EMBO/sd-character-level-ner huggingface dataset:
Figure 3Schematic of allelic recombination between two chromosomes III. A DSB is induced at MATa and repaired by recombination with the homologous chromosome carrying MATα-inc. Chromosome ends are marked with TRP1, LEU2, and KANMX to distinguish different repair products or chromosome loss. Trp+, Leu-, G418R colonies are scored as BIR products. Reciprocal sectored colonies (Trp+, Leu-/+ G418R/S) represent gene conversion with crossover. Percentage of BIR product among all cells are shown. Data information: χ2 test is used to determine the p-value and , number of colonies tested per mutant is indicated in Appendix Table S1. Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1. Comparison of BIR percentage among the products in different assays shown in figures 1-3 in WT and rad52-R70A mutant cells. Percentage of repair products are measured at 6 hrs after break induction (H-150, H-1400, and H-2100 assays) or scored among plated cells (allelic assay) were pooled together. Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013). Data information: χ Mutation rates are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.
Figure 6mRNA (top) and protein (bottom) levels of the indicated genes in cells transfected with the indicated siRNAs. n=8. Error bars are s.e.m. P-values computed two-sided t-test. ** P≤0.01 **** P≤0.0001. Differences in the Nuc/Cyto (log2) ratios for tiles containing the SIRLOIN element (up to 6 mismatches) and other NucLibB tiles (only WT sequence tiles were considered). Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test. Same differences as in (B) for tiles with the indicated protein binding pattern (+: log2(IP/Input)≥0.5; -: log2(IP/Input)≥0.5). Number in parentheses indicates the P value when comparing the indicated group and the "- SLTM - hnRNPK" group (C) or "- SNRNP70 - hnRNPK" group (D)). P-values computed using two-sided Wilcoxon rank-sum test. IP/Input (top) and changes in Nuc/Cyto (log2) ratios upon siRNA targeting of the indicated factor (top) for WT NucLibB tiles with the indicated number of the indicated motifs. Number of tiles in each group is indicated in parentheses. *- P<0.05 ** P<0.005 *** P<0.0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count. Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test. * - P<0.05, ** - P<0.005, *** - P<0.0005.
Figure 8When ZIKA virus infects the pregnant woman, the virus can reach the placenta and transmit to the fetus. In the ZIKV replication cycle, it starts with the virus binding to host cell surface receptors, leading to endocytosis of the virus. Internalized viral particles release the viral RNA into the cytoplasm of the host cell and start replication. During its replication, rISG20-Fc degrades ZIKV RNA and inhibits the subsequent transcription and translation. Therefore, less new virus particles are released from the placenta to fetus.
TLR8 agonists inhibit HIV replication.Figure 5TLR8 agonists inhibit HIV replication.Macrophages were incubated with (A) ssRNA40 or (B) CL097 for 24 h before infection with HIV for 3 h. Cells were then washed and incubated with ssRNA40 or CL097 for 10 d. Extracellular release of HIV p24 antigen into the cell supernatant at days 0, 3, 5, 7, 10 was detected by ELISA. Results are reported as mean ± s.e.m., n = 5. * p<0.05.
Figure 6A, B In vitro ubiquitination of Flag-TRAF2 (A) and Flag-TAK1 (B) in the presence of Ub-conjugated UbcH7 (catalyzed by PknG) and PknG or its mutants, with or without ATP as an energy source. The reaction products were analyzed by immunoblotting with antibodies against Flag-tagged TRAF2 and TAK1. C, D In vivo ubiquitination assay of TRAF2 (C) and TAK1 (D) in U937 cells infected with WT, ΔpknG, ΔpknG:pknG, ΔpknG:pknG K181M, or ΔpknG:pknG E190A Mtb strains at an MOI of 1 for 6 hours. Endogenous ubiquitinated proteins were enriched using TUBE reagents and immunoblotted with antibodies against TRAF2 (C, top panel) or TAK1 (D, top panel). E, In vitro ubiquitination of TRAF2 in the presence of UbcH7, Ub, and PknG or its mutants. The reaction products were immunoblotted using antibodies against TRAF2 or TAK1. F In vitro ubiquitination of TAK1 in the presence of UbcH7, Ub, and PknG or its mutants. The reaction products were immunoblotted using antibodies against TRAF2 or TAK1. G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.
Figure 7(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm. (C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.
