We found that ngn-1 is required for axon outgrowth, nerve ring architecture, and neuronal cell fate specification. We also showed that ngn-1 may have roles in neuroblast migration and epithelial integrity during embryonic development. Using RNA sequencing and comparative transcriptome analysis, we identified eight transcription factors  whose transcription is activated, either directly or indirectly, by ngn-1. Our results show that ngn-1 has a role in transcribing known terminal regulators that establish and maintain cell fate of differentiated neural subtypes and confirms that ngn-1 functions as a proneural transcription factor in C. elegans neurogenesis.Proper nervous system development is required for an organism’s survival and function. Defects in neurogenesis have been linked to neurodevelopmental disorders such as schizophrenia and autism. Understanding the gene regulatory networks that orchestrate neural development, specifically cascades of proneural transcription factors, can better elucidate which genes are most important during early neurogenesis. Neurogenins are a family of deeply conserved factors shown to be both necessary and sufficient for the development of neural subtypes. However, the immediate downstream targets of neurogenin are not well characterized. The objective of this study was to further elucidate the role of  Defects during nervous system development are implicated in numerous neurological diseases that are polygenic in nature, such as autism spectrum disorder (ASD) and schizophrenia  transcription factors . These p“CANNTG” . This inXenopus embryos caused ectopic neurogenesis in ectodermal tissue  required for the development of pancreatic tissue and insulin-secreting cells . These aIn humans, neurogenin 1 is associated with multiple neurodevelopmental disorders. Two single nucleotide polymorphisms associated with schizophrenia fall within the regulatory region of the neurogenin 1 locus . ngn-1 also has roles in transcriptional repression, suppressing the transcription of almost 500 target genes, although its role here may be indirect and via non-cell autonomous mechanisms. These data establish ngn-1 as a key proneural gene in C. elegans nervous system development and demonstrate the value of comparative transcriptomics for identifying transcription factor down-stream targets.The association of neurogenins with multiple human neurological disorders demonstrates the need for greater understanding of neurogenin function during nervous system development. Neurogenins are highly conserved across phyla . The nemC. elegans strains were grown on nematode growth medium plates (NGM Lite) at 20° as described previously (dpy-5(e907) and mab-20(bx24), LGII vab-1(dx31), LGIII cnd-1(ju29), cnd-1(gk718) and unc-119(ed3), LGIV daf-18(ok480), ngn-1(ok2200) and efn-4(bx80), LGV him-5(e1490), LGX sax-3(ky123) and sdn-1(zh20). Integrated transgenes used in the course of this study were juIs76 [unc-25p::GFP + lin-15(+)], mgIs18 [ttx-3p::RFP], nIs394 [ngn-1::GFP + lin-15(+)], otIs33 [kal-1p::GFP], sIs14542 [hlh-34p::GFP + dpy-5(+)], and ujIs113 [pie-1::mCherry::Histone H2B + nhr-2p::mCherry::HIS-24 + unc-119(+)]. Extrachromosomal arrays used in this study were leEx1829 [unc-42p::GFP + unc-119(+)], quEx99 [sax-3(minigene) + odr-1p::RFP], and wwEx37 [ngn-1p::GFP + unc-119(+)]. The chromosomal rearrangement mIn1 [mIs14dpy-10(e128)] II was used to balance a lethal construct. All mutants were outcrossed at least four times except cnd-1(gk718) (2x), vab-1(dx31) (1x), daf-18(ok480) (2x), and sax-3(ky123) (3x). Double and triple mutant strains bearing reporter genes were constructed using standard genetic techniques. See Table S1 for details of strains generated during the course of this study including strain numbers and extra-chromosomal array construction.eviously . N2 (BrileEx1829 [unc-42p::GFP + unc-119(+)] and ngn-1(ok2200); leEx1829 [unc-42p::GFP + unc-119(+)] animals, the head regions of L1 larvae (collected within an hour of hatching) were captured as an image stack using a Zeiss LSM 700 confocal microscope at 40x magnification under identical settings, ensuring no detector saturation. Images were processed in Fiji using a Z-project - Sum Slices workflow, which rendered the summated stacks as 32-bit images. The head, from the nose tip to the posterior end of the pharynx was outlined using a segmented line and the region outside the line deleted using the Edit – Clear Outside function. Pixel values and counts were obtained using the Analyze - Histogram function  then processed in Microsoft Excel  and expressed as arbitrary fluorescence units.Well-fed worms grown under normal conditions were used to characterize expression patterns. Images were captured using either a Zeiss LSM 700 confocal microscope with Zen Black imaging software, a Zeiss AxioImager Z2 fluorescent microscope with Zen Blue imaging software, or an Olympus BX61 fluorescence microscope with CellSenseDimension software. Adult worms were staged for imaging by transferring 10-20 L4 hermaphrodites to a new plate and imaging those worms 24 hr later. Worms were mounted on 2% agarose pads, anesthetized with 3.5 μl of 10 mg/ml sodium azide solution, and then immobilized under an 18 × 18mm cover slip. Images were analyzed using Fiji software  z-stack images  were collected using a 63x magnification oil immersion objective on a Zeiss AxioImager Z2 microscope equipped with motorized z-axis stage. Data stacks were captured every 2 min for 300 cycles using Zen Blue software and analyzed using Fiji. The following developmental time points were scored: Ea/Ep cell ingression, gastrulation cleft opening, gastrulation cleft closure, ventral enclosure, comma stage, and elongation. Scores for mutant strains were compared to wild-type worms and statistical analysis was performed using the student’s kal-1p::GFP lineaging strain was constructed by crossing otIs33 [kal-1p::GFP] with a mCherry histone marker line, ujIs113 [pie-1::mCherry::Histone H2B + nhr-2p::mCherry::HIS-24 + unc-119(+)]. Embryo preparation and microscopy were performed according to previous studies (A  studies . 4D timengn-1(ok2200) mutants and were grown in liquid culture in M9 buffer supplemented with 1 mg/ml cholesterol, and 5 mls of 50% OP50 E. coli slurry as described previously  according to the vendor’s protocol and the aqueous phase separated by centrifugation in MaXtract High Density tubes . The aqueous phase was precipitated using an equal volume of 100% isopropanol plus 3 μl of 20 ng/ml glycogen, the RNA pellet washed once in isopropanol, once in 70% ethanol then resuspended in 50μl of diethylpyrocarbonate (DEPC) treated water. RNA concentration was estimated using a Thermo Scientific NanoDropTM Lite. Samples were then diluted to less than 10 ng/μl and analyzed on an Agilent 4200 Tapestation using High Sensitivity RNA ScreenTape. Only samples with an RNA integrity number (RIN) number of 8.9 or greater were sequenced. Four biological replicates of wild-type and ngn-1(ok2200) embryonic total RNA were sent to the University of Kansas Genome Sequencing Core for sequencing via an Illumina NextSeq 550 system .Wild-type and C. elegans reference genome (wbcel235/ce11) using Hisat2 ver.2.1.0 with Samtools 1.4. Aligned reads were assembled and transcripts quantified using Stringtie 1.3.3.2 via Samtools 1.6. Differential expression analysis was performed using DESeq2 ver.3.4.1 . Tissue Enrichment and Gene Ontology analyses were performed using a web-based implementation of the https://www.wormbase.org/tools/enrichment/tea/tea.cgi.RNA sequencing data analysis was performed using the Galaxy platform (ngn-1(ok2200) mutants. Figure S2 shows a representative kal-1p::GFP cell lineage. Figure S3 shows ngn-1; guidance cue double-mutant analysis of AIY neuron axon outgrowth. Table S1 shows strains generated in this study. Table S2 shows the inferred terminal cell fates identified in the kal-1p::GFP cell lineage. Tables S3 and S4 show the up-regulated and down-regulated lists of ngn-1 transcriptome hits identified in this work. Transcriptome files generated in this study are publicly available via the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE143599). Supplemental material available at figshare: https://doi.org/10.25387/g3.12034392.Reagents generated in this study are available on request. Supplementary tables and figures are available via the GSA Figshare portal. Figure S1 shows the precocious embryo laying phenotype of ngn-1 in defining MI pharyngeal motorneuron cell fate, little is known about this deeply conserved gene in C. elegans nervous system development  mutants showed 45% embryonic lethality (ngn-1(ok2200) animals bearing a ngn-1::GFP translational transgene , indicating that the embryonic lethal phenotype is caused by the ngn-1(ok2200) allele and not a closely linked second-site mutation. ngn-1 mutant animals that survived to adulthood showed sluggish, uncoordinated movement, aberrant head movement, and precocious embryo laying . Overall, these observations suggest that ngn-1 is required for a broad array of neuromuscular functions.Beyond the role of mutation . In our ethality , Table 1ngn-1 in embryonic development, we scored whole broods of ngn-1 mutants for embryonic lethality and larvae that died before reaching adulthood .To further investigate the role of dulthood . In addingn-1 mutants were dying during embryonic development, we used 4-dimensional (4D) time-lapse microscopy to observe developing ngn-1(ok2200) embryos from the 2-4 cell stage through just prior to hatching  showed defects in gastrulation consistent with errors in neuroblast migration when compared to wild-type embryos (0/29 observed). Instead, the main arrest stage observed (25% of embryos) was due to a posterior ventral rupture during elongation (6/24 ngn-1(ok2200) embryos compared to 0/29 wild type). While only 33% of ngn-1(ok2200) embryos arrested during 4D imaging when compared to 46% when measured by whole brood analysis, this discrepancy is likely a result of the small sample size (n = 24) used in this assay. These data suggest that ngn-1 plays only a minor role in neuroblast migration but may have a non-cell autonomous role in posterior epithelial integrity.To determine when ngn-1 in development of the nervous system, ngn-1(ok2200) mutants were crossed with a kal-1p::GFP reporter gene, which labels a subset of neurons and glia in the head region  appears as a distinct narrowing of fluorescence around the isthmus of the pharynx  and 12.8 cell bodies (70%) were located in the isthmus region, with the remaining 5.7 (30%) clustered around the terminal bulb . However, many neurons were anteriorly displaced, with an average of 7.5 neurons (42%) now located in the corpus region (P < 0.001), 8.7 neurons in the isthmus  and