EMBO/sd-character-level-ner huggingface dataset:
Figure 7Figure 7. Rpp30 mutation leads to tRNA processing and piRNA transcription defects.Top. In wild-type ovaries, tRNA genes (violet boxes) are transcribed by pol-III. tRNAs are thenprocessed by RNase P and Z ribozymes that cleave tRNAs precursors (pre-tRNAs, dark violet). tRNA gene clusters can be localized near piRNA clusters (turquoise), which are characterized by H3K9me3epigenetic mark (green) required for piRNA transcription. piRNAs arising from piRNA precursors guide Piwi proteins to Transcriptionally Silence Transposable Element expression (TGS), protecting genome integrity. Bottom. In Rpp30 ovaries, tRNA processing defects [1] lead to Transcription-Replication Conflicts (orange) [2]. These conflicts activate both Replication Stress and DNA damage checkpoint proteins, Claspin and Chk2 [3], which induce piRNA transcription defects [4] and Replication fork stalling and PCNA collapse [5]. Downregulation of piRNAs results in TEs overexpression [6], which also activate DNA damage checkpoint [3].
Structural and functional analysis of artificial muscle and underlying TA control muscle.Figure 3A Electron microscopy with Bluo-Gal staining reveals a LacZ-positive donor nucleus (arrow) peripheral to transversely sectioned sarcomeres of the artificial muscle. Scale bar: 1 μm. B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization. Scale bar: 0.5 μm. C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre. Scale bar: 10 μm. D X-Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ-positive donor nuclei. Scale bar: 100 μm. E Immunofluorescence analysis of LacZ-positive nuclei (green) and dystrophin (red) in an individual muscle fibre after functional analysis. Scale bar: 10 μm.F The same fibre shown in (E) is counterstained with DAPI to show that the interstitial nuclei (blue) are of donor origin. Scale bar: 10 μm. G-I Specific force (Po/CSA) (G), maximum shortening velocity (Vo) (H), and cross-sectional area (CSA) (I) of single fibres (n = 153) from artificial muscles and control (CTRL) muscles (n = 103). J Silver-stained SDS-PAGE (8%) gel revealing myosin heavy chain (MyHC)-2B isoform expression in two single fibres of artificial muscles (lanes 1, 2) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in (K) (lane 3). K Comassie blue-stained SDS-PAGE (8%) of bulk samples of two artificial muscles (lanes 1, 2) and of a bulk control muscle sample (lane 3), as in (J), showing different MyHC isoform expression.
Figure 7WRNIP1 interacts with the BRCA2/RAD51 complex and stabilizes RAD51 on ssDNA at stalled forks, counteracting the dissolution of the RAD51 filament by FBH1. After stalled fork stabilization, the ATPase activity of WRNIP1, in collaboration with other proteins, could be required for stimulating the restart of DNA synthesis, which ensures genome stability. Loss of WRNIP1 or its catalytic activity leads to DNA damage accumulation and enhanced chromosomal instability. See the text for more details.
Figure 6(A) Representative H&E stained liver, adipose tissue, and skeletal muscle sections from CTRL and BAM15 treated animals. Scale bars (black) = 100 μm. (B) Representative immunohistochemical analysis of pancreatic insulin (red) content counterstained with diaminobenzidine (blue). Scale bars (black) = 100 μm. C-F (C) Quantitative analysis of liver steatosis (μm2) (N=6 per group; P<0.0001), (D) gastrocnemius muscle cross sectional area (μm2)(CTRL N=7, BAM15 N=8), (E) pancreatic insulin positive area (% of total area) (N=6 per group; P=0.0420), (F) and β-cell mass (N=4 per group; P=0.0005). (G-I) Size (area; μm2) and distributional (number) analysis of (G) iWAT, (H) gWAT, and (I) BAT from CTRL and BAM15 treated animals (N=8 per group).
Figure 4A) Fold changes in the H/L ratios ( ) measured for each protein in the experiments described in Fig. 3 (expressed as log2(H/L)Lactacystin - log2(H/L)control). H/L ratios used here are the average of ratios obtained in two experiments for each protein and each condition. Proteins were sorted according to the fold change in H/L ratios - from the proteins whose ratios were most strongly reduced (~8-fold reduction) to proteins whose H/L ratios increased to the greatest extent (>8-fold increase). Red circles indicate the location of synaptic proteins along this sorted list. The names of 34 synaptic proteins located at the left most region of the sorted list are provided in the order of their appearance. B) The apparent fold-change in protein half-lives under certain assumptions described in Results. See Materials and Methods for further details on the calculation of the apparent fold change in half-lives.
