cues in the form of graded expression of Fj and Ds are required for cellular planar polarization and there is an absence of non-local  feedbacks able to drive spontaneous cell polarization; (2) weak local feedback interactions partially sort Ft-Ds heterodimers within puncta and most likely amplify cellular polarity. The presence of a significant stable fraction of Ds on the wrong side of puncta may favor such local feedback interactions being “positive” and stabilizing Ft-Ds heterodimers of the same orientation, although we cannot rule out “negative” interactions destabilizing heterodimers of opposite orientation.trans-heterodimerization between cells and cis-homodimerization within cells. A comparable combination of cis- and trans-interactions allows E-cadherin to form stable domains at cell-cell contacts through a phase transition mechanism leading to a lattice-like arrangement , potentially followed by amplification of these asymmetries.It may be biologically advantageous during development to have available these two different circuit logics of strong feedback/weak global cues versus strong global cues/weak feedback. The former logic most likely requires more complex cellular machinery to implement positive and negative feedbacks at different scales , but canThe aim of this work has been to examine the mechanisms of polarization used by the core and Ft-Ds pathways under as similar conditions as possible. To this end, we have compared their behaviors at the same stage of development in the same region of the wing, using measures of polarity that we have tested to be insensitive to differences in cell shape/size and image parameters . Neverthde novo polarization experiments.A further caveat is that the global cues that promote core pathway proximodistal polarity are poorly understood .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, David Strutt  y w P1; P FRT40/ft-EGFP FRT40 y w P1; P FRT80/fz-EGFP FRT80 y w P1; ft-EGFP FRT40/ds-mApple FRT40(A) y w P1; P FRT40/ds-EGFP FRT40 y w P1; P FRT80/fz-EGFP FRT80 y w P1; ds-mApple FRT40/ds-EGFP FRT40 y w P1; P[acman]-EGFP-stbm FRT40 stbm6/P[acman]-mApple-stbm FRT40 stbm6 w P; P/+; fzP21(A-D) w P; dsUA071P/dsUA071(E-H) w P; dsUA071P fjd1/dsUA071fjd1; P/+(I-L) y w P1; P[acman]-mCherry-Fbxl7 FRT40/P[acman]-Fbxl7-mEGFP FRT40 w; ds38KP[acman]-mCherry-Fbxl7 FRT40 fjd1/dsUA071P[acman]-Fbxl7-mEGFP FRT40 fjd1; P/P, P3 y w P1; FRT82 fbxl7Q201/FRT82 P(A-D) ds-EGFP/ds-EGFP (controls), ds-EGFP/ds-EGFP; FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (E) ft-EGFP/ft-EGFP (controls), ft-EGFP/ft-EGFP; FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (F) w; ds-EGFP P/P(G) w; ds-EGFP/P, P; P FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (induction experiments), w; ds-EGFP/+; P FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (no induction controls)(H) y w P1; P FRT80/fz-EGFP FRT80 y w P1; P FRT40/ds-EGFP FRT40 y w P1; P FRT40/ft-EGFP FRT40 y w P1; ft-EGFP FRT40/ds-mApple FRT40(A) y w P1; ds-mApple FRT40/ds-EGFP FRT40(B) w[1118](B) w P; dsUA071fjd1P/dsUA071fjd1; Act5C-fj/+ (with no heat-shock so arm-ds not expressed)(C) w P; P/+; fzP21(D-G) w[1118](H) w; dsUA071P/dsUA071(I) w P; dsUA071P/dsUA071(J-M) w P; dsUA071P fjd1/dsUA071fjd1; P/+(N-Q) ds-EGFP/ds-EGFP (controls), ds-EGFP/ds-EGFP; FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (A) ft-EGFP/ft-EGFP (controls), ft-EGFP/ft-EGFP; FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (B) w; ds-EGFP P/P(C) w; ds-EGFP/P, P; P FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (induction experiments), w; ds-EGFP/+; P FRT82 fbxl7Q201X/FRT82 fbxl7Q201X (no induction controls)(D) ds expression was achieved by cloning the ds CDS downstream of the arm promoter and randomly integrating into the genome by P-element transgenesis , and for inducible expression a removable stop cassette was included between the promoter and the CDS . Uniform fj expression was achieved by cloning the fj CDS into the vector attB-Actin5C>stop>polyA vector . Pupae were heat-shocked for 2 h at 38°C at the indicated times. On the basis of when trichomes subsequently