Germ cells are absent in the gonads of PCKO newborn pups A Schematic design of the targeting vector.Figure 2A Schematic design of the targeting vector. Prmt53flox−2frt founders were mated with Actin‐Flpe to excise the FRT flanked cassette to obtain Prmt52flox or Prmt5fl/+ mice. Prmt5fl/+ mice were intercrossed to obtain Prmt5fl/fl mice. B Recombination rate of Blimp1‐Cre (BC). BC was crossed to YFP lox‐stop‐lox mice, and recombination rate was calculated based on the fraction of YFP+ cells in the STELLA+ (E9.0) or MVH+ (E13.5) fraction. C, D P1‐2 male gonad (C) and P1‐2 female gonad (D) in control (Ctrl) and PCKO embryos. Arrows indicate germ cells. Scale bar, 100 μm. E, F IF for PRMT5 (green) and MVH (red) in (E) P1‐2 male and (F) P1‐2 female gonads. L, Leydig cell. Arrows indicate germ cells. Scale bar, 20 μm.
De novo DNA methyltransferases control maturation dynamics, dendritic outgrowth and spine formation of adult-born neurons in the hippocampus.Figure 5A, Dnmt3a/b-KO mice show reduced percentages of mature neurons (Calb1-positive, Dcx-negative) among Gfp-positive cells compared to WT mice (n = 3 mice, WT; n = 3 mice, KO). Immature neurons were identified as Dcx-positive and Calb1-negative. Experimental scheme as shown in Fig. 4E. B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm. C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus. D, No difference in long-term survival of new-born cells (BrdU-positive) was observed between WT and KO mice. BrdU was injected three months before analysis (experimental scheme depicted in Fig. EV3D). E, Long-term deficit in maturation of adult-born neurons in KO mice. Mature neurons were identified as BrdU-positive, Calb1-positive and Dcx-negative (n = 6 mice, WT; n = 8 mice, KO). F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm. G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E. H, Representative neuronal traces of WT and KO mice. I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test). J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse). K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice. L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.
d mice.Figure 4(A) Clustering of gene expression by genotype in RNAseq of islets isolated of cre control and Abca12tm1d mice at 8 weeks of age. (B) Scatter plot and heat map of differential gene expression between cre control and Abca12tm1d mice at 8 weeks of age. Legend indicates Log2 fold change values. (C) Circulating glucose levels after administration of intraperitoneal L-arginine in mice at 8 weeks of age, mean±SEM, n =3 mice per genotype, *p=<0.05 of cre relative to tm1d, Students t-test). (D) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test). (E) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 20mM KCl (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01 of cre relative to tm1d, Students t-test). (F) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals. Fluorescence responses were corrected for basal fill (F/F0). (mean±SEM, each data point represents the average of 10 cells/coverslip for 13-16 coverslips for each of 4 wild type and 5 Abca12tm1d mice) (G) Expression of GLUT2 in isolated mouse islets at 8 weeks of age. The upper panel depicts a representative Western blot and the lower graph the quantitation of protein levels from 3 mice of each genotype, normalised to β-actin (mean±SEM). (H) Insulin content of islets of Abca12tm1d mice (mean±SEM, n=3 mice of each genotype, normalised to total protein content).
Cleavage site of PPCA in lamp2a.Figure 7(A) Left: amino acid sequences of the C‐terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse. The proposed cleavage site for PPCA in lamp2a is highlighted. Right: immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 (mouse; upper panel) and NIH 3T3 cells (human; lower panel) stably transfected with wild‐type (FL) and mutated (GL) HA‐lamp2a. The arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region. (B) Total rates of protein degradation in the presence and absence of serum in non‐transfected HEK293 cells (WT) and in the transfected HEK293 clones described in (A). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different experiments. (C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.
Figure 4A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses. B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.
Electrophysiological classification of secretagogin+ parvocellular neurons A–A3 Biocytin‐filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple‐labeling experiments).Figure 3A-A3 Biocytin‐filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple‐labeling experiments). Scale bars: 50 μm (A), 8 μm (A3). lv, lateral ventricle; PVN, paraventricular nucleus. B-B2 Secretagogin+ biocytin‐filled neuron (arrow) lacking AVP/oxytocin immunosignal. Scale bars: 8 μm. C, C1 Dendritic reconstruction of secretagogin− and secretagogin+ neurons at their actual location in the paraventricular nucleus of the hypothalamus. D Electrophysiology characteristics of magnocellular and parvocellular neurons. The waveform of repetitive action potential firing is shown on the left, while differential channel characteristics are depicted on the right. Examples of secretagogin+neurons are shown in red. E Pie diagrams showing the grouping of neurons in the PVN (upper row) and in adjacent pre‐autonomic areas (lower row) cumulatively based on electrophysiological criteria listed in Supplementary Table S1. Secretagogin+ neurons typically belonged to Ia and IIa subtypes.
