EMBO/sd-character-level-ner huggingface dataset:
a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX.f1a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3) P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3) P, yielded virtually no labelling in PtdIns(3) P-containing liposomes. Scale bar, 0.2 μm. (b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3) P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3) P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).
Figure 1(A) Phylogenetic relationship among human and putative nematode NSUN proteins. Unrooted phylogenetic tree of NSUN homologues in Homo sapiens and C. elegans using entire protein sequence. Phylogenetic tree reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0). (B, C) Knockdown of nsun genes through RNAi by feeding. Representative images of wild type adult animals after silencing of nsun genes via RNAi by feeding. Widefield DIC images are 10x magnification (B). Percentage of fertile adults after gene silencing by RNAi (C). n = 2 independent experiments, 3 biological replicates each. Data information: In (C data are presented as mean ± SEM. In (C), a representative plot of two independent experiments is shown. (D) Mutant alleles used in this study. CRISPR-Cas9 strategy for creation and screening of catalytically inactive alleles of nsun-1 (mj473), nsun-2 (mj458) and nsun-4 (mj457). A homologous recombination template bearing a point mutation to convert the catalytic cysteine into alanine whilst creating a restriction site for HaeIII was co-injected. For the study of nsun-5, a 928 bp deletion allele (tm3898) was used. Image not to scale. (E, F) Mass spectrometry quantification of m5C (D) and hm5Cm (E) levels in total RNA from nsun mutants. RNA was extracted from populations of L1 animals synchronised by starvation, digested to nucleosides and analysed via LC-MS. n = 3 independent biological replicates. n.d. = not detected. Data information: In E, F data are presented as mean ± SEM. (G) Fold change in total RNA modification levels upon loss of m5C. Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples. n = 3 independent biological replicates. Multiple t-tests. Data information: In G), data are presented as mean ± SEM.
Figure 4A-E The top 30 Age-TFs regulating the ageing-induced transcriptomic changes control a substantial part of Age-DEGs and Age-BPs in analysed tissues. Only the core Age-TFs targets (leading-edge targets), which accounts for the gene sets enrichment signal, were analysed for the Age-DEGs and Age-BPs regulated by corresponding Age-TFs. Hypergeometric test. F-J Many Age-TFs function in a synergetic manner. Synergetic interactions networks were generated from top 30 Age-TFs by synergy analysis, filtered by P<0.001. The hub genes selected from the synergetic network using the MCC algorithm of CytoHubba plugin in Cytoscape. The Age-TFs are ranked by the P-values from MARINa. The Age-TFs in the synergetic network are coloured by their MCC values. proAge-TFs are defined as those promoting the transcriptomic changes (up- and down-regulation) during ageing, whereas antiAge-TFs are those inhibiting these changes. proAge-TFs promote whereas antiAge-TFs suppress the ageing-induced transcriptomic changes. Please note that these two classes of TFs are not necessarily pro- or anti-aging by their names. The transcriptomic changes triggered by a proAge-TF could be adaptive for ageing and induce longevity.
Figure 1A. Western blot analysis of Tace using lysates from WT and TaceFl/Fl;P0-Cre mousesciatic nerves at P10 (65ug of total protein lysate loaded). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments. B. Western blot analysis (50ug of total protein lysate loaded) shows that Tace expression in axons is maximal at P5-P10 and then progressively declines in the adult. Representative of two independent experiments. C. Western blot analysis of Tace in Mtmr2-/- sciatic nerves at P5, P10, and P20. Representative of two independent experiments. D. Western blot analysis of Tace using lysates from Mtmr2-/- mouse sciatic nerves at 5 months, with quantification, p=0.10, two-tailed Mann Whitney U test. Representative of two independent experiments. E. Phosphorylation of ErbB2 in Mtmr2-/- sciatic nerves at P2, P10 and P15. At P2, each lane is a pool of n=7 animals per genotype, representative of six independent experiments using n=6 different nerve pools per genotype, and quantification using WT values arbitrary set to1, p=0.0313, Wilcoxon Rank Sum test. At P10 and P15, mean values of two samples are shown. At P10, each lane is a pool of n=3/4 animals per genotype, representative of three independent experiments using n=6 different pools per genotype. Nf-l is the neurofilament light chain and Clx is calnexin. F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.
Figure 1A. Quantification of spore formation in the wild-type (WT) and the indicated PHSL1-CDC20 strains at 23 h in sporulation medium (SPM). Error bars extend to data from two independent experiments (n = 200 cells per strain). B. Cells at 23 h in SPM. Nuclei are labeled with TetR-RFP. Scale bar, 2 μm. C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of four independent experiments. D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.
Figure 1A. Scheme of the strategy used to assay HP1γ association with RNA on chromatin. B. Genome views of RNA read density profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D. C. Genome-wide scatter plot of IP and input normalized (norm.) RNA read counts per gene for the mean of the triplicates; n=21,754; r, Pearson's correlation coefficient between IP and input. D. Relative quantities of RNA in the RNAchIP samples, detected by RT-qPCR with primers aligning on the intron1 of the Fosl1 gene as depicted in B (orange arrow). n=3 independent experiments. Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test). E. Heat maps of RNA IP and input signal centered on the summit of RNAchIP peaks of IP versus input detected by MACS2 analysis (10,013 and 2609 peaks) for a representative replicate (replicate 1). Black arrows represent peak summit. F. Percentage of RNAchIP peaks overlapping with indicated chromatin features in MEF samples from ENCODE database (green bars). Overlaps was evaluated by comparison with the list of merged peaks whose genomic location was randomized among genes with one hundred permutations (random, gray bars). Data information: Histograms represent mean and s.d. Dots represent individual data points. P-values indicate significantly higher difference between IP in HP1γ and in KO cells (***P < 0.001; two-tailed Student's t-test).