emerge, we observe that development is halted for the period of the heat shock. The time of induction of protein expression was varied but the tissue fixed at the same developmental time of 28 h APF.ts system  and labeled with their orientation  dependent upon the angle of the line (in 90° bins) relative to vein 4 which was oriented as 0°. Clone regions lacking EGFP were measured and used to subtract background signal. Mean intensity for the oriented junctions were calculated per wing image.2  or 0.16 μm2 (wing disc) that covered the width of the junctions. Mean intensity per wing was then calculated.To measure intensity in puncta and non-puncta regions, samples were co-immunolabelled with Fmi or Ds and the images analysed to select puncta by total area using a Matlab script . Selecti∗Power3.1).Mean intensities were compared using a one-way ANOVA. Tukey-Kramer’s multiple comparison test was used to compare between all categories of junction within an experiment and Sidak’s multiple comparison test was used to compare genotypes pairwise. Where a post hoc test was used this is described in the figure legends, and multiplicity adjusted p values calculated in GraphPad Prism are indicated on the graphs. Based on the mean junctional asymmetry of Ds-EGFP in a pilot experiment, we aimed for a sample size of at least 6 wings per timepoint. This would allow detection of differences of 20% in the means, in a pairwise comparison, with a power of 0.8 and α 0.05 . This was achieved for all but one timepoint where we lacked 1 sample.For the polarity analysis following induction of gene expression, a cropped 300 × 300 pixel (31.5 × 31.5 μm) region was selected in the centre of the image at least 5 cells away from vein 4. Based on the mean cell polarity measurements of a pilot experiment, we aimed for a sample size of at least 6 wings per timepoint. This would allow detection of differences of 20% in the means, in a pairwise comparison, with a power of 0.8 and α 0.05  was used to fit a one-phase exponential association curve for each wing. Data from several wings were then used to fit a final exponential association curve, and an extra sum-of-squares F test was performed to compare curve plateaux (Ymax) between puncta and non-puncta or between genotypes. To determine the stable amount of EGFP in the ROIs, the mean intensity of the ROIs from the three pre-bleach images was measured in ImageJ, and averaged per wing. The intensity was then corrected for distance from the coverslip as previously described , and thi


Mater. Chem. A, 2019, 7, 20369–20382, https://doi.org/10.1039/C9TA07361B.Correction for ‘Blade coated P3HT:non-fullerene acceptor solar cells: a high-throughput parameter study with a focus on up-scalability’ by Enrique Pascual-San-José  The authors regret that the labels for IDTBR and IDFBR in A corrected version of The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Staphylococcus aureus (MRSA), has become a serious clinical and public safety concern with rising incidence in hospitals. Polyketides with diverse chemical structures harbor many antimicrobial activities, including those of rifampin and rapamycin against MRSA. Streptomyces sp. QHH-9511 was isolated from a niche habitat in the Qinghai-Tibet Plateau and used to produce antibacterial metabolites. Herein, an integrated approach combining genome mining and metabolic analysis were employed to decipher the chemical origin of the antibacterial components with pigmented properties in strain QHH-9511, a novel Streptomyces species from a lichen symbiont on the Qinghai-Tibet Plateau. Genomic phylogeny assembled at the chromosome level revealed its unique evolutionary state. Further genome mining uncovered 36 candidate gene clusters, most of which were uncharacterized. Meanwhile, based on liquid chromatography coupled to diode array detection mass spectrometry, a series of granaticins, BSMs, chromones, phaeochromycins, and related molecules were discovered by using the Global Natural Product Social molecular networking platform. Subsequently, several pigment compounds were isolated and identified