Human S100A9 negatively regulates HIV-1 reverse transcription(A) MoLC were transfected with siCTR or siS100A9 and infected with HIV-GFP vectors for 24h.Figure 9MoLC were transfected with siCTR or siS100A9 and infected with HIV-GFP vectors for 24h. DNA was extracted and copies of late reverse transcripts were quantified by qPCR. Shown data were collected from experimental triplicates ± SD of four different donors (n=4). MoLC or eLC previously transfected with control siRNA (siCtrl) or S100A9-targetting siRNA (siS100A9) were challenged with 200 ng P24 of HIV-1-R5 for 24h. Nevirapine (10μM) was used as a control of RT inhibition. Total DNA was then extracted and late RT products were quantified by qPCR and normalized as copies/100ng DNA using a standard curve based on the serial dilution of a defined and linearized HIV-1 plasmid. Duplicates from three different donors are shown (n=3). Nevirapine (10μM) treatment was performed once on each donor to control RT inhibition. Data were represented as means ± SD. The same experimental procedure as above was done with HIV-1 primary isolates. Data in triplicates ± SD from three different donors (n=3) were collected and represented as bar charts. E2C-expressing (upper dot-plot) and S100A9-expressing (lower dot-plot) THP-1 cells were controlled for E2C and S100A9 expression by flow cytometry upon analysis of fluorescence as indicated in the X axis label. Parental untransduced THP-1 cells were used as control cells (gray dots). The experiment shown is representative of 3. E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown. Means ± SD of duplicates from two independent experiments are presented in the graph (n=2). When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).
Analysis of AP-3 interactors by proximity labelling.Figure 5(A) Scheme of Apl5 with C-terminal TurboID tag. (B) Model of possible biotinylation events along the AP-3 pathway. AP-3 has to be recruited to the TGN where it concentrates cargo and forms a vesicle. This is transported to the vacuole where it is tethered and subsequently undergoes fusion. During these steps several interactors can come in close proximity to the TurboID Biotin Ligase (grey shade). (C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots, (D) Model of possible biotinylation events with AP-3 vesicles tethered to the mitochondrial surface by mitochondrially targeted Vps41. (E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots
Figure 2(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band. (B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm. (C) Quantification of the nuclear translocation of CCNB1 in RPE cells treated with 25 μM P22077 for 1-2 hrs measured by high throughput microscopy in three different experiments (** p<0.01, ***p<0.005, t-test). Bars represent mean values ± SEM. N>2000 cells were analysed per condition. (D, E) Flow cytometry profiles representing the levels of MPM-2 (D) and H3S10P (E) in WT and CDK2/4/6 knockout MEF in control conditions (Control, black) and after treatment with 50 μM P22077 for 4 hrs (USP7i, red). The experiment was performed twice using two different wild type and CDK2/4/6 KO MEF and one representative result is shown. (F, G) Flow cytometry profiles representing the levels of MPM-2 (F) and H3S10P (G) in RPE cells in control conditions (Control, black), after treatment with 25 μM P22077 for 4 hrs (USP7i, red) or after treatment with 10 μM RO3306 for 8 hrs followed by incubation with 25 μM P22077 and 10 μM RO3306 for 4 additional hrs (CDK1i+USP7i, blue). DNA content was measured with Propidium Iodide (PI). One representative experiment is shown, out of five.
Figure 1A Representative 3D tile scan images of young adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland stained with the antibodies indicated in the panel. Scale bars are 200 µm. The white and dashed lines represent the outlines of each organ. B Representative 3D images at single-cell resolution level of adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland. Insets (*) indicate higher magnification images of regions with single-cell resolution and surface rendering for all markers. Scale bars for tile scan 3D images are 200 µm, for single-cell resolution insets are 5 µm. The dashed lines represent the outlines of each organ. C Exemplar image illustrates the cell-cell interactome analysis with the distance estimates of CD68+ macrophages to blood vessels (Endoglin) in cortex, medulla and the transition zone (TZ) of an aged adrenal gland. The colour code indicates the distance which is very close in violet and very far in red. The scale bar is 50 µm. The white and dashed lines represent the outline of the transition zone. D Illustration of the EC interactome in adrenal glands and changes in the interactome with ageing. Cells ≤18 μm of distance from the blood vessels were considered to be within the interactome of blood vessels. Analysis of endothelium in the different region showed that they have distinct marker expressions. CD102pos / VCAM-1lo / Vimentinneg, CD102neg / VCAM-1hi / Vimentinneg and CD102neg / VCAM-1hi / Vimentinpos ECs were located in cortex, transition and medulla regions respectively in young and aged adrenal glands. Their interactions in young and aged adrenal glands with PDGFRβ+ and NG2+ pericytes, PDGFRα+ mesenchymal cells, α-SMA+ pericytes, Desmin+ stromal cells, Decorin+ stromal cells, CXCR4+ stromal cells, FSP1+ fibroblasts, CD34+ hematopoietic cells, F4/80+ macrophages and CD68+ macrophages are shown. Interaction with Decorin expressing stromal cells is only observed in ageing. E Schematic illustration of the study design with 3D spatial proteomic data and cell-cell interactome database strategy, analysis of age-dependent changes and validation of protein expression followed by functional analysis.