only 1.7 neurons  in line with the terminal bulb. These data indicate the ngn-1 has roles in controlling both nerve ring architecture and neuronal cell body positioning.To further understand the role of nal bulb . ngn-1 mngn-1 function at the single cell level, we used a ttx-3p::GFP reporter gene to examine axon outgrowth in the AIY interneurons . In ngn-1 mutants, 98% of animals (49/50) show anteriorly displaced AIY cell bodies  showed defective dorsal axon extension, such that the AIYL and R axons fail to meet on the dorsal side  and 10.2 μm  compared with 28 μm  in wild type  showed such severe outgrowth defects that the axons remained below the pharynx, appearing as a tangle  animals carrying the ngn-1::GFP translational reporter gene showed complete rescue of cell body displacement and axon outgrowth defects, further confirming that the phenotypes observed were due to ngn-1 loss-of-function and not a closely-linked mutation  mutants  alone, suggesting that the ok2200 phenotype masks defects seen in each single mutant . Based on these data, it was unclear if ngn-1 has any role in the transcriptional regulation of these axon guidance cues.To further examine the genetic interactions between candidate axon guidance genes and ngn-1 regulatory targets was unsuccessful, we used RNAseq to identify transcript differences between wild type and ngn-1(ok2200) mutant embryos. This unbiased approach was employed to characterize components of the ngn-1 gene regulatory network downstream of ngn-1. To generate a comparative transcriptome of wild type and ngn-1(ok2200) null mutants (n = 4 replicates each), RNA was isolated from mixed-stage embryos, sequenced, and analyzed. This generated a list of 587 differentially expressed genes with a p-value ≤ 0.05 . We found that the majority of these genes  were upregulated in the ngn-1 mutant background, suggesting that ngn-1 acts primarily as a transcriptional repressor during embryogenesis (ngn-1 (as expected from the comparative transcriptome of a deletion mutant), ceh-17, and hlh-34 , but were outside the 40 most significant hits (ngn-1 controls the transcription of these genes directly or indirectly (via transcription of one or more intermediate factors or via non-cell autonomous mechanisms) is not known.RNA sequencing (RNAseq) is a powerful genetic tool to both identify and quantify gene expression .Our RNAseq assay generated an unbiased list of all l tissue . The SIAed genes . This rangn-1 having a role in activating gene transcription. Conversely, genes in the up-regulated dataset were mostly associated with cuticle and innate immune response terms. This suggests that ngn-1 may suppress transcription of target genes associated with the hypodermis including the innate immune response, possibly via non-cell autonomous mechanism(s).We corroborated our tissue enrichment analysis by performing a gene ontology (GO) term enrichment analysis on our up- and down-regulated target genes . Most ofngn-1 might be regulating transcription at the cellular level, we performed 4D-timelapse video analysis of ngn-1p::GFP expression in the early embryo  . unc-42 positively regulates transcription of multiple genes including nuclear receptor 2E fax-1, which is also significantly down-regulated in our transcriptome (P = 0.009), and the glutamate receptor glr-1 , suggesting that NGN-1 is required to activate transcription of this gene.The paired-like homeodomain transcription factor unc-42 transcriptome result, we crossed an unc-42p::GFP transgene  into the ngn-1(ok2200) mutant background and imaged L1  larva via confocal microscopy. Wild type worms had an average of 13 unc-42p::GFP expressing head neurons (unc-42p::GFP expression was imaged in the ngn-1(ok2200) background, GFP expression was significantly lower (P < 0.05) and restricted to less than 10 cells  mutants, hlh-34p::GFP expression was strongly suppressed, with only 5% of animals showing any GFP expression (P < 0.001). To confirm and validate the identity of the ngn-1(ok2200); hlh-34p::GFP strain, we crossed hermaphrodites with wild type males and scored F1 male progeny for the presence of GFP. All F1 males (anticipated genotype ngn-1(ok2200)/+; hlh-34p::GFP/+) were GFP-positive, confirming that our original strain was indeed homozygous at both the ngn-1(ok2200) and hlh-34p::GFP loci. We conclude that ngn-1 is almost exclusively required for hlh-34 transcription during development, but that there is a low-level, partial redundancy in the transcriptional activation of hlh-34 that can stochastically activate transcription in around 5% of animals.We performed a similar GFP reporter gene analysis on rneurons  – J. In ngn-1 in neural development and to identify downstream transcriptional targets using candidate gene and comparative transcriptomic approaches. Our data demonstrate roles for ngn-1 in embryonic epithelial integrity, fate specification, cell body positioning, and overall morphology of multiple neuron classes , making it a useful reporter for nerve ring structure  is not known. Work is ongoing to identify early acting GFP reporter genes that can be used to investigate NGN-1 transcriptional activation vs. repression at the single cell level during early embryogenesis.One of the surprising discoveries from our transcriptome study was NGN-1’s extensive role as a transcriptional repressor ( imaging . WhetherC. elegans using multiple techniques including genetic approaches (both forward and reverse), yeast-1 and 2-hybrid assays, chromatin immunoprecipitation, and protein binding microarrays , we were still able to identify over 500 targets whose transcript levels changed significantly. In particular, we identified hlh-34 as a ngn-1 target (hlh-34 is only expressed in a single pair of cells (the AVJL/R interneurons), we still had the statistical resolution to identify this ngn-1 target. Overall, this suggests that comparative transcriptomics offer a powerful general approach to identifying transcription factor targets during embryogenesis. Future work will refine our transcriptomics approach by taking this to a single cell or single embryo level to provide tighter analysis of either cell lineage or developmental timing.Transcription factors and gene regulatory networks have been investigated in 1 target . Althoug


Another SNP (rs9532931) is tentatively associated with a specific sub-group of autism patients . The C. elegans VWA-8 long isoform shares 38% and 55% amino acid sequence identity and similarity, respectively, with human VWA8 long isoform. We showed that endogenous VWA-8 is expressed in mitochondria of somatic tissues, except neurons . To determine the function of C. elegansvwa-8, we generated a null allele vwa-8(ju1659) by CRISPR-Cas9. vwa-8(ju1659) mutants are homozygous viable, and indistinguishable from wild type in gross phenotypes such as body size, brood size, growth rate and movement.VWA8 proteins, named for von Willebrand factor A (VWA) domain containing 8, are conserved from worm to mammals . In human, two SNPs (rs9566845 and rs9566867) in vwa8 are found to be associated with bipolar disorder with comorbid migraine (Oedegaard vwa-8(ju1659) using two CRISPR RNA (crRNAs), 5’-AGTGAAACCCGTGTGATCAT-3’ and 5’-CTACAACGAGAGTTGCCTGT-3’ (Integrated DNA Technologies) targeting the start and stop codons of vwa-8, respectively. The crRNAs were injected into wild type hermaphrodites with purified Cas9 protein , trans-activating crRNA (tracrRNA) and dpy-10 crRNA, as described . The dumpy F1 progeny of the injected P0 wild type animals were singled to separate plates. F1 dumpy worms were then genotyped for the presence of potential vwa-8 deletions using the following primers: 5’-CCTCGAGGGCCCCATATTTT-3’ and 5’-TGCTCTCGAACACCTTGCTT-3’. Several independent vwa-8 deletions were identified. All deletion mutants behaved similarly. ju1659 is a 7989bp deletion of vwa-8 whichremoves nearly all the coding sequence of vwa-8, except the last 82bp of the last exon. CZ26606 vwa-8(ju1659) was generated after outcrossing with N2 for 3 times to remove the dumpy mutation.We generated vwa-8(ju1659) will be available at the CGC.CZ26606


Preservation of the mTOR/DNMT binding mode of oleacein was dispensable for suppression of the ALDH+-CSC functional phenotype in hydroxytyrosol-unrelated mimetics. The anti-CSC chemistry of complex EVOO phenols such as oleacein can be phenocopied through the use of mimetics capturing its physico-chemical properties.The extra virgin olive oil (EVOO) dihydroxy-phenol oleacein is a natural inhibitor of multiple metabolic and epigenetic enzymes capable of suppressing the functional traits of cancer stem cells (CSC). Here, we used a natural product-inspired drug discovery approach to identify new compounds that phenotypically mimic the anti-CSC activity of oleacein. We coupled 3D quantitative structure-activity relationship-based virtual profiling with phenotypic analysis using 3D tumorsphere formation as a gold standard for assessing the presence of CSC. Among the top 20 computationally-predicted oleacein mimetics, four fulfilled the phenotypic endpoint of specifically suppressing the tumorsphere-initiating capacity of CSC, in the absence of significant cytotoxicity against differentiated cancer cells growing in 2D cultures in the same low micromolar concentration range. Of these, 3,4-dihydrophenetyl butyrate –a lipophilic ester conjugate of the hydroxytyrosol moiety of oleacein– and  Extra virgin olive oil (EVOO) is a unique functional food with a major contribution to the health-promoting effects of the so-called Mediterranean diet. EVOO contains a group of complex phenol-conjugated compounds named oleosidic secoiridoids or oleosides that exert nutritional and beneficial effects on major aging-driven diseases including cancer –10. UsinThe anti-CSC effects of oleacein are most likely related to its chemical structure, largely due to the presence of two hydroxyl groups in the hydroxytyrosol moiety , 19–21. Here, we used a natural-product-inspired drug discovery approach to identify new small molecules capable of phenotypically mimicking the anti-CSC actions of oleacein. Using the structure of oleacein as a “seed”, we coupled 3D quantitative structure-activity relationship (3D-QSAR)-based virtual profiling (VP) with laboratory-based phenotypic testing using tumorsphere-formation potential as a gold standard for evaluating the presence of CSC . We provWhen a 2D similarity, ligand-based VP program was executed over the Chembl(v19) database using the Tanimoto coefficient and 2D (Morgan/circular) fingerprints, only the closely related secoiridoid molecule oleocanthal (CHEMBL2172394) was identified. The execution of a comparative molecular similarity indices analysis (CoMSIA)-based 3D VP program, however, identified several compounds with physico-chemical similarity scores greater than 0.75 . Taking The binding mode of oleacein to mTOR was predicted to share key amino acid residues with the binding modes of second-generation ATP-competitive TORKinhibs and, consequently, partially mimicked the binding behavior of PP242 and Torin 2 to the ATP-binding catalytic pocket . But, thThe binding mode of oleacein to DNMT was predicted to closely resemble that of DNMT inhibitors such as 5-azacytidine, SGI-110, and curcumin . In the in vitro tumorsphere formation in low-density non-adherent serum-free medium supplemented with growth factors  against CSC-driven mammosphere formation), 4 out of the 14 oleacein mimetics tested specifically suppressed mammosphere formation, namely CHEMBL1621113 (N’-[4-nitro-2-(trifluoromethyl)phenyl]propane-1,3-diamine), CHEMBL1632504 ((E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide), CHEMBL126593 (N-(4-nitrobenzyl)ethenesulfonamide), and CHEMBL1950046 , while not exerting significant cytotoxic effects against differentiated cancer cells growing in 2D in the same low micromolar range (N-(4-chloro-5-nitrothiazol-2-yl)hexanamide) exhibited anti-CSC activity due to unspecific cytotoxicity against CSC and non-CSC cells  –40 and tar range . CHEMBL1SC cells .