by high-resolution mass spectrometry and/or nuclear magnetic resonance, among which the structure-activity relationships of seven aromatic polyketides showed that the fused lactone ring of the C-2 carboxyl group could increase antibacterial activity. Genetic experiments indicated that all seven aromatic polyketides are a series of metabolic shunts produced by a single type II polyketide synthase (PKS) cluster. Comparative genomic analysis of granaticin producers showed that the granaticin gene cluster is widely distributed. This study provides an efficient method to combine genome mining and metabolic profiling techniques to uncover bioactive metabolites derived from specific habitats, while deepening our understanding of aromatic polyketide biosynthesis.The prevalence of superbugs, represented by methicillin-resistant IMPORTANCE Undescribed microorganisms from special habitats are being screened for anti-superbug drug molecules. In a project to screen actinomycetes for anti-MRSA activity, we isolated a Streptomyces strain from Qinghai Lake lichens. The phylogeny based on the genome assembled at the chromosome level revealed this strain's unique evolutionary state. The chemical origins of the antibacterial components with pigment properties in strain QHH-9511 were determined using an integrated approach combining genome mining and metabolic analysis. Further genome mining uncovered 36 secondary metabolite gene clusters, the majority of which were previously unknown. A series of aromatic compounds were discovered using molecular network analysis, separation, and extraction. Genetic experiments revealed that all seven aromatic polyketides are a series of metabolic shunts produced by a single cluster of type II PKSs. This study describes a method for identifying novel Streptomyces from specific habitats by combining genome mining with metabolic profiling techniques. Staphylococcus aureus (MRSA), has triggered a radical revolution in natural product discovery strategies  domains catalyzing consecutive Claisen condensations between enzyme-bound thioesters of the growing polyketide chain and extender units typically originating from malonyl-coenzyme A    and loadnyl-CoA) , 22. Thenyl-CoA) , qinimycnyl-CoA) , medermynyl-CoA) , and actnyl-CoA)  as well nyl-CoA) , are rep enzymes . The comStreptomyces sp. QHH-9511, isolated from the brown lichen symbionts on the Qinghai-Tibet Plateau, whose unique metabolic profile caught our attention. The genome was assembled at the chromosome level using a combination of Illumina Hiseq with PacBio RSII sequencing technologies, while phylogenomic and average nucleotide identity analyses based on chromosomal sequences confirmed its unique evolutionary status. Genome mining using bioinformatics identified 36 candidate biosynthetic gene clusters (BGCs), with large parts being undescribed BGCs. BIG-SCAPE-based gene cluster family analysis further revealed the uniqueness of these BGCs. On the other hand, molecular network analysis of metabolites and the isolation and structural identification of monomeric compounds demonstrated the ability to produce secondary metabolites of this strain. The structure-activity investigation of aromatic compounds demonstrated the importance of specific functional groups. Inactivation of orf26-28 in cluster 20 suggested that seven compounds come from the same gene cluster. Comparative genomic analysis of granaticin producers revealed a varied distribution of granaticin BGCs. This study combines genome and metabolite profiling to characterize novel lichen-derived Streptomyces on the Tibetan Plateau and add new insights into aromatic polyketide biosynthesis.Here, we report a new strain, Streptomyces sp. QHH-9511, a strain isolated from yellow lichen on a rocky surface on the west coast of Qinghai Lake . Specifically, 3 days after inoculating mycelium on a Gause’s agar plate, a blue-violet pigment diffused through the medium . An agar plug bioassay revealed anti-MRSA properties , and its absorption spectrum was clearly pH dependent . These findings prompted a whole-genome sequencing to dissect