Figure 7A. Experimental outline. Anti-miR-mix of the 3 microRNAs was injected into the dorsal hippocampus of the mice as previously described. RNAseq data were generated from dorsal hippocampal tissues and compared to those treated with control scramble oligonucleotides. B. Weighted gene co-expression analysis of hippocampal RNA-seq data identified the MEblue gene cluster that is decreased when comparing 3-month old mice (young-control) to cognitively impaired 16,5-month old mice (old -control) with a scramble control oligonucleotide injected. Treating old mice with the miR-inhibitor mix (old miR-inhibitor mix) reinstated gene-expression of this cluster, at least in part (n = 6-7, Kruskal-wallis test). C. Gene ontology reveals that the MEblue cluster is linked to cognition and synapse organization. D. qPCR assay for several synaptic genes (LRKK2, Cadm3 and Slc6a11) confirms reinstatement of gene expression with anti-miR-mix (n = 5-7, Kruskal-wallis test). E. Weighted gene co-expression analysis of hippocampal RNA-seq data identified a MElightgreen gene cluster that is decreased when comparing wilt type control (WT control) to APPPS1-21 mice (APP-control) and was reinstated in APPPS1-21 mice treated miR-inhibitor mix (n = 6-7, Kruskal-wallis test). F. Gene ontology reveals that the MElightgreen cluster is linked to cognition and synapse organization. G. qPCR data shows rescue of AFF2/FMR2 and Hivep3 expression in APP/PS1 mice treated with inhibitor cocktail (n = 6-7, Kruskal-wallis test).
Figure 2(A) Schematics of two rounds of directed evolution to improve potency of dCas9-AsiA. Pie charts show frequencies of dCas9-AsiA variants identified from each round. (B) Mutations found in enriched AsiA variants and their positions along the AsiA secondary structure (left). Crystal structure of wild-type AsiA (blue) interfaced with region 4 of σ70 (orange) are shown (right). (C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom). (D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.
Pgs1 depletion enhances respiration in wild type and Opa1-deficient MEFs.Figure 7(A) Mitochondrial respiration measured in adherent MEFs of the indicated genotypes using Seahorse FluxAnalyzer. Oxygen consumption rate (OCR) normalized to protein concentration. Following basal respiration, cells were treated sequentially with 1μM Oligomycin (Omy), 2μM CCCP, Antimycin A 1μM + 1μM Rotenone. Bar graphs of (A) representing basal (B) and maximum (C) respiration. Data represent mean ± SEM of 7-12 independent OCR measurements, One-way ANOVA; *p < 0.05, ** p < 0.01, ***p < 0.001, ns; not significant. Mitochondrial membrane potential measured by fluorescence microscopy in WT, Opa1Crispr, Opa1Crispr + pLenti-Opa1, Opa1CrisprPgs1Crispr, Opa1CrisprPgs1Crispr + pLenti-Pgs1, Pgs1Crispr and Pgs1Crispr MEFs + pLenti-Pgs1. Membrane potential is represented as the ratio between TMRE/mitoYFP. WT MEFs treated with 20μM CCCP serve as a negative control for TMRE. Data represent mean ± SD of three independent experiments, number of analyzed cells indicated in inset, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant. mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant. mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant. (G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.
Figure 2HEK293 cells: (A) Schematic outline of the experiment shown in (B). HA-Nrxn1α or HA-Nrxn1α (S>A), a mutant of the modified serine were expressed alone or in combination with V5-tagged CA10 in HEK293 cells. Neurexins were immunoprecipitated for HA and treated with heparinases ('Heps'). HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5. HEK293 cells: (C) Schematic outline of the experiment shown in (D). HA-Nrxn1α was expressed alone or in combination with CA10-V5 in HEK293 cells, lysates were immunoprecipitated for HA (for Nrxn1α) or V5 (for CA10). HEK293 cells: (D) Representative immunoblots (of three independent experiments) analyzed as described for (B). HEK293 cells: (E) Schematic illustration of how endogenous neurexin-HS was analyzed. HS-modified α-neurexin migrates at higher apparent molecular weight (HMW) than non-modified low molecular weight (LMW; white arrowheads) species (left). Following treatment with heparinases (right), both HS-modified and non-modified α-neurexin migrates as LMW species, but HS-modified α-neurexin can be detected by the 3G10 monoclonal. (F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3). (I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.
Figure 6A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate. B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies. C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).