(E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide), to evaluate their capacity to target epithelial-like CSC cells with high levels of ALDH1 (ALDH1+). To do this, we used the Aldefluor® reagent, which quantifies ALDH activity by measuring the conversion of the ALDH substrate BODIPY aminoacetaldehyde to the fluorescent product BODIPY aminoacetate  in the number of ALDH1+ cells when BT-474 cells were treated with a non-cytotoxic concentration (10 μmol/L) of CHEMBL1950046 (hydroxytyrosol butyrate). A more pronounced effect was seen with CHEMBL1632504 ((E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide), which significantly decreased the proportion of ALDH+ cells from 40±2% in untreated BT-474 cells to levels as low as 2±1% (96% reduction). To corroborate the ability of oleacein mimetics to target ALDH1+ epithelial-like CSC irrespective of the mutational landscape of cancer cells, we employed triple-negative MDA-MB-436 cells as a second breast cancer model naturally enriched with ALDH1+ cells. Treatment with hydroxytyrosol butyrate decreased the ALDH1+ cell content of MDA-MB-436 by approximately 40%. Remarkably, the large population of ALDH1+ cells in untreated MDA-MB-436 cultures (42±8%) was drastically reduced by 93% (from 42±8% to 3±1%) in the presence of (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide.Oleacein selectively suppresses functional traits of CSC such as the expression of aldehyde dehydrogenase (ALDH) , a well-oacetate . Using H(E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide preserved the dual anti-mTOR/DNMTactivity of the parental oleacein.Preservation of the oleacein binding mode is required for a dual mTOR/DNMT inhibitory activity but not for their anti-CSC behavior of oleacein mimetics. We finally evaluated whether the selected mimetics hydroxytyrosol butyrate and (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide spanning over five logarithmic decades were selected. 50 of ~39 μmol/L; (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide was unable to decrease mTOR activity even at the maximum concentration tested.We first employed the FRET-based Z-LYTE™ Kinase Assay to test the ability of the selected oleacein mimetics to inhibit mTOR activity. Ten concentrations of hydroxytyrosol butyrate and 3H]methionine (SAM[3H]) into DNA (DNA 5-[methyl-3H]-cytosine) in the absence or presence of oleacein mimetics. The selected oleacein mimetics were tested in 10-dose IC50 mode with 2-fold serial dilution and reactions were carried out at 1 μmol/L SAM. Although hydroxytyrosol butyrate decreased DNMT3A activity in a dose-dependent manner, concentrations higher than 150 μmol/L were necessary to reach the IC50 value. (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide did not reach the half maximal inhibitory concentration of DNMT3a activity even at the highest concentration tested.We finally carried out a radioisotope-based methyltransferase profiling measuring the DNMT3A-catalyzed incorporation of S-adenosyl-Lhexanoic acid), CHEMBL1545778 ([2-(methylcarbamoylamino)-2-oxo-ethyl] (E)-3-(3-bromophenyl)prop-2-enoate), CHEMBL1366164 (ethyl 2-[(2-methyl-5-nitro-phenyl)amino]-2-oxoacetate), and CHEMBL1642794 ([2-(tert-butylamino)-2-oxo-ethyl] 4-nitrobenzoate) were purchased from Enamine . CHEMBL1440472 (2- [(6- chloro- 3- nitro- 2-pyridinyl)amino]-3-methylbutanoic acid) was purchased from Key Organics . CHEMBL1621113 (N-[4-nitro-2-(trifluoromethyl)phenyl]propane-1,3-diamine) and CHEMBL1079062 ((Z)-4-[(4-nitrophenyl)amino]-4-oxobut-2-enoic acid) were purchased from ABCR GmbH .1H at 400.10 MHz). HPLC-MS. HPLC-MS were performed with a High-Performance Liquid Chromatography Thermo Ultimate 3000SD (Thermo Scientific Dionex) coupled to a photodiode array detector and a mass spectrometer LTQ XL ESI-ion trap (Thermo Scientific); 5μl of sample MeOH were injected (c=0.5mg/mL). Data from mass spectra were analyzed by electrospray ionization in positive and negative mode and peaks are given m/z (% of basis peak). The mobile phase used was a mixture of A = water + 0.05 formic acid and B = Acetonitrile + 0.05 formic acid with method described as follows: flow 0.5 mL/min; 5% B for 0.5 min; 5%-100% B in 5 min, 100% B for 2min.NMR spectra were recorded on an Agilent VNMRS-400 . Graded concentrations of oleacein mimetics were added to triplicate sets of wells on day 1 and the number of 6-day-old mammospheres was recorded as a measurement of CSC content. Images were recorded using a BioTek Cytation 5 image cytometer at 2.5× magnification. Prior to image acquisition, spheroid cultures were stained with a fluorescent vital dye to increase the accuracy of spheroid detection and analysis. The system was then set to count number, size, and aspect ratio of the objects. Thresholds were set to >100 μm in size and 0.4 as aspect ratio (with 1 being the aspect ratio of a perfect circle).® assay  was performed with or without the addition of hydroxytyrosol butyrate and (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide for 48 h.The ALDEFLUOR50 determinations for FRAP1 (mTOR) of oleacein mimetics were outsourced to Invitrogen (Life Technologies) using the FRET-based Z-LYTE™ SelectScreen Kinase Profiling Service. The effect of oleacein mimetics on the enzymatic activities of the recombinant human DNMT3A was outsourced to Reaction Biology Corp.  using HotSpotSM, a nanoliter-scale radioisotope filter binding platform.ICStatistical analysisF values at ANOVA, were assessed by multiple contrasts. P values < 0.05 and <0.005 were considered to be statistically significant . All statistical tests were two-sided.All statistical analyses were performed using GraphPad Prism software . Data are presented as mean ± S.D. Comparisons of means of ≥ 3 groups were performed by analysis of variance (ANOVA) and the existence of individual differences, in case of significant Supplementary FiguresSupplementary Tables 1, 2, 3, 4 and 5Supplementary Table 6Supplementary Table 7


Gipr andGlp1r are expressed in the hypothalamus in both rodentsand human. To better understand incretin receptor-expressing cellpopulations, we compared the cell types and expression profiles ofGipr- and Glp1r- expressinghypothalamic cells using single-cell RNA sequencing.The hypothalamus is a key region of the brain implicated inhomeostatic regulation, and is an integral centre for the control of feedingbehaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependentinsulinotropic polypeptide (GIP) are incretin hormones with potentglucoregulatory function through engagement of their respective cognatereceptors, GLP-1R and GIPR. Recent evidence indicates that there is asynergistic effect of combining GIP- and GLP-1-based pharmacology onappetite and body weight. The mechanisms underlying the enhanced weight lossexhibited by GIPR/GLP-1R co-agonism are unknown. Glp1r-Cre or Gipr-Cretransgenic mouse lines, fluorescent reporters were introduced into eitherGlp1r- or Gipr-expressing cells,respectively, upon crossing with a ROSA26-EYFP reporterstrain. From the hypothalami of these mice, fluorescentGlp1rEYFP+ orGiprEYFP+ cells were FACS purified andsequenced using single-cell RNA sequencing. Transcriptomic analysis provideda survey of both non-neuronal and neuronal cells, and comparisons betweenGlp1rEYFP+ andGiprEYFP+ populations were made.Using Glp1rEYFP+ andGiprEYFP+ cells were isolated, sequenced andtaken forward for bioinformatic analysis. BothGlp1rEYFP+ andGiprEYFP+ hypothalamic populations weretranscriptomically highly heterogeneous, representing vascular cell types,oligodendrocytes, astrocytes, microglia, and neurons. The majority ofGiprEYFP+ cells were non-neuronal, whereasthe Glp1rEYFP+ population was evenly splitbetween neuronal and non-neuronal cell types. BothGlp1rEYFP+ andGiprEYFP+ oligodendrocytes express markersfor mature, myelin-forming oligodendrocytes. While mural cells arerepresented in both Glp1rEYFP+ andGiprEYFP+ populations,Glp1rEYFP+ mural cells are largely smoothmuscle cells, while the majority of GiprEYFP+mural cells are pericytes. The co-expression of regional markers indicatethat clusters of Glp1rEYFP+ andGiprEYFP+ neurons have been isolated fromthe arcuate, ventromedial, lateral, tuberal, suprachiasmatic, andpremammillary nuclei of the hypothalamus.A total of 14,091 Glp1r andGipr cells of the hypothalamus with single-cellresolution. This resource will provide mechanistic insight into how engagingGipr and Glp1r cells of thehypothalamus may result in changes in feeding behaviour and energybalance.We have provided a detailed comparison of  Studies in preclinical models have identified key circuits within thehypothalamus that integrate nutritive and metabolic cues from the periphery withneural networks that regulate appetite, feeding behaviour, and energy expenditure. ThisciGlucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropicpolypeptide (GIP) are incretin hormones with potent stimulatory effects onpostprandial insulin release .GLP-1 rThe next generation of therapeutics for the treatment of metabolic diseaseincludes multifunctional GLP-1RA-based drugs designed with the aim to simultaneouslytarget multiple signalling pathways to improve the therapeutic index. The GIPreceptor (GIPR) signalling axis has proven to be an effective co-target, with bothGIPR agonists and antagonists demonstrating enhanced weight loss or prevention ofweight gain when combined with GLP-1R agonism in preclinical models . The advGipr and Glp1r are expressed in cellpopulations in the hypothalamus in both rodents and