the genetic basis of biosynthesis of these compounds in Streptomyces sp. QHH-9511.As part of a screening aimed at identifying extracts of natural products with MRSA inhibition activity, we identified the candidate Streptomyces sp. QHH-9511 consists of a linear chromosome of 7,524,079 bp with 71.34% GC content , and KEGG  categories revealed that the number of genes involved in metabolic processes was higher than the number of function-related genes annotated in GO  and KEGG , which implies that the producer possesses powerful metabolic capacities. abolites . There aStreptomyces species with complete genome assemblies revealed these strains share 156 single-copy orthologous genes. Phylogenomic analyses revealed that strain QHH-9511 is in a distinct clade  of strain QHH-9511 and its closely related strains on the phylogenomic tree. The result revealed that the ANI value of strain QHH-9511 and is value . As the is value , strain in silico analysis, with up to 36 putative BGCs (clusters 1 to 36)  (The genome of strain QHH-9511 has a considerable number of candidate BGCs for various metabolites based on 1 to 36) , Table 1Biosynthetic gene similarity clustering and prospecting engine (BiG-SCAPE), a bioinformatic software package, was subsequently used to analyze these 36 predicted BGCs  in detaiAntibiotic-resistant target seeker (ARTS) 2.0 was used to further understand the genome, and the characterization of the BGCs was predicted by antiSMASH. A total of 42 resistance models were identified, including genes coding for ATP-binding cassette antibiotic efflux pumps, biotin-dependent carboxylases, DNA topoisomerase, pentapeptide repeats, carboxyl transferase, glyceraldehyde 3-phosphate dehydrogenase, etc. Analysis of BGC proximity revealed that 20 BGCs harbored either neighboring putative core genes (clusters 9 and 17), known resistance genes , both genes , or unknown resistance genes (Table S3).The antiSMASH prediction results showed that 30 of the 37 predicted BGCs were less than 50% similar to known BGCs, indicating that strain QHH-9511 contains many undescribed BGCs responsible for novel compounds. The high number of antibiotic resistance genes implies that the strain is equipped with complex defense systems and some of the BGCs are involved in resistance mechanisms. Therefore, additional studies are needed to further characterize these clusters, as well as to identify and characterize the compounds produced and match them with the corresponding BGCs.4, 8, and 9, klavuzon compound 16, anthraquinone compound 18, and chromone compound 20. In addition, benzoic acid derivative 27 was detected  fingerprint analysis showed that the metabolites of solid fermentation were more abundant than those of liquid fermentation . The detection and analysis of concentrated crude samples of ethyl acetate extracts from solid fermentation of strain QHH-9511 by GNPS indicated that the metabolites contained large numbers of unknown small molecules, and at least six pigment molecules were present among the identified molecules (detected . The fac1), 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticin A (2), dihydrogranaticin A (3), MM44785 (4), diglycosyl-MM44785 (5), 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticin B (6), dihydrogranaticin B (7), metenaticin A (8), granaticin A (9), metenaticin B (10), granaticin B (11), dehydromutacin (12), phaeochromycin E (13), phaeochromycin D (14), phaeochromycin B (15), aloesaponarin II (16), 9′-hydroxyaloesaponarin II (17), oxanthroquinone (18), BSM2 (19), BSM1 (20), fogacin (21), phaeochromycin I (22), n-butyl isobutyl terephalate (23), dibutyl phthalate (24), dehydroxy-GTRI-02 (25), GTRI-02 (26), aloesaponol I (27), and 5,5′-dibutoxy-2,2′-bifuran (28) on the basis of comparisons of their MS and/or NMR data with literature values  to obtain relatively pure monomeric compounds. Next, these compounds were purified by semipreparative HPLC to afford 29 known compounds, which were identified as granaticin acid , Pseudomonas syringae pv. actinidiae, and Ralstonia solanacearum. The MIC results corresponded with