Hdac3 and Nr5a2/Dax1 are genetically and biochemically distinct pathways repressing PrE differentiation.Figure 4Z-scores of high-confidence interactors coIPed by Hdac3, Dax1, Nr5a2, and Esrrb (indicated in red) in 2iL mESCs. Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones. Gene expression changes in Dax1-/-, Nr5a2-/- and Dax1-/-;Nr5a2-/- cells compared to WT cells in 2iL (upper) and after 2d in SLRA (lower).
signaling pathway in DC.Figure 4DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH. DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA. DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control. DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.
Figure 2Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot. (B) Associations between the plasma level of individual CMA and the 10 most significantly correlated plasma metabolites are presented. Asterisks indicate statistical significance (FDR < 0.05) based on Spearman correlation analysis. Only metabolites detected in more than 50% of samples are included in the analysis. Cor.Coeff: correlation coefficient.
Loss of NDST3 promotes the assembly of the V-ATPase V1-V0 holoenzyme.Figure 3(A) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively. (B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786). Data information: Error bars represent ± standard deviation. (C, D) Ratios of ATP6V1A (C) and ATP6V1C1 (D) to ATP6V0D in the membrane fraction from (A) to represent the assembly of the V-ATPase V1-V0 holoenzyme (n = 3 independent experiments, ***P =0.0005, ****P < 0.0001). Data information: Error bars represent ± standard deviation.
Endocrine composition and function of FI, CI, PI and PI+MVF.Figure 3A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF. Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.
Figure 7A Western blot of ER-GATA-1, Bach1, and b-actin in untreated or b-estradiol-treated (48 hours) WT clone 1 and double-mutant clone 1 incubated with or without 5-ALA. The upper band of the ER-GATA-1 blot represents sumoylated ER-GATA-1, which increases as active ER-GATA-1 becomes nuclear-localized. A darker exposure of the Bach1 blot is also shown. B Real-time RT-PCR analysis of Bach1 mRNA levels (left panel) and primary transcripts (right panel) (n = 4, mean +/- SE). C Real-time RT-PCR quantitation of Hmox1 mRNA levels (left panel) and primary transcripts (right panel) (n = 4, mean +/- SE). D Bach1 knockdown in b-estradiol-treated double-mutant clone 1 (n = 4, mean +/- SE). Western blot analysis of ER-GATA-1, Bach1, and b-actin. A darker exposure of the Bach1 blot is also shown. E Real-time RT-PCR analysis of mRNA levels of GATA-1/heme-activated genes in b-estradiol-treated WT1 or double-mutant clone 1 expressing shRNA targeting Bach1. P values were calculated with respect to b-estradiol-treated control double-mutant clone 1 (n = 4, mean +/- SE). F Real-time RT-PCR analysis of primary transcripts of GATA-1/heme-activated genes in b-estradiol-treated WT1 or Bach1-knockdown double-mutant clone 1. P values were calculated with respect to b-estradiol-treated control double-mutant clone 1 (n = 4, mean +/- SE). G Real-time RT-PCR analysis of mRNA levels of GATA-1/heme-activated genes in untreated or b-estradiol-treated WT1 and double-mutant clones. P values were calculated with respect to b-estradiol-treated WT1 (n = 4, mean +/- SE).
Fig.Figure 4(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample. (B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01) (C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).
A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation.Figure 1.(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP. (D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.
Figure 1(A) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress (200μM H2O2). Biological triplicate data is depicted at each time point relative to control cells. (B) MCF10A cells were exposed to oxidative stress (200μM H2O2) and processed for small RNA-sequencing. The log2-fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted. (C) Schematic depicts the overlap of tRNAs in (A) that decreased over time with tRFs from (B) that were induced.
Departure of Armi from Yb bodies depends on Piwi.Figure 3Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei. The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05. The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31. In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.
Figure 5A Amino acid sequences of the mutations introduced in LUBEL by CRISPR/Cas9. Two LUBEL mutant lines were created for in vivo studies; w- parental line, C2690S/C2693S (CC/SS), and delR2, which has an indel mutation with an early stop codon at amino acid 2719 due to a frame shift. Mutations are indicated in red. B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band. C The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant. D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&amp;amp;E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 μm.