humans  to purify Gipr- andGlp1r- expressing hypothalamic cells and perform single-cellRNA sequencing (scRNAseq). Through downstream bioinformatic analysis, we havedetailed and compared the composition and expression profiles ofGipr- and Glp1r- expressing hypothalamic cellpopulations, providing an in-depth analysis of both non-neuronal and neuronal celltypes expressing these incretin receptors.Recent advances in single-cell transcriptomics have enhanced ourunderstanding of the heterogeneity of brain cells acting within molecularly-definedneurocircuits as well as non-neuronal cells that contribute to brain tissuemaintenance and function. In this report, to better characterise hypothalamic22.1All animal procedures were approved by the University of CambridgeAnimal Welfare and Ethical Review Body and conformed to the Animals (ScientificProcedures) Act 1986 Amendment Regulations (SI 2012/3039). The work wasperformed under the UK Home Office Project License PE50F6065. All mice weregroup-housed and maintained under SPF health/immune status in individuallyventilated cages with standard bedding and enrichment unless otherwise stated.Mice were housed in a temperature (22°C) and humidity-controlled room ona 12 h light/dark cycle  with ad libitumaccess to water and standard laboratory chow diet .Gipr-Cre  was dissected into Hibernate-A medium without calcium (BrainBits). Thetissue was digested with 20 U/ml Papain (Worthington) for 30 min at 37°C,followed by trituration in Hibernate-A medium (Thermo Fisher Scientific)containing 0.005% (w/v) DNase 1 (Worthington). The cell suspension was filteredthrough a 40 μm cell strainer into a fresh tube.Single cell suspensions were prepared from hypothalamic tissue pooledfrom two to six eviously . Both maGiprEYFP+ andGlp1rEYFP+ cells prepared fromGiprEYFP orGlp1rEYFP mice, respectively. Cells were gatedaccording to cell size (FSC), cell granularity (SSC), FSC pulse-width forsinglets, fluorescence at 488 nm/532 nm for EYFP and 647/670 nm for nuclearstain with DraQ5 (Biostatus).Fluorescence-activated cell sorting was performed using a BD Influx CellSorter  to isolate2.3GiprEYFP+ orGlp1rEYFP+ cells were generated using the10× Genomics Chromium Instrument and single-cell 3’ Reagent kit. Pooled libraries were sequenced on an Illumina NovaSeq6000 instrument . Library preparation andsequencing was performed by the Genomics Core, Cancer Research UK CambridgeInstitute. For downstream analysis, previously published sequencing data fromhypothalamic GiprEYFP+ cells . The 3’ untranslated regions (UTRs) ofyA_DB v3 , and extyA_DB v3 . DownstryA_DB v3 . Each saXist reads. Uniform manifoldapproximation and projection (UMAP) were generated using the top 30 principlecomponents from principle component analysis (PCA), run on variable featuresdetected by SCT. Clustering was performed using the Louvain algorithm withresolution 0.8-1.0, and the k-parameter (k=15-30) for neighbour detection wasreduced to the point where the smallest cluster visible was detected. Genemarkers for each cluster were cross referenced against other bulk and singlecell RNA sequencing databases RNAseq and in situ hybridisation (ISH) databasesto assign cell type identities for each cluster -normaliseddata, regressed for percentage of mitochondrial reads, percentage of ribosomalreads, and percentage of  cluster .Pdgfra > 1 | Pdgfrb> 2 | Cldn5 > 1 |Tagln > 3) & (Mbp < 1| Snap25 < 1); for oligodendrocytes,(Olig1 > 2 | Sox10 >1 | Mog > 2) & (Aqp4< 1); for neurons, (Syt1 > 1 |Snap25 > 2) & . Vascular cells were further subdividedinto pericytes, smooth muscle cells (SMCs), endothelial cells (ECs), andvascular leptomeningal cells (VLMCs) based on the expression of known markersfrom previous studies   . FiltereWhere differential expression analysis was performed, marker genes wereidentified for all clusters using a negative binomial regression model,implemented by Seurat’s FindAllMarkers function. Datasets were integratedusing canonical correlation analysis (CCA), approximate nearest neighbours(ANN), and n = 2,000 integration features.33.1Gipr+ andGlp1r+ populations represented in these studies are low innumber, likely owing to difficulties in detecting lowly expressed transcripts atthe sequencing depth afforded by single-cell and single-nucleus sequencingapproaches. We therefore used Gipr-Cre andGlp1r-Cre transgenic mice crossed with aROSA26-EYFP reporter strain to produceGiprEYFP andGlp1rEYFP mice, allowing for the purificationand enrichment of GiprEYFP+ andGlp1rEYFP+ cells based on EYFP expression fordetailed transcriptomic characterisation of these hypothalamic cell types.While multiple reports have used scRNAseq to survey and characteriseneurons within the hypothalamus, GiprEYFP orGlp1rEYFP mice were FACS purified andencapsulated to create sequencing libraries forGiprEYFP+ andGlp1rEYFP+ cells, respectively. We integratedGiprEYFP+-andGlp1rEYFP+ data with a previously-publishedGiprEYFP+ dataset to increase power forcomparative analysis. Three sequencing libraries forGiprEYFP+ cells and one sequencing library forGlp1rEYFP+ cells yielded a total of 11,351GiprEYFP+ cells and 2,740Glp1rEYFP+cells for subsequent scRNAseq analysis, vascular leptomeningeal (VLMCs), andendothelial cells (ECs), formed the majority of the detected cells 70% (9879),while neurons and oligodendrocytes (ODs) accounted for 11%  and 17% of the remaining cells, respectively. A small number of astrocytes(174), macrophages (26) and ependymocytes (43) were also found.Among the detected cell types, neurons, oligodendrocytes (ODs), andmultiple vascular cell types were identified within theGipr andGlp1r transcript in each cell type cluster , smooth muscle cells (SMCs)(Tagln), vascular leptomeningal cells (VLMCs)(Pdgfra), and endothelial cells (ECs)(Cldn5). Neurons and ODs were excluded using markersSnap25 and Mbp, respectively.Dimensionality reduction for the 8,512 vascular cells reproduced the separationof pericytes, SMCs, VLMCs and ECs based on known cell type marker expression   (CampbelCs: Lum) . DimensiCs: Lum) . SmallnCs: Lum) .Kcnj8 expression, formed eightclusters (Peri-1 to Peri-8)  in cluster Peri-1 and Peri-4  .Signifind Prrx1 .Gene exd Peri-4 . Despiter Peri-7 .Acta2expression, SMC-6 expressed significantly higher levels of cell adhesion andproliferation gene Ccn1, and higher levels of transcriptionfactors Atf3, Fos, Jun,Junb and Jund  and venous and capillarymarkers (Slc38a5 and Mfsd2a)  . Though P+cells .Lum expression, separatedinto two clusters   .Nnat ann VLMC-2 .VLMC-1  and Dcn. AdditioePdgfra , purinerlin Edn3 . VLMC-2 r Nkain4 . In VLMCf Pgrmc1.3.3GiprEYFP+/Glp1rEYFP+ODs further, cells were filtered forpositive expression of general OD gene markers , and negatively againstastrocytes (Aqp4). Filtering left 2,323 cells, which afterre-clustering revealed 10 clusters of ODs (OD-1 to OD-10) , whileclusters OD-7, OD-8 and OD-9 expressed higher levels of myelinating OD markers  . Cluster Opalin) . The mater Ttyh1. OD-10, r Slc4a4 . The 32clusters .3.4Syt1 andSnap25. Contaminating pericytes, SMCs, and ODs wereeliminated based on expression of Abcc9,Acta2, and Mal, respectively. 397GiprEYFP+ and 944Glp1rEYFP+ resultant neurons clustered into 12distinct sub-populations following dimensionality reduction . Expression ofSlc17a6 (encoding a vesicular transporter for glutamate)and Slc32a1 (encoding GABA vesicular transporter) indicatedthat ARH.1 and ARH.2 were both hybrid glutamateric/GABAergic cell populations.Enrichment of Pnoc (encoding prepronociceptin) expressiondistinguished ARH.1, with increasedCartpt/Serpina3n  expression identifying ARH.2. ARH.3 was highly enrichedfor Pomc and Cga, . Neurons in NK.1 were principallyGiprEYFP+, and express bothSlc17a6 and Slc32a1, with enrichedexpression of arginine vasopressin transcript (Avp).In addition to regional markers, the neuronal clusters were stratifiedby their relative enrichment for neurotransmitters and genes encoding secretedproducts and cell surface receptors . All thrSlc17a6+). VHM.2 was composed of equal proportion ofGiprEYFP+ andGlp1rEYFP+ neurons, whereas the majority ofVMH.1 neurons are Glp1rEYFP+. VMH.1 is enriched forprodynorphin transcript (Pdyn) and the cannabinoid receptortranscript, Cnr1, with VMH.2 expressing high levels of tachykinin precursor,Tac1 and the alpha-2-adrenergic receptor,Adra2a. Though not significantly enriched, the majority ofneurons in the LH cluster expressed Cck (encodingcholecystokinin). The PMd cluster also expressed Cck, with asignificant enrichment of neurexophilin4 (Nxph4). In contrastto PMd, PMv was principally GiprEYFP+, and enrichedfor Tac1 and the serotonin receptor,Htr2c.VMH.1, VMH.2, LH, PMd, and PMv were all principally glutamatergic(Scl32a1+). Both SCN clusters were enriched for neuromedinS transcript (Nms). SCN.1 expressed higher levels of thevasoactive intestinal peptide receptor 2 (Vipr2), anddemonstrated a relatively higher percentage ofGlp1rEYFP+ neurons. MTu was 95%GiprEYFP+, and was significantly enriched forsomatostatin (Sst) and parathyroid hormone-like hormone(Phtlh) transcripts.The MTu, SCN.1, and SCN.2 clusters were GABAergic, potentially before myelination is complete. Whether the relativerepresentation of oligodendrocyte precursor cells verses mature oligodendrocytesexpressing incretin receptors changes with maturity should be evaluated.Glp1r. While these astrocytes demonstrate lowGfap expression, they instead express Aqp4, auniversal marker for astrocytes encoding the water channel, aquaporin 4 that islocated on astrocyte vascular end-feet  or dorsomedialhypothalamus (DMH). Previous immunohistochemistry studies indicate thatGipr+ and Glp1r+ neurons are present in thesenuclei  for immunohistochemical (IHC) analysisof mouse brain tissue. In this study the authors compared the distribution ofGLP-1R mAb staining to a report by Cork et al where theGlp1rEYFP mouse model we present here was usedto map Cre-dependent EYFP expression in mouse brain . Twoaddpromotor . Discreppromotor . While t4.2Glp1rEYFP+andGiprEYFP+ will be an important resource forunderstanding the molecular fingerprint of these cells, and provides insight forfuture functional studies.Here we provide transcriptomic molecular identification of neuronal andnon-neuronal cells in the hypothalamus that express receptors for the incretinhormones, GIP and GLP-1. This census of cell types expressingSupplementary Figures


Correction to: Eur Radiol Exp 4, 33 (2020)https://doi.org/10.1186/s41747-020-00163-4The originally published  article FundingThis study has received funding by European Commission (Horizon 2020) under the grant agreement no. 761031 (nTRACK — H2020-NMBP-2016-2017), the Carlos III Health Institute and the European Regional Development Fund (PI16/02064) and AGAUR from Generalitat de Catalunya (2017SGR1303).