structural complexity. The more inhibitory compounds, 2, 9, and 11, displayed lower MICs (Table S5), with compound 11 having a MIC of 0.125 μg/mL against both Bacillus cereus and Erwinia carotovora pv. carotovora, indicating their antimicrobial potential.With the monomeric compounds obtained, the retention times and spectral features of seven aromatic polyketide compounds with significant UV-Vis absorption were characterized . These c2 relative to 24). Second, the γ-lactone linking the C-4 hydroxyl and the C-2 carboxyl may moderately increase antimicrobial activity of compound 9 relative to that of compound 2  among granaticin analogs. First, the tetracyclic scaffold is clearly essential for antibacterial activity , with thmpound 2 . Althoug strains .Compound separation and identification, as well as antimicrobial activity assays, indicated that the strain QHH-9511 has a distinct metabolic profile due to its seven aromatic polyketides. To our knowledge, this is the first time that these seven aromatic polyketides –21, 26 h–33–Streptomyces violaceoruber Tü22 . To verify the importance of the putative cluster, an in-frame deletion of orf26-28 (putative keto-acyl synthase and acyl carrier protein) was constructed  in strain QHH-9511 responsible for these seven compounds, cluster 20  specifically indicated that target ions could not be detected in the mutant  in negative ion mode gave total ion current (TIC) profiles showing abolition of product peaks in the e mutant . These fS. olivaceus (9 and 11 have been discovered in S. violaceoruber (S. thermoviolaceus subsp. pingens var. WR-141(S. litmogenes (S. vietnamensis GIMV4.0001(9 and 11 has been established in S. violaceoruber (S. violaceoruber S21. The BGC for granaticins (9 and 11) were in the region with the highest similarity between strains QHH-9511 and GIM4.0001, whereas the corresponding region in strain S21 did not contain the BGC for granaticins , was initially isolated from Streptomyces sp. CPCC 200532  (Although the aromatic polyketide compounds C 200532 . Compounor CH999 . The onl Tü 6319 . Compounp. SA246  and was or A3(2) .26 and related polyketides as metabolic shunt products of the biosynthesis of 9 and 11 were revealed in S. vietnamensis GIMV4.0001 by time course metabolic profiling  assembled 33,853,158 readings from scratch. The PacBio platform sequencing data were corrected using BLASR26 (https://github.com/jcombs1/blasr) to map the Illumina sequencing reads and then assembled using the Celera assembler (http://wgs-assembler.sourceforge.net). Following the generation of a valid scaffold, the correction of sequencing reads was performed again using the Illumina data. The final finished assembly contained the entire genome sequence. Majorbio Cloud web platform was used to examine the basic genetic features. BLAST searches of nonredundant protein sequences from the NCBI, Swiss-Prot, COG, and KEGG databases were used to annotate the gene products' functions.The whole-genome sequencing of QHH-9511 was performed using a combined strategy of Illumina Hiseq and PacBio RSII high-throughput sequencing technologies by Majorbio Corporation , and we obtained 592-fold average genome coverage, with a paired-end library. SOAPdenovo 2.0425  was completed by running OrthoFinder 2.5.4 (http://tree.bio.ed.ac.uk/software/figtree/). ANI values between the genomes of QHH-9511 and other species were calculated by FastANI (The phylogenomic comparison of the strain QHH-9511 genome with 624 annotated genomes of er 2.5.4  on a dua FastANI .http://jvenn.toulouse.inra.fr/app/example.html). Chromosomal genome and plasmid map visualization and genomic synteny analysis were achieved through MCScanX  was used to predict metabolite biosynthetic clusters, and further protein alignment and manual rechecking of the core genes within BGCs were performed. The predicted BGCs were subsequently detected as core or essential genes or antibiotic resistance genes by ARTS 2.0, and the predicted BGCs were further analyzed by BIG-SCAPE (The website antiSMASH 6.1 (IG-SCAPE . Cutoffs2(SO4)3·4H2O, 40 mg; CaCO3, 1 g; KBr, 100 mg; H2O, 