Figure 2(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647 in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.
LDM chemotherapy selectively offsets HIF-1α levels in colon cancer metastases to the liver A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size.Figure 2A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle). B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. C: Correlation between metastatic and peri-metastatic HIF-1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,57) = 150.7 and P< 0.0001; slope ≠ 0 by F-test. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test.. D: Correlation between metastatic and peri-metastatic HIF-2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-2α expression. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test. n, number of nodules.
USP45 localises at sites of DNA damageRepresentative immunofluorescence images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h).Figure 7A Representative immunofluorescence microscopy images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h). Cells were fixed 10 min after laser micro‐irradiation and stained for localisation of endogenous USP45, phosphorylated γ‐H2AX and DAPI. The right‐hand panel shows magnification of one micro‐irradiated cell. Similar results were obtained in at least three independent experiments. Scale bar, 10 μm.
Figure 9a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5) P2, PI(4,5) P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel. b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5) P2.
Figure 9Left image: SOCE mediated by STIM1 and ORAI1 leads to Ca2+ influx, recruitment of the AKAP79/NFAT signaling complex and the phosophodiesterase PDE8. Locally recruited PDE8 reduces cAMP at ER-PM junctions. The AKAP79 signalosome recruitment is instable due to limited membrane PIP2 levels. CAN: Calcineurin. Right image: SOCE mediated by STIM1A and ORAI1 leads to reduced Ca2+ influx due to destabilized gating and hampers recruitment of PDE8. Reduced recruitment leads to reduced local cAMP degradation, which enhances PIP2 levels by activating PIPkinases. Increased levels of PIP2 stabilize the AKAP79 signalosome, enabling more efficient NFAT translocation.
VpT6SS1 is functional in V.Figure 1A Schematic representation of the V. parahaemolyticus RIMD 2210633 T6SS1 gene cluster. Genes are represented by arrows indicating the direction of transcription. Locus numbers (vpxxxx) are denoted above. Notable T6SS components are denoted below. B Expression (cells) and secretion (media) of VgrG1 from V. parahaemolyticus RIMD 2210633 derivative POR1 (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from V. natriegens (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in Vnat samples. C Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. D Illustration of interbacterial competition mediated by VpT6SS1 when introduced into V. natriegens (yellow) on a plasmid (pT6SS1), as shown in C. Prey cells are denoted in blue.
Biochemical assessment of patient fibroblasts and Uqcrh-/- mice show impaired complex III activity and stability A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts.Figure 3A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity. B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison). C Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]) E Western blot analysis of liver lysates derived from 6 wild-type (WT) and 6 Uqcrh-/- animals at 4 weeks of age. The upper band (97 kDa) refers to loading control VCP protein, the lower band (11 kDa) indicates UQCRH. F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).
Figure 2A, A diagram indicating the position of DSBs generated by sgRNAs/Cas9 on the EGFP-SSA reporter. B, RMD efficiency is decreased when the distance from a DSB to one repeat is increased. RMD assays were performed in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is shown. C and D, Knockdown of POLD3 impairs RMD when a DSB is close to one repeat. RMD frequency was determined in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9 with or without depletion of POLD3 by lentivirus-expressed shRNA. Cells infected with a lentiviral empty vector were used as control. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.
mtON affects whole-animal energy sensing.Figure 4Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation. Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation. Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.
Figure 3A, FGFR inhibition upregulates peroxisome and lysosome signatures. Gene set enrichment analysis (GSEA) is based on the GEO dataset GSE73024, whereby H1581 and H520 cells treated with CH5183284/Debio 1347, a selective FGFR inhibitor. B, Flow cytometry-based measure of ROS in H358, A549 and MIA PaCa-2 cells treated for 24 h with AZD4547 (5 µM) and BI2536 (5 nM) in the presence or absence of NAC (5mM). Quantification of relative ROS levels was shown in the right. Data are presented as the mean ± SD (n=3). ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. C, Apoptotic assay of H358 and A549 cells treated for 48 h with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5nM), in the presence or absence of NAC (5 mM). Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated. E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM). F, Immunoblots of H358 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Apoptotic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test.
Function and structure of FRH.Figure 1(A) In the Neurospora crassa circadian core clock oscillator FRH associates with FRQ and CK1a to form a repressor complex (FFC) that inhibits the positive-acting white-collar transcription factors (WC-1 and WC-2) which form the white-collar complex (WCC). Stoichiometries of the components are not represented in the schematic. FFC composition has been estimated as 1 FRH molecule: 2 FRQ molecules (Cheng et al, 2005; Hurley et al., 2013)), or 2 FRH molecules: 2 FRQ molecules (Lauinger et al, 2014). (B) Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.
Figure 5Expression and binding of Ronin to structural and regulatory factors of the mitochondrial cristae. Binding of Ronin to the promoters of mitochondrial cristae genes. Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition. Flow cytometry analysis of Ronin fl/del and Ronin del/del stained for MitoTracker and TMRE. Quantification of the TMRE signal determined by the flow cytometry analysis. Ordinary one-way Anova, data represent mean ± SEM, n = three independent experiments. OCR measurement using the Seahorse mitochondrial stress test assay. FCCP - Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Rot - Rotenone; AA - Antimycin, data represent mean ± SD, three independent experiments. Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.