Scientific Reports 10.1038/s41598-018-26347-y, published online 21 May 2018Correction to: This Article contains an error. In Figure 3C, the panels for tau treated wild-type and spinal muscular atrophy motor neurons are reversed. The correct Figure 3C appears below as Figure


Cancer has several phenotypic subtypes where the responsiveness towards drugs or capacity of migration or recurrence are different. The molecular networks are dynamically altered in various phenotypes of cancer. To reveal the network pathways in epithelial-mesenchymal transition (EMT), we have profiled gene expression in mesenchymal stem cells and diffuse-type gastric cancer (GC), as well as intestinal-type GC. Gene expression signatures revealed that the molecular pathway networks were altered in intestinal- and diffuse-type GC. The artificial intelligence (AI) recognized the differences in molecular network pictures of intestinal- and diffuse-type GC.male-specific lethal 3 homolog (Drosophila) pseudogene 1 (MSL3P1), CDC28 protein kinase regulatory subunit 1B (CKS1B), DEAD-box helicase 27 (DDX27), golgi to ER traffic protein 4 (GET4), chromosome segregation 1 like (CSE1L), translocase of outer mitochondrial membrane 34 (TOMM34), YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), ribonucleic acid export 1 (RAE1), par-6 family cell polarity regulator beta (PARD6B), and MRG domain binding protein (MRGBP), which have differences in gene expression between intestinal- and diffuse-type GC. A total of 463 direct relationships with three molecules  were found in the biomarker-filtered network generated by network pathway analysis. The networks and features in intestinal- and diffuse-type GC have been investigated and profiled in bioinformatics. Our results revealed the signaling pathway networks in intestinal- and diffuse-type GC, bringing new light for the elucidation of drug resistance mechanisms in CSCs.Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance, which are the main hallmarks of cancer malignancy. Although previous findings have shown that several signaling pathways are activated in cancer progression, the precise mechanism of signaling pathways in EMT and CSCs are not fully understood. In this study, we focused on the intestinal and diffuse-type gastric cancer (GC) and analyzed the gene expression of public RNAseq data to understand the molecular pathway regulation in different subtypes of gastric cancer. Network pathway analysis was performed by Ingenuity Pathway Analysis (IPA). A total of 2815 probe set IDs were significantly different between intestinal- and diffuse-type GC data in cBioPortal Cancer Genomics. Our analysis uncovered 10 genes including  CDH1 and RHOA distinguished GC from colorectal and esophageal tumors, and these mutations were specific to diffuse-type GC, it is still challenging to discriminate the intestinal-type and diffuse-type GC in molecular gene expression networks [CDH2 to CDH1 distinguish the intestinal- and diffuse-type GC [Different cell types show a variety of molecular networks. Gastric cancer (GC) has several subtypes, which includes intestinal- and diffuse-type GC ,2. Intesnetworks . We haveEpithelial-mesenchymal transition (EMT) is associated with the malignancy of GC and diffuse-type GC . EMT is The importance and potential to use the molecular network profile to distinguish diffuse- and intestinal-type GC are increasing in the digital era to reveal the EMT mechanism . The premale-specific lethal 3 homolog (Drosophila) pseudogene 1 (MSL3P1), CDC28 protein kinase regulatory subunit 1B (CKS1B), DEAD-box helicase 27 (DDX27), golgi to ER traffic protein 4 (GET4), chromosome segregation 1 like (CSE1L), translocase of outer mitochondrial membrane 34 (TOMM34), YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), ribonucleic acid export 1 (RAE1), par-6 family cell polarity regulator beta (PARD6B), and MRG domain binding protein (MRGBP). Gene expression profile of the top 10 genes in intestinal- and diffuse-type GC are shown in t-test, p < 0.00001)  type and genomically stable (GS) type samples in TCGA RNAseq data, respectively. 0.00001) .t-test, p < 0.00001). A total of 25 networks generated from genes that have significant difference between intestinal- and diffuse-type GC are shown in Networks of genes altered in intestinal- and diffuse-type GC were analyzed using Ingenuity Pathway Analysis (IPA). A total of 2815 IDs that had significant difference between intestinal- and diffuse-type GC were analyzed in IPA -related regulator effect networks were analyzed in intestinal- and diffuse-type GC . Table 5Upstream regulators of genes altered in intestinal- and diffuse-type GC were defined by IPA analysis. The top 25 upstream regulators of the altered genes in intestinal- and diffuse-type GC are shown in p value < 10−6, p < 0.005 in Bonferroni statistics). In the 2815 IDs, EMT-related genes, which have GO  term “epithelial to mesenchymal transition”, included BMP and activin membrane bound inhibitor (BAMBI), EPH receptor A3 (EPHA3), GLI pathogenesis related 2 (GLIPR2), MAD2 mitotic arrest deficient-like 2 (yeast) (MAD2L2), SMAD family member 4 (SMAD4), SRY-box 9 (SOX9), Wnt family member 11 (WNT11), adiponectin receptor 1 (ADIPOR1), bone morphogenetic protein 7 (BMP7), ephrin A1 (EFNA1), forkhead box A2 (FOXA2), hepatocyte growth factor (HGF), histone deacetylase 2 (HDAC2), low density lipoprotein receptor class A domain containing 4 (LDLRAD4), msh homeobox 2 (MSX2), ovo like zinc finger 2 (OVOL2), pleckstrin homology like domain family B member 1 (PHLDB1), transforming growth factor beta receptor 2 (TGFBR2), and tripartite motif containing 28 (TRIM28).Gene Ontology (GO) was analyzed in genes regulated in intestinal- and diffuse-type GC. A total of 2815 IDs were analyzed for enrichment analysis in the Database for Annotations, Visualization and Integrated Discovery (DAVID) database, which resulted in 2762 DAVID gene IDs analyzed in GO Biological Process. The top 21 GOs are shown in CCR4-NOT transcription complex subunit 3 (CNOT3), CD34 molecule (CD34), ETS variant 4 (ETV4), Fanconi anemia complementation group D2 (FANCD2), GATA binding protein 2 (GATA2), LDL receptor related protein 5 (LRP5), LIM domain binding 2 (LDB2), RNA polymerase II subunit C (POLR2C), RNA polymerase II subunit H (POLR2H), RNA polymerase II subunit J (POLR2J), SMAD family member 4 (SMAD4), SRY-box 2 (SOX2), SRY-box 9 (SOX9), Wnt family member 2B (WNT2B), abnormal spindle microtubule assembly (ASPM), alpha-2-macroglobulin (A2M), cullin 4A (CUL4A), cyclin dependent kinase inhibitor 2A (CDKN2A), endothelial PAS domain protein 1 (EPAS1), epithelial cell adhesion molecule (EPCAM), fibulin 1 (FBLN1), frizzled class receptor 1 (FZD1), growth arrest specific 6 (GAS6), hes family bHLH transcription factor 1 (HES1), insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1), keratinocyte differentiation factor 1 (KDF1), lysine demethylase 1A (KDM1A), mediator complex subunit 10 (MED10), mediator complex subunit 24 (MED24), mediator complex subunit 30 (MED30), mesenchyme homeobox 1 (MEOX1), msh homeobox 2 (MSX2), notchless homolog 1 (NLE1), ovo like zinc finger 2 (OVOL2), paired related homeobox 1 (PRRX1), platelet activating factor acetylhydrolase 1b regulatory subunit 1 (PAFAH1B1), platelet derived growth factor receptor alpha (PDGFRA), programmed cell death 2 (PDCD2), proteasome 26S subunit, non-ATPase 11 (PSMD11), spalt like transcription factor 4 (SALL4), squamous cell carcinoma antigen recognized by T-cells 3 (SART3), ubiquitin associated protein 2 like (UBAP2L), and vacuolar protein sorting 72 homolog (VPS72).EMT may be related to the stemness of cancer. Genes related to stem cells have been investigated in the 2815 IDs, which have significant differences between intestinal- and diffuse-type GC. Genes which have the “stem cell” term in GO  included CTNNBL1, ZEB2, ERBB3, TGFBR2, SMAD4, CDH5, STAT5A, AKT3, STAT5A, AKT3, STAT5B, WNT11, STAT1, WNT2B, ERBB2, and TWIST2 [Metabolism is one of the possible EMT-modified processes. Genes which have “metabolism” term in KEGG PATHWAY in the analysis of DAVID of the 2815 IDs are listed in d TWIST2 .The results of upstream analysis of intestinal- and diffuse-type GC data were analyzed in DataRobot Automated Machine Learning version 6.0 for creating prediction models. The list of upstream regulators was up-loaded and linked with network picture data, followed by the target prediction setting as subtype differences in intestinal- and diffuse-type GC . Among vThe canonical pathways for Regulation of the EMT pathway include TGF-beta pathway, Wnt pathway, Notch pathway, and Receptor Tyrosine Kinase pathway . In eachCDH2 to CDH1 [CKS1B, CSE1L, DDX27, GET4, MRGBP, MSL3P1, PARD6B, RAE1, TOMM34, and YTHDF1. The network analysis of altered genes in intestinal- and diffuse-type GC generated networks related to cancer, gastrointestinal disease, organismal injury and abnormalities, amino acid metabolism, molecular transport, small molecule biochemistry, and so on. Several miRNAs including miR-205-5p, miR-21-5p, let-7a-5p, let-7, miR-24-3p, and miR-291a-3p were identified to regulate networks involved in intestinal- and diffuse-type GC. Since previous studies have revealed the involvement of miR-200s in promoting metastatic colonization by inhibiting EMT and promoting mesenchymal-epithelial transition (MET), it may be an intriguing approach to reveal miRNA networks in EMT [It is critical to distinguish the intestinal- and diffuse-type GC for effective therapeutic strategies, since the pathogenesis and prognosis are quite different in these subtypes. We previously revealed the gene signature of intestinal- and diffuse-type GC, which is indicated by the ratio of gene expression in  to CDH1 ,14,15,16s in EMT ,18. The s in EMT ,20,21. DCDH2 to CDH1, as previously shown [The intestinal- and diffuse-type GC can be distinguished with the mRNA ratios of The RefSeq data of intestinal- and diffuse-type GC are publicly available in The Cancer Genome Atlas (TCGA) of The cBioPortal for Cancer Genomics database ,14,15 inData of intestinal- and diffuse-type GC in TCGA cBioPortal Cancer Genomics were uploaded and analyzed through the use of Ingenuity Pathway Analysis (IPA)  .Gene Ontology (GO) was analyzed in the Database for Annotations, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.8 (Laboratory of Human Retrovirology and Immunoinformatics) ,25.To create a prediction model by using multi-modal data including images and text description of molecular networks, an enterprise AI platform  was used. For the modeling, the 116 molecular networks of IPA upstream analysis in intestinal- and diffuse-type GC were collected and input as image data in the DataRobot (58 images in each subtype), which automatically created and tuned prediction models using various machine learning algorithms  ,27. FinaThe results of gene expression data of RefSeq and network analysis were visualized by Tableau software.p value < 0.00001. For DAVID Gene Ontology (GO) enrichment analysis, data was analyzed in the default setting. GO enrichment was considered significant in modified Fischer Exact p value < 10−6. Bonferroni statistics showed p value < 0.005.The RefSeq data were analyzed by Student’s t-test. Z-score in intestinal- and diffuse-type GC samples were compared, and the difference was considered to be significant in The regulatory molecular networks are altered in intestinal- and diffuse-type GC. Networks generated from genes altered in intestinal- and diffuse-type GC included a network related to cancer, gastrointestinal disease, and organismal injury and abnormalities. We demonstrated that several miRNAs regulated the networks in intestinal- and diffuse-type GC. Machine learning of network image data created prediction models to distinguish the subtypes of the GC. The molecular mapping of intestinal- and diffuse-type GC may reveal the EMT mechanism. The miRNAs identified in the study may be regulated in EMT, which would be critical for progression and metastasis process. Our results support further identification of GC subtypes through visual changes in molecular networks.


Correction to: Trials 21, 530 (2020).https://doi.org/10.1186/s13063-020-04349-4Following publication of the original article , we wereThe first sentence is incomplete. It should read: “There were four phases of data collection for 31 time points (in months): (1) November 2016 to June 2017 (pre-intervention), (2) August 2017 to June 2019 (post-intervention).” The original article has been corrected.


Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes angiogenesis. It has been established that regulatory loops exist between angiogenic growth factors and distinct pro or anti-angiogenic miRNAs, but the nature and the roles of Ang-1-regulated miRNAs remain unclear. In this study, we assessed the role of miR-640 in Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Exposure to Ang-1 (300 ng/mL) from 6 to 72 h significantly decreased expression of mature miR-640, a response that was mediated by Tie-2 receptors and was also observed in response to Ang-2, the vascular endothelial growth factor, and transforming growth factor β. Increasing miR-640 levels using a mimic inhibited Ang-1-induced cell migration and capillary-like tube formation whereas inhibition of miR-640 enhanced these responses. Pull down assays of biotinylated miR-640 revealed that miR-640 directly targets Zinc Finger Protein 91 (ZFP91), an atypical E3-ubiquitin ligase. Ang-1 exposure induced ZFP91 expression through down-regulation of miR-640. Silencing of ZFP91 significantly inhibited Ang-1-induced cell migration and tube formation. We conclude that Ang-1 upregulates ZFP91 expression through transcriptional down-regulation of miR-640 and that ZFP91 plays important roles in the promotion of Ang-1-induced endothelial cell migration and differentiation. Angiopoietin-1 (Ang-1) and its receptor, Tie-2, are major regulators of physiological and pathological angiogenesis. In mouse embryos, knocking down Ang-1 gene is associated with major vascular abnormalities ,4,5, andIt is increasingly evident that miRNAs play important roles in angiogenesis. Angiogenesis is strongly inhibited when Dicer and Drosha, enzymes that are essential to miRNA biogenesis, are deleted in mice . AngiogeWe recently conducted pilot experiments to identify miRNAs whose expression in human umbilical vein endothelial cells (HUVECs) are downregulated in response to 24 h exposure to Ang-1. These experiments indicated that Ang-1 exposure elicits a significant decrease in the expression of miR-640. Little information is available regarding the expression and functional roles of miR-640 in various cells. To our knowledge, Zhou et al.  is the oRecombinant human Ang-1 and Ang-2 were purchased from R&D Systems . Vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and transforming growth factor beta (TGF-β) were purchased from Bioshop . Antibodies for zinc finger protein 91 (ZFP91) and β-ACTIN were purchased from Sigma-Aldrich . Cleaved caspase-3 antibody was purchased from Cell Signaling Technology . Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza .2. In experiments where EC migration, survival, and activation of caspase-3 activity were measured, cells were cultured in basal MCDB131 medium containing gentamicin sulfate and 2% FBS.HUVECs were maintained in complete MCDB 131 medium  supplemented with 20% fetal bovine serum (FBS), EC growth supplement, 2 mM glutamine, heparin, and gentamicin sulfate, and incubated at 37 °C and 5% COHUVECs were cultured for 6 h in basal medium and were then maintained for 2, 4, 6, 12, 24, 48, and 72 h in basal medium containing aliquots of Ang-1 (300 ng/mL) or phosphate buffered saline PBS (control). To assess whether other angiogenesis factors regulate miRNA expression, we exposed HUVECs to VEGF (40 ng/mL), FGF-2 (10 ng/mL), TGF-β (2 ng/mL), or Ang-2 (300 ng/mL) for 24 h. Total RNA was extracted and miRNA expression was detected as described below.®  and miRNeasy Mini Kit . miRNAs were detected using an NCode™ miRNA qRT-PCR Kit , and real-time PCR using specific primers, SYBR® green , and a 7500 Real-Time PCR System . All experiments were performed in triplicate. Relative miRNA expression was calculated on the basis of the CT method where CT values of individual miRNA data were normalized to CT values of U6 snRNA as previously described [Total RNA was extracted using Qiazolescribed . When HUVECS reached 60–70% confluency they were then exposed for 6 h to a serum-free medium containing 100 multiplicity of infection (MOI) virus units of adenoviruses expressing GFP or EX-TEK  . Cells wHUVECs were cultured in complete MCDB131 medium until they reached full confluence. The medium was then changed to basal MCDB131 medium for 6 h, cells were then exposed to basal medium containing aliquots of Ang-1 (300 ng/mL) or PBS for 24 h. The Exiqon miRCURY™ Exosome Isolation Kit  was used to isolate exosomes secreted from cells into media. Total RNA was extracted from isolated exosomes and miRNA expression was measured as described below. HUVECs were transfected with synthetic siRNA oligos (10 nM of scrambled or specific to ZFP91), synthetic mature miRNA mimic , or LNA- miRNA Inhibitor  using Lipofectamine™ RNAiMAX (Invitrogen Inc.) according to the manufacturers’ instructions. After 48 h of recovery in complete MCDB131 medium, cells underwent specific experimental procedures. 4/cm2). Cells were cultured for 24 h in complete medium or basal medium containing aliquots of PBS or Ang-1 (300 ng/mL). Cells were then counted using a hemocytometer and underwent immunoblotting for cleaved caspase-3 measurements.HUVECs transfected with siRNA oligos, miRNA mimics, or miRNA inhibitors were seeded onto 12-well plates  healing assay as in our previous study . In brie®  (1 × 104 cells per well). Cells were cultured for 24 h in basal medium containing PBS (control) or Ang-1 (300 ng/mL). Olympus inverted microscope (40×) was used to capture whole-well images at 40× magnification. Images were analyzed using Image-Pro Plus™ software. Angiogenic tube formation was determined by counting total branches in each field, as previously described [Cells transfected with siRNA-, miRNA mimic-, or inhibitor-transfected were seeded onto 96-well plates pre-coated with growth factor-reduced Matrigelescribed .® green, and a 7500 Real-Time PCR System (T method (2−ΔΔCT) as previously described [Total RNA was extracted using PureLink RNA mini kit  according to the manufacturer’s protocol. mRNA levels were detected with real-time PCR using specific primers, SYBRR System . GAPDH aescribed .HUVECs were lysed in RIPA buffer (Santa Cruz Biotechnologies) and protein concentration was measured with the Bio-Rad assay using bovine serum albumin (BSA) as a standard. Cell lysates were diluted in Laemmli sample buffer, boiled for 5 min, and loaded onto Tris-glycine SDS-polyacrylamide gels. After SDS-PAGE, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, blocked with 5% non-fat dry milk and then incubated with the specific primary antibodies overnight at 4 °C. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies and ECL reagents . ™ dual reporter plasmids containing 2000 bp of wild-type (wt) ZFP91 3’UTR cloned downstream of a SV40-driven Firefly Luciferase cassette and a CMV-driven Renilla Luciferase . The 2000 bp region of ZFP91 3’UTR was predicted to have two miR-640 binding regions  and Firefly luciferase activity was normalized for Renilla activity to account for differences in transfection efficiencies.pEZX-MT06 regions . The mut2, 20 mM Tris-Cl pH 7.5, 5 mM DTT, 0.3% NP-40, 60 U/mL RNase OUT, and 1× Complete Mini protease inhibitor ). Simultaneously, magnetic streptavidin beads  were coated with bovine serum albumin (1 μg/μL) and yeast tRNA  and incubated with supernatants while rotating at 4 °C for 2 h. Cell debris was cleared by centrifugation  and cleared lysates were then incubated with the pre-coated beads rotating overnight at 4 °C. Beads were then washed with hypotonic lysis buffer and RNA was then released by adding 750 µL of TRIzol (Invitrogen) and 250 µL of RNase-free water. RNA was then precipitated using a standard chloroform-isopropanol method and was then subjected to reverse transcription and qPCR for detection of specific mRNA transcripts. Biotinylated miR-640 pull-down assays with target mRNAs were performed as described earlier ,24. Briep values < 0.05 were considered statistically significant.Data are expressed as means ± SEM. Differences between experimental groups were determined using a two-way analysis of variance followed by a Student–Newman–Keuls post-hoc test. The effects of Ang-1 on miR-640 (mature form) expression were assessed in HUVECs maintained for 24, 48, and 72 h in basal medium containing PBS or Ang-1 (300 ng/mL). Ang-1 significantly decreased miR-640 expression at the three time points relative to PBS A. miR-64Exosomes are mediators of intercellular communication and incorporate and transfer mRNAs and miRNAs. miR-640 levels in exosomes isolated from HUVECs pre-incubated with PBS or Ang-1 for 24 h were measured to evaluate whether increased excretion of miR-640 in the exosomes may account for the decrease in its levels in cells exposed to Ang-1. Exposure to Ang-1 for 24 h significantly decreased exosomal miR-640 levels relative to PBS suggesting that Ang-1-induced decrease in miR-640 levels was not due to increased excretion of this miRNA in the exosomes C.The essential role of Tie-2 receptors in mediating the inhibitory effect of Ang-1 on miR-640 expression was assessed by infecting HUVECs with Ad-GFP (control) or Ad-Ex Tek (express recombinant soluble Tie-2 receptors). With Ad-GFP, Ang-1 significantly decreased miR-640 levels relative to PBS D. With AThe miR-640 gene is located within an intron of the GATA zinc finger containing 2A (GATAD2A) gene. GATAD2A mRNA and miR-640 levels were measured 2, 4, 6, and 12 h post PBS or Ang-1 exposure to assess whether Ang-1-induced decreases in miR-640 expression correlate with that of GATAD2A. Ang-1 significantly decreased GATAD2A mRNA levels after 6, and 12 h, time points at which miR-640 expression was also significantly decreased E.HUVECs were pre-incubated for 24 h with PBS, Ang-2, FGF2, VEGF, or TGFβ to assess whether the inhibitory effect of Ang-1 on miR-640 expression is unique to this angiogenesis factor. Ang-2, VEGF, and TGFβ but not FGF-2 significantly decreased miR-640 relative to PBS F. It shoHUVECs were transfected with control or miR-640 mimics. After a 48 h period of recovery, cells were maintained in complete medium, basal medium containing aliquots of PBS (control), or Ang-1 and survival, proliferation, migration, and differentiation were then measured. Transfection with the miR-640 mimic significantly increased miR-640 levels . With thWith the control mimic, Ang-1 increased EC migration and capillary-like tube formation relative to PBS  or miR-640 (LNA-anti-miR-640) inhibitors to assess the functional importance of endogenous miR-640 on angiogenic processes. With control inhibitor, incubation with basal medium containing PBS significantly decreased cell counts and increased cleaved caspase-3 levels relative to the complete medium A,B. IncuWith the control inhibitor, Ang-1 significantly increased cell migration and capillary-like tube formation relative to PBS C,D. WithThese results suggest that endogenous miR-640 inhibits basal EC migration and differentiation and removal of this effect using the miR-640 inhibitor enhances the pro-migration and pro-differentiation effects of Ang-1.www.targetscan.org), miRSearch3.0 (www.exiqon.com/mirsearch), and miRanda (www.microrna.org) algorithms to identify potential targets of miR-640. Fifteen genes were commonly identified by the three algorithms with ZFP91 having the highest score (We used TargetScan (st score A. ZFP91 st score . We measst score B–D whilest score B–F.The binding of miR-640 to a luciferase reporter expressing a 2000 bp portion of ZFP91 3′UTR (wt) was assessed to confirm that miR-640 selectively targets ZFP91 mRNA. This portion is predicted to have two miR-640 binding regions  Ang-1 induced Tie-2-dependent decreases in miR-640 levels; (2) miR-640 inhibited pro-migration and pro-differentiation effects of Ang-1 but had no effects on cell survival; (3) ZFP91 was a direct target of miR-640; (4) Ang-1 upregulated ZFP91 expression by decreasing miR-640 binding to ZFP91 mRNA; and (5) ZFP91 was required for Ang-1-induced cell migration and capillary-like tube formation.The expression and functional roles of miR-640 in various cellular processes are poorly documented. miR-640 expression is elevated in cholangiocarcinoma, ovarian carcinoma, and lymphocytic leukemia cells ,26,27. DmiR-640 is located within an intron of the GATAD2A protein coding gene. We assessed whether Ang-1 regulates miR-640 and GATAD2A expression in a similar fashion since many intronic miRNAs are regulated in a similar fashion to their host genes. Ang-1 decreased GATAD2A mRNA levels in a similar time frame (6 and 12 h) to that of miR-640 . These rLittle information is available regarding the physiological roles of miR-640. In ovarian cancer, high levels of miR-640 are associated with better survival rates . In nuclThe current study demonstrated that miR-640 has anti-angiogenic properties, as indicated by the inhibitory effects of its mimic and the enhancing effects of its inhibitor on EC migration and differentiation. The mechanisms behind miR-640 anti-angiogenic effects remain unclear. In the current study, we identified miR-640 intracellular targets using three algorithms commonly used for predicting miRNA targets. ZFP91 is considered one of the top targets of miR-640 because of the presence of three putative miR-640 binding regions in the ZFP91 3′UTR. Here we provide three sets of observations that strongly support the notion that ZFP91 is a direct target of miR-640 in ECs. First, the miR-640 inhibitor increased while the miR-640 mimic decreased ZFP91 mRNA and protein levels. Second, luciferase reporter activity of 2000 bp of wild type ZFP 3’UTR decreased significantly by the miR-640 mimic. This inhibitory effect was not detected when miR-640 binding regions 1 (nucleotide 112–121) and 2 (nucleotide (335–347) of the ZFP91 3’UTR were mutated indicating that miR-640 binds to these two regions and destabilizes ZFP91 mRNA. Third, ZFP91 mRNA was significantly enriched in the pull-down materials of the biotinylated miR-640 mimic indicating that miR-640 directly binds to ZFP91 mRNA.63-linked ubiquitination in the non-canonical NF-κB signaling pathway [ZFP91 is an atypical ubiquitin E3 ligase that reportedly activates NF-κB-inducing kinase (NIK) via Lys pathway . ZFP91 h pathway ,36. It i pathway .The role of ZFP91 in angiogenesis is unknown. In the present study, we reported that ZFP91 expression in ECs was significantly induced by Ang-1, Ang-2, and VEGF. In the case of Ang-1, upregulation of ZFP91 appeared to be mediated through downregulation of miR-640 expression and attenuation of the binding of this miRNA to two regions of the ZFP91 3’UTR . Our stuIn summary, we reported that miR-640 directly targeted ZFP91 and that exposure of ECs to Ang-1 elicited upregulation of ZFP91 expression through transcriptional down-regulation of miR-640. We also identified important roles for ZFP91 protein in the regulation of Ang-1-induced endothelial cell migration and differentiation.