1,000 mL). Cultures were grown at 28°C for 7 days with shaking at 220 rpm. The violet-blue plate or liquid medium was collected separately, extracted three times with an equal volume of ethyl acetate (vol/vol), and the organic layer was concentrated in vacuo to give the crude extract.A seed culture containing QHH-9511 spores was kept at 30°C for 3 days in liquid TSB medium with 220 rpm shaking. Two different fermentation media were evaluated to optimize the chemical diversity of secondary metabolites produced by strain QHH-9511, including Gauze's plate medium and liquid medium  for molecular network formation. To generate consensus spectra, the data were clustered with MS-cluster with a parent mass tolerance of 0.02 Da and an MS/MS fragment ion tolerance of 0.02 Da. The network file based on negative-ion mode MS data can be found and accessed at in vacuo to yield a brown ointment. The resulting extracts were then separated and purified with different types of chromatography, including silica gel, reversed-phase C18 column, and Sephadex LH-20. A detailed diagram of the separation process is shown in Fig. S12. Finally, totals of 29 compounds were separated on a semipreparative HPLC equipped with a Thermo Fisher Hypersil GOLD column . The product from the wild type and mutant were detected with an Agilent HP1100 HPLC system equipped with a Phenomenex Luna C18 column , with a linear gradient of protocol of 0 to 15 min, 5 to 30% B; 15 to 45 min, 30 to 75% B, at a flow rate of 0.6 mL/min . UV absorption of HPLC-PDA was monitored at 254 nm. LC-ESI-HRMS analysis of the wild type and mutant were carried out on an LTQ XL Orbitrap (Thermo Fisher Scientific) coupled to an HPLC system, operated in negative-ion mode with electrospray ionization. HPLC separation was performed using the same Phenomenex column mentioned above. HPLC was equilibrated with H2O containing 20 mM ammonium formate (eluent A) and MeOH (eluent B), with the following program: 0 to 10 min, 5 to 25%B; 10 to 40 min, 25 to 80%B. The flow rate was 0.6 mL/min. Structural data for the compounds were obtained by using an AB Sciex TripleTOF 6600 mass spectrometer and/or Bruker Advance IIIHD 500-MHz NMR spectrometer.The violet-blue plate media were collected and extracted three times with an equal volume of ethyl acetate (vol/vol), and the organic layers were concentrated Staphylococcus aureus ATCC 29231, Bacillus cereus IOP 01, Erwinia carotovora pv. carotovora NPCB 01, Erwinia carotovora subsp. carotovora (Jone) NPCB A3, Pseudomonas syringae NPCB 201, and Ralstonia solanacearum NPCB 002. The tests were done by taking 20 μL stock solution of each compound at 1 mg/mL in MeOH. Assays were conducted in 96-well microtiter plates in EBS medium  for bacteria.MICs were determined in a dilution assay with the seven compounds using a series of pathogens, including methicillin-resistant 18 column , with a linear gradient of protocol of 0 to 15 min, 5 to 30% B; 15 to 45 min, 30 to 75% B, at a flow rate of 0.6 mL/min (H2O for eluent A and MeOH for eluent B). UV absorption of HPLC-PDA was monitored at 254 nm.The retention time characterization of seven aromatic compounds and the detection of mutant strains were performed by Agilent HP1100 HPLC equipped with a Phenomenex Luna C2O containing 20 mM ammonium formate (eluent A) and MeOH (eluent B), with the following program: 0 to 10 min, 5 to 25% B; 10 to 40 min, 25 to 80% B. The flow rate was 0.6 mL/min.LC-ESI-HRMS analyses of the mutant were carried out on an LTQ XL Orbitrap (Thermo Fisher Scientific) coupled to an HPLC system, operated in negative-ion mode with electrospray ionization. HPLC separation was performed using the same Phenomenex column mentioned above. HPLC was equilibrated with Horf26  with genomic DNA and the primer pairs Larm-F (5′-AGGTCGACCAAAGCGGCCATCGTGCCTCCCCACTCCTGCAtgctcaccgcgtaagggatc-3′)/Larm-R (5′-GGTGCGTCGGACGGCTTGGTCAGGCCGCCTCGGCCTGGGCGgcggtgagcaggtcccaga-3′) and Rarm-F (5′-gcccaggccgaggcggcctg-3′)/Rarm-R (5′-GTTATGCAGCGGAAAAGATCCGTCGACCTGCAGGCATGCAggagtgggggcggacgtgga-3′). The shuttle digest pYH7 was digested by BglII and HindIII, treated