To the Editor:Handling over 100 million COVID-19 cases, frontline medical personnel are threatened due to the high risk of cross-infection. Several studies have recommended that medical personnel with suspicious symptoms  clearly receive SARS-CoV-2 testing and at least 14 days of quarantine (close contact with COVID-19 patients without appropriate infection prevention) [From 1 March to 30 June 2020, a total of 1272 patients were reported to the Taiwan CDC for testing COVID-19 (SARS-CoV-2) in our hospital. Among them, 115 (9%) were frontline medical personnel (handling or facing patients). In addition, 105 of them (91.3%) received rapid respiratory panel test (BIOFIRE® FILMARRAY® Respiratory Panels) in the emergency department (ED) , our results demonstrated that 24.7% tested positive for non-COVID pathogens. Rhinoviruses and enteroviruses were the leading non-COVID-19 pathogens during the pandemic period. When facing workforce insufficiency, long-term quarantine for medical personnel might not be necessary when their COVID-19 and non-COVID-19 pathogens are both confirmed early. In one testing model, the chance of post-quarantine transmission might obviously decrease after 7 days of quarantine [


HSPG2 is implicated in the human diseases Schwartz-Jampel Syndrome type 1 and Dyssegmental Dysplasia, Silverman-Handmaker type . The mutated unc-52 gene expresses the phenotype for uncoordinated movement (“Unc”), which involves progressive paralysis and retarded sarcomere construction . The UNC-52 protein is localized in the striated muscle dense bodies and the basement membrane, where it plays an important role in developmental processes such as cell adhesion, cell migration, and signal transduction . Within the unc-52 gene, an RGD (Arg- Gly- Asp) sequence is located at amino acid locations 746, 747, and 748 in exon 7. Exon 7, containing RGD748, is included in all three major isoforms, short (S), medium (M), and long (L) . The RGD sequence is part of the Laminin IV type A domain and primarily functions as a cell attachment and adhesion site for integrins . In this study, two separate mutations were performed on this unc-52 RGD sequence using CRISPR-Cas9 technology. The unc-52(kq748) mutation replaced the aspartic acid (D) located at the 748 amino acid position with a glutamic acid (E) . The unc-52(kq745) mutation removed the RGD sequence by deleting amino acids 746, 747, and 748. Previous studies have shown that unc-52 gene mutations cause the disorganized distribution of pat-3 β integrin, which is a receptor for extracellular matrix proteins . In order to study the cellular phenotypes of the unc-52(kq748) mutation, a double mutant was created: pat-3::GFP; unc-52 (kq748). Staining showed that localization of the pat-3::GFP reporter in the double RGD mutant appeared normal, with dense bodies and M-lines alternating along the muscle filaments . In order to visualize the actin filaments in the body wall muscles of N2 wild type and unc-52(kq748) mutants, staining was performed using 0.4 U/mL rhodamine-conjugated phalloidin, Thermo Fisher Scientific, Waltham, MA . This unc-52(kq748) staining showed no obvious abnormalities outside of interruptions due to fixation and slicing procedures (unc-52(kq748) mutants compared to the N2 worms, and a 33% decrease in unc-52(kq745) mutants  and unc-52(kq745) groups differed significantly from the N2 wild-type group, as shown by p-values of 7.1×10-6 and 4.63×10-8 respectively. These results show that there was a statistically significant decrease in motility for both unc-52(kq748) and unc-52(kq745) mutants.The ocedures . Additio mutants . A one-wunc-52(kq748) and unc-52(kq745) mutations. For the unc-52(kq748) mutation, the DNA repair template (UNC52RGE748), crRNA (UNC52RGE748), tracrRNA (cat. #1072532), Cas9 nuclease (cat. #1081058), and a dpy-10 crRNA co-CRISPR marker were mixed and micro-injected into wild-type N2 worms, which served as the parent (P0) generation. F1 worms that carried the mutation were then identified and isolated using the dpy-10 marker  and underwent PCR genotyping using mutant specific primers (UNC52RGE748SEQF and UNC52RGE748SEQR) to screen for the RGE mutation. The F2 generation then also underwent PCR screening using both a mutant specific primer (UNC52RGE748SEQF) and a wild-type specific primer (UNC52RGD748WTF) to isolate non-dumpy homozygous mutants. These homozygous mutant PCR products were sequenced to validate the success of the mutation . This process was repeated for the unc-52(kq745) mutation using the DNA repair template (UNC52RGD748D) and the primers (UNC52RGD748DF and UNC52RGDWTF). The DNA repair template and all other oligos were designed at IDT Inc., Coralville, IA. A double mutant was then created by crossing unc-52(kq748) II males with NK358 pat-3::GFP III hermaphrodites. Homozygous double mutants from the F2 generation were identified as green-fluorescent progeny and confirmed through PCR genotyping. Staining was performed by collecting mutants and fixing them with ice-cold methanol for 5 minutes. Images were captured using a Nikon Eclipse Ni-U epifluorescence microscope and processed with NIS Elements (version 5.02). Once the homozygous mutants were identified and isolated, phenotypic assays were conducted on 50 young adult worms from both homozygous mutants and N2 wild-type worms in order to characterize the phenotypes for both the unc-52(kq748) and unc-52(kq745) mutations. A one-way ANOVA test was then run to confirm statistical significance of the results. Since the p-value for the ANOVA test was less than 0.05, post hoc tests were run to determine which groups differed .CRISPR-Cas9 technology was used to create both the crRNA sequences (5’ to 3’)UNC52RGD748 TTTCTATTGATTATGCTCGT(dpy-10) ZQDP10A GCUACCAUAGGCACCACGAGRepair templates (5’ to 3’)PrimersUNC52RGE748SEQF TTCCTTGCTTCTGCTCAGGTUNC52RGE748SEQR TGATCGGAGTTGCCATTTCCAUNC52RGD748WTF TTCTATTGATTATGCTCGTGGGGATUNC52RGE748F TCAATCGACTACGCAAGAGGCUNC52RGD748DF GTCAATCGACTACGCAAGAGATCAAunc-52(kq748) II, BU745 unc-52(kq745) II, and BU111 unc-52(kq748); pat-3::GFP are available upon request. NK358 pat-3::GFP III was purchased from Caenorhabditis Genetics Center, Minneapolis, MN.BU748


A 24-year-old patient accepted laparoscopic radical trachelectomy for stage IB1 cervical squamous cell carcinoma. During the surgery, after r


Obesity is an important public-health problem worldwide. This study aimed to determine effects of porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on adipocytes injuries and explore associated mechanisms. Adipocytes were isolated from SD rats. pLVX-XBP1 (XBP1 over-expression) and pLVX-XBP1-RNAi (silencing XBP1) were structured and transfected into adipocytes. All adipocytes were divided into pLVX-NC, pLVX-XBP1, pLVX-NC+Pg-LPS and pLVX-XBP1+ Pg-LPS group. Oil-Red O staining was employed to identify isolated adipocytes. Quantitative real-time PCR (qRT-PCR) was used to examine gene transcription of IL-6, TNF-α, leptin, adiponectin. Western blotting was used to detect Bax and caspase-3 expression. Adipocytes were successfully isolated and identified with Oil-Red O staining. Both XBP1 mimic and XBP1 RNAi were effectively transfected into adipocytes with higher expressing efficacy. XBP1 over-expression significantly aggravated Pg-LPS induced inflammatory response compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS significantly enhanced leptin and inhibited adiponectin expression by up-regulating XBP1 expression (p<0.05). XBP1 silence significantly alleviated Pg-LPS induced inflammatory response and reduced leptin, enhanced adiponectin expression in Pg-LPS treated adipocytes compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS induced apoptosis of adipocytes by enhancing XBP1 expression and modulating Bcl-2/Bax pathway associated molecules. In conclusion, Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) induces adipocytes injuries through modulating XBP1 expression and initialling mitochondria-mediated apoptosis. In recent years, obesity has been become an important public-health problem affecting billion peoples in the whole world ,2. ObesiPorphyromonas gingivalis  is a kind of Gram-negative (G−) anaerobic bacterium and considered to be a major pathogen causing chronic inflammation in periodontal disorders ) approach was used to analyse and calculate the qRT-PCR findings [Total RNAs of adipocytes were extracted with TRIzol kits  as manufacturer’s instruction described. The obtained total RNAs were reversely transcripted to generate complementary DNA (cDNA) using RNA Transcription Kit . The obtained cDNAs were utilized for conducting the qRT-PCR assay. The PCR primers for were generated by Western Biotech.  and the associated sequences are listed in findings .Table 1.The adipocytes were lysed with RIPA Lysis Buffer  as instructed by protocol of manufacturer. The lysates were separated with 15% sodium dodecylsulfate polyacrylamide gel electrophoresis , and electro-transferred onto PVDF . PVDF membranes were blocked with 5% defatted milk in PBS containing 0.05% Tween-20 , with pH 7.5. Then, the PVDF membranes were incubated using rabbit anti-rat Bax polyclonal antibody , rabbit anti-rat caspase 3 polyclonal antibody  and rabbit anti-rat GAPDH polyclonal antibody  at 4 °C overnight. Subsequently, the PVDF membranes were treated using 1:3000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG . Western blot bands were visualized using BeyoECL Plus kit . Eventually, western blot bands were imaged and analysed using LabworksTM Analysis Software .p < 0.05. All the experiments or the tests were conducted at least for 6 repeats.Data were analysed using professional SPSS software . The continuous variables were represented as mean±SD. Tukey’s post hoc test was used to validate ANOVA test for comparing continuous data among multiple groups. A statistical significance was defined when