with alkaline phosphatase to obtain the linear fragment, and then purified by gel electrophoresis. According to the manufacturer’s protocol, the ligation between linear pYH7 and the amplification was carried out with the isothermal Gibson assembly method. Plasmid pYH7 was used for conjugational transfer after confirmation by sequencing. The recombinant plasmids were transformed into Escherichia coli ET12567/pUZ8002 cells, and pYH7-G01 was introduced into the wild type by conjugation on a serum-free medium containing 50 mg/mL apramycin and 25 mg/mL nalidixic acid. The double-crossover mutants were created by removing pYH7-G01 from the exconconjugants by numerous rounds of nonselective growth. PCR analysis was used to assess the integrity of the mutant orf26-28 using the primer pair id-F(5′-GTCTCCCTTGGGTGGGGTGCG-3′) and idR (5′-GCCTCCTCGATACGGGCCCCGG-3′).First, the target gene region disruption plasmid pYH7-G01 was created in order to obtain target mutants to find BGCs for aromatic polyketides. The homologous arms were created by amplifying areas upstream and downstream of Streptomyces sp. QHH-9511 genome has been deposited in NCBI GenBank under accession numbers CP046905 and CP046906. All other data generated or analyzed during this study are included in this published article .The


Taken together, our in vitro findings demonstrate that soluble HLA-G5 suppresses markers of B cell activation, suggesting that HLA-G5 has an impact on splenic B cell differentiation and activation. Based on these results, further investigation regarding the role of HLA-G as a prognostic factor and a potential therapeutic agent with respect to B cell function appears reasonable.The human leukocyte antigen G (HLA-G) is an immune checkpoint molecule with a complex network of interactions with several inhibitory receptors. Although the effect of HLA-G on T cells and NK cells is well studied, the effect of HLA-G on B cells is still largely elusive. B cells are of particular interest in the context of the HLA-G-ILT-2 interaction because the ILT-2 receptor is constitutively expressed on most B cells, whereas it is only present on some subsets of T and NK cells. To characterize the effect of HLA-G5 molecules on B cells, we studied splenic B cells derived from cytomegalovirus (CMV) sero-positive donors after CMV stimulation with antigens in the presence and absence of soluble HLA-G5. In the presence of HLA-G5, increased expression of the ITIM-bearing Ig-like transcript (ILT-2) was observed on B cells, but its expression was not affected by stimulation with CMV antigens. Moreover, it became evident that HLA-G5 exposure resulted in a decreased expression of CD27 and CD38 and, accordingly, in lower proportions of CD19 The non-classic HLA class I molecule G (HLA-G) is a potent immunosuppressive molecule that negatively influences the effector functions of various immune cells belonging to the innate and adaptive immune system and thereby induces immune inhibition , 2. Due Several immunosuppressive mechanisms mediated by cell-associated and soluble HLA-G (sHLA-G) molecules have been described , 4. HLA-β2-microglobulin (β2m) and a nonapeptide  is not significantly different between CD19 B cells . Of note+ splenic B cells primed overnight with HLA-G5 exhibited significantly lower CD27 expression whether they were stimulated with CMV (P = 0.04) or not (P = 0.009). In addition to modulating CD27 expression, HLA-G5 treatment resulted in lower proportions of CD19+CD27+ cells (P = 0.04). Stimulation with CMV antigen alone exerted a significant, albeit marginal, effect on the CD27 expression in CD19+ cells . These findings show that HLA-G5 mediates an alteration in CD27 expression and in subset frequencies of splenic B cells.Next, we elucidated the effect of HLA-G5 on the costimulatory receptor CD27, which is involved in effector and memory differentiation of B cells. As shown in + cells increased significantly (P < 0.00001) after stimulation with CMV antigens (9.28 ± 0.82) compared to non-stimulated cells (4.50 ± 0.52). Remarkably, although overnight priming with HLA-G5 alone  