Drosophila, we identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). We showed that NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. We showed that NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in  Nt-acetylation is often regarded as a constitutive, irreversible, and static modification that is not suited to serve regulatory functions. Our observation that Nt-acetylation by NatB coordinately regulates some of the highly conserved positive and negative components of the EGFR/MAPK pathway suggest that Nt-acetylation and N-end rule pathways can play important roles regulating important signaling pathways. As these positive and negative pathway components are conserved and shared by multiple growth factor signaling pathways and as Acetyl-CoA level, which is influenced by cell metabolism, can be rate limiting for Nt-acetylation, our results also suggest a potentially new mechanism by which cellular metabolic status can regulate growth factor signaling. Mutant clones were marked by the lack of red pigment. (K-P) Large clones (white patches) of additional psidin alleles  were observed when mutant clones were generated in a Minute background. In contrast, rbf psidin double mutant clones generated in the Minute background were mostly lost, resulting in smaller eyes . (Q-S) Cell death level (caspase 3 staining) in third instar eye/antenna discs with single or double RNAi of Rbf or NAA20. Genotype of flies used in S1 Fig: w, eyFLP /Y; FRT82B, Ubi-GFP /FRT82B (panel A), rbf15aΔ,w, eyFLP /Y; FRT82B, RBF-G3, Ubi-GFP/FRT82B (panel B), w, eyFLP /Y; FRT82B, Ubi-GFP /FRT82B, psid  , rbf15aΔ,w, eyFLP /Y; FRT82B, RBF-G3, Ubi-GFP / FRT82B, psid  , w, eyFLP/Y; FRT82B,Rps3-, Ubi-GFP/FRT82B, psid  , rbf15aΔ,w, eyFLP/Y; FRT82B, RBF-G3, Rps3-, Ubi-GFP/ FRT82B, psid  , eyFLP, Act>CD2>Gal4; UAS-Rbf RNAi / + or UAS-NAA20 RNAi (panel R-S), eyFLP, Act>CD2>Gal4; UAS-NAA20 RNAi (panel Q).(A-J) Adult eyes with single mutant clones of (TIF)Click here for additional data file.S2 Figpsidin alleles marked by the absence of GFP. (D-F') NAA20 RNAi clones generated with EyeFlp-CoinGal4 and marked by GFP expression were stained with pErk (D-D’), and neuronal differentiation markers Elav (E and E') and Senseless (F and F'). Genotype of flies used in S2 Fig: w, eyFLP /Y; FRT82B, Ubi-GFP / aos-lacz, FRT82B, psdin  (panels A-C), eyFLP, UAS-Dcr2 / +; CoinFLP-Gal4-UAS-GFP; UAS-NAA20 RNAi (panels D-F).(A-C’) Expression level of aos-lacZ was reduced in mutant clones of additional (TIF)Click here for additional data file.S3 Figpsid-D4 mutation on activated Ras (A-B) or activated Raf (C-D) induced MAPK activation in eye discs were detected by pERK staining (red). Activated Ras-induced pERK (white arrowhead in A) was much higher than the endogenous pERK observed in the morphogenetic furrow (Yellow arrowhead in A). psid-D4 mutation significantly reduced activated Ras-induced pERK (white arrowhead pointed in B), which was similar to the endogenous pERK level in WT tissues (yellow arrow pointed in B). Activated Raf-induced pERK (white arrow pointed in C) was not obviously affected by psid-D4 mutation (white arrow pointed in D). Genotype of flies used in S3 Fig: HsFLP; Act > y >Gal4, UAS-GFP/UAS-Rasv12; FRT82B, tub-Gal80/ FRT82B or  (panels A-B), eyFLP; Act > y >Gal4, UAS-GFP/UAS-Rafgof; FRT82B, tub-Gal80/ FRT82B or  (panels C-D).(A-D) The effects of (TIF)Click here for additional data file.S4 Figpsid-D1 MARCM clones in wing discs, marked by GFP expression (pointed by arrowheads), showed decreased Drk level. (C) Ubr1 RNAi did not rescue the decreased Drk level in psid-D1 clones marked by GFP expression (pointed by arrowheads). Genotype of flies used in S4 Fig: eyFLP; Act > y >Gal4, UAS-LexA-RNAi (or NAA20-RNAi) (panel A), HsFLP; Act > y >Gal4, UAS-GFP; FRT82B, tub-Gal80/ FRT82B, psid D1 or  (panels B-C).(A) mRNA levels from eye antenna discs expressing LexA control RNAi or NAA20-RNAi were determined by qRT-PCR. # indicates no significant statistical difference. (B) (TIF)Click here for additional data file.S5 FigCG1317/Teb4 Δ6–1, generated from imprecise excision of CG1317 P element (BL20646). DNA sequencing data revealed a 2078 bp deletion, which starts from 14 bp upstream of CG1317. The numbers in the diagram indicate the precise location of deletion in the fly genome. (B) CG1317/Teb4 Δ6–1 mutant clones (pointed by white arrowhead) in eye disc, marked by lack of GFP, did not affect PP2AC levels. (C) Quantitative RT-PCR results of RNA isolated from 3rd instar eye/antenna discs expressing LexA control RNAi and NAA20 RNAi, or from Cnot4 RNAi and control W RNAi expressing. # indicates no significant difference was observed in PP2AC mRNA levels. (D-E) Clones of cells (pointed by white arrowheads) with GFP and Ubr1 RNAi (D) or Ubr5 RNAi (E) expression did not affect PP2AC levels in eye discs. (F) CG1317/Teb4 Δ6–1 mutant clones (pointed by white arrowhead) in eye disc, marked by lack of GFP, did not affect Drk levels. Genotype of flies used in S5 Fig: w, eyFLP; Ubi-GFP, FRT80B/ Teb4, FRT80B , eyFLP; Act > y >Gal4, UAS-LexA-RNAi  (panel C), HsFLP; tub-Gal80, FRT40A/ FRT40A; Act > y >Gal4, UAS-GFP / UAS-Ubr5 RNAi (panel E), HsFLP, Act>CD2>Gal4/Y; UAS-Ubr1 RNAi (panel D).(A) Genomic structure of a deletion allele of (TIF)Click here for additional data file.S6 Figpsid-D1 mutant clones . (C-D) expression of HA tagged Spry (shown in red) with GFP (shown in green) in control (C-C”) or psid mutant (D-D”) MARCM clones. (E) Spry-HA levels normalized by GFP signal in FRT control or psid mutant clones were shown. (F-G’) Images of wing discs with PTP-ER-RNAi flip-out clones  stained by two PTP-ER monoclonal antibodies (26E4C7 and 2D7F8). Genotype of flies used in S6 Fig: eyFLP, UAS-Dcr2 / +; CoinFLP-Gal4-UAS-GFP; UAS-Sprouty RNAi (panel A), HsFLP; FRT82B,Ubi-GFP / FRT82B, psid D1 (panel B), HsFLP; Act > y >Gal4, UAS-GFP / UAS-Spry; FRT82B, tub-Gal80/ FRT82B or  (panel C-D).Reduced levels of Spry were observed in clones of cells expressing Spry RNAi . White and yellow arrowheads in (A) point to RNAi cells located in the posterior or the anterior region of eye disc. Reduced levels of Spry were also observed in (TIF)Click here for additional data file.S7 Fig(A) when NatB is inhibited or when Ac-CoA level is low, most Drk, MAPK and PP2AC will not be Nt-acetylated, which results in the inhibition of EGFR signaling due to the accumulation of PP2AC, a negative component of the pathway, and the loss of Drk and MAPK, the positive components of the pathway. (B) When NatB activity is high or when Ac-CoA level is high, more Drk, MAPK, and PP2AC will be Nt-acetylated, which results in the higher levels of EGFR signaling due to the accumulation of acetylated Drk and MAPK and the loss of PP2AC.(TIF)Click here for additional data file.


Nasopharyngeal swab is the reference sampling method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as recommended by the World Health Organization (WHO) . HoweverPatients who were suspected of having coronavirus disease 2019 (COVID-19) attending the Hôpital Européen Georges Pompidou, Paris, France, were for their own care according to medical decision prospectively included and subjected to SARS-CoV-2 molecular testing using nasopharyngeal swab  and nasal swab .Nasal and nasopharyngeal swabs were inserted in the nostril until they hit an obstacle , rotated five times, and removed. The test was conducted in only one nostril per patient. After sampling, the nasopharyngeal swab was inserted into a vial containing 3 ml of virus transport medium , and the nasal swab was placed in a 15-ml tube containing 3 ml of saline solution (0.9% NaCl). SARS CoV-2 was detected using Allplex 2019-nCoV assay .A total of 44 patients were prospectively included up to the end of March 2020. Their median age was 63.0 years, ranging from 18 to 94 years. There were 23 (52.3%) male and 21 female patients. A total of 37 (84.1%) patients showed laboratory-confirmed SARS-CoV-2 infection using nasopharyngeal swab, with 7 patients giving negative results (15.9%) .n = 7) gave negative test results using nasal swabs  true SARS-CoV-2-negative specimens, 4 (9.1%) false-negative specimens, and 33 (75.0%) true SARS-CoV-2-positive specimens. Thus, the sensitivity of SARS-CoV-2 RNA detection by multiplex real-time PCR from nasal secretions was 89.2% , and its specificity was 100.0% . The κ index was 0.72, indicating substantial concordance between nasal and nasopharyngeal swabbing to detect SARS-CoV-2 according to Landlis and Koch rank. The Youden J index was calculated at 89.2%, demonstrating good efficiency to detect SARS-CoV-2 RNA.TC) (mean ± standard deviation [SD]) values for E (envelope), RdRP (RNA-dependent RNA polymerase) and N (nucleocapsid) genes by nasopharyngeal  and nasal  swab testing were similar. Differences in TC values for the E, RdRP, and N genes were not statistically significant .Threshold cycle (We herein report that the molecular detection of SARS-CoV-2 using nasal swab specimens was nearly equivalent to the detection using nasopharyngeal swab considered the gold standard. SARS-CoV-2 detection from nasal samples showed high sensitivity and specificity. Agreement and accuracy of test results using nasal sampling by reference to gold standard nasopharyngeal sampling were estimated as substantial and good, respectively. Taken together, these observations demonstrate that nasal sampling could be used to screen SARS-CoV-2 in times of nasopharyngeal swab shortage.