had no effect on CD38 expression, HLA-G counteracted the effect induced by CMV antigen stimulation. As a result, CD38 expression was significantly decreased (P = 0.004) as measured by MFI in CD19+ cells, as well as decreased in the proportion of CD19+CD38+ cells in the total population of CD19+ cells (P = 0.0006)  .P = 0.046) [P = 0.0004) under the influence of CMV antigen stimulation  . This fimulation . In para+CD38+ B cells and increases the expression of the inhibitory molecule ILT-2. These effects may promote B cell inhibition.Taken together, the combined results demonstrate that HLA-G5 affects the proportion of B cell subpopulations because it reduces the number of activated CD27in vivo, in accordance with in vitro experiments [HLA-G is an important immune modulator belonging to the group of non-classical HLA class Ib molecules. Because of its complex interaction network with specific inhibitory receptors on immune effector cells, it is considered a relevant immune checkpoint molecule. Over the past decades, aberrant HLA-G expression has been reported in various pathological situations, such as malignancy, infections, and transplantation , 3. Multeriments .in vitro. Overall, our work found that HLA-G5 (i) increases the expression of the inhibitory molecule ILT-2 on splenic B cells and (ii) decreases the proportion of activated CD19+CD27+CD38+ while increasing the proportion of CD19+CD27−CD38− cells, which are considered plasmablasts or naïve B cells.In the present study, we investigated the effect of HLA-G5 on B cells derived from the spleen Immunosuppressive functions of HLA-G are mediated by inhibitory ILT-2 and ILT-4 signaling. The importance of this interaction has been well described, rendering the HLA-G/ILT-2 ligand-receptor axis one of the most promising new therapeutic targets for immune checkpoint inhibitors , 57. ILTin vitro study that demonstrated that HLA-G aggregates on-to nanoparticles and suppresses B cell function through ILT-2 receptor interactions [in vitro. In the present work, we chose a more physiologic approach: We used free biologically active HLA-G5 molecules associated with β2-microglobulin. These molecules have previously been shown to functionally interact with the ILT-2 receptor [The upregulation of inhibitory receptors in immune cells may increase their activation thresholds; thus, this upregulation is an important mechanism of immune defense. Previous studies have shown that HLA-G1 and its soluble isoform HLA-G5 can induce transcriptional and phenotypical changes in immune cells as described by the upregulation of ILT-2, ILT-3, ILT-4, and KIR2DL4 on antigen-presenting cells, NK cells, and T cells . This upractions . This inractions . Howeverractions . This direceptor .Betaherpesvirinae subfamily, establishes lifelong persistence after primary infection and has a high sero-prevalence [The HLA-G5 isoform has been shown to upregulate the inhibitory receptors on the cell surface more potently than does the membrane-bound HLA-G1, although these two molecules differ only slightly in their structure . Thus, wevalence . In immuevalence . CMV hasevalence .After nonspecific stimulation with pokeweed mitogen and specific stimulation with tetanus toxin, the interaction of HLA-G and ILT-2 leads to reduced antibody production . Our stu+CD27+CD38+ B cells and an increase of CD19+CD27−CD38− B cells. CD27 is a member of the tumor necrosis factor receptor superfamily that regulates lymphocyte function; it has been identified as a hallmark of memory B cells [− B cell compartment comprises mainly naïve B cells, and CD27+ B cells comprise memory or plasma cells [Co-cultivation with HLA-G5 and CMV antigens resulted in a significant decrease in the number of CD19 B cells . After C B cells , 63. In  B cells . Thus, tma cells . CD38 isma cells . Becausema cells . In thisma cells .in vitro data show that HLA-G plays a crucial role in regulating splenic B cell fate decisions.Consequently, all of these effects of HLA-G5 on B cells converge to activate inhibitors or inhibit activators of cell survival and proliferation. Altogether, our