EMBO/sd-character-level-ner huggingface dataset:
Transient CDK4/6 inhibition emphasises the cell cycle dependency of paclitaxel cytotoxicity in the human hair follicleA) Schematic of experimental design.Figure 6A) Schematic of experimental design. Hair follicles (HFs) were pre-treated with palbociclib for 18h then further incubated either with or without paclitaxel for a further 24h. Subsequently, drug-containing media was washed out and HFs were left in organ culture for an additional 24-48h period. B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error. C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error. D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error. E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; "wo" - washout. 20 µm scale.
Figure 4(A-B) Relative Pseudomonas abundance in rhizosphere samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test). (C-D) Relative Bacillus abundance in rhizosphere samples after (C), Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (E-F) Relative Geodermatophilus abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (G-H) Relative Methylobacillus abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).
Figure 4A. Dot blot of tau levels in media of cells overexpressing the phospho-deficient FLAG-DnaJC5 S10A compared to FLAG-DnaJC5 WT. Intracellular levels shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. B. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated tau release in HEK293T cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody. C. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated α-synuclein release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 by one-way ANOVA with a Tukey's post-hoc analysis. DnaJC5 is detected by FLAG antibody. D. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated TDP-43 release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody. E. Schematic representation of Hsp70 control of DnaJC5-mediated tau release. DnaJ selection on Hsc70 dictates client fate: if a pro-degradation DnaJ binds to Hsc70, clients are sent for degradation. If DnaJC5 binds to Hsc70, client proteins are sent for release by a SNAP23-dependent pathway.
a) SAR405 induces late endosome-lysosome swelling.figf3(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm. (b) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14. (c) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.
Figure 4(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test). (C) Schematic representation of the experimental workflow for Figs. 4D and 4E. (D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test). (E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test). (F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test). (G) A proposed model for a role of SIRT5 in mediating oxidative stress to increase IDH2 and G6PD activities, NADPH production, and cellular protection. SIRT5 is activated by oxidative stress by a yet unknown mechanism. The active SIRT5 stimulates IDH2 and G6PD by desuccinylation and deglutarylation, respectively, thereby increasing NADPH production and protecting cells from ROS damage.
Figure 7(A-C) Reduced immuno-reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4-AP treatment, revealed by conventional fluorescence microscopy (KS test, p < 0.001 in B1 and p < 0.0001 in B2, p < 0.0001 in C). Total gephyrin was probed with polyclonal rabbit primary antibody (rbGPHN), and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody (m7a). Number of synapses: n = 4040 in TTX and n = 3818 in 4-AP conditions from two independent experiments. Scale bar: 2 µm. (D-F) Reduced numbers of pS270 gephyrin (m7a) detections were recorded by dSTORM for the entire synaptic area (D, KS test, p < 0.01). The number of detections of pS270 gephyrin per SSD (E, KS, p = 0.18) and the number of SSDs (F, MW, p = 0.11) were not changed by 4-AP treatment. Gephyrin was probed with mAb7a antibody and Alexa 647 dye. Number of synapses: n = 810 in TTX and n = 727 in 4-AP conditions from three independent experiments; (F) mean ± SEM. Scale bar: 100 nm.
HOTAIR expression changes H3K27 acetylation and LSD1 occupancy of gene promoters.Figure 4Venn diagram representing intersections of gene promoters possessing H3K27ac peaks within the 2kb window around their TSS in Epi cells. Metagene profiling within the 10 kb window around TSS of H3K27ac, LSD1 and H3K4me2 over IgG in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs. Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups. Biological process pathways identified by DAVID as enriched for Class 1 or Class 2 genes hyperacetylated in low- or high-migrating cells, respectively (adj. P-value < 0.05 for Class 1 and P-value < 0.05 for Class 2). Dots represent GO term enrichment with color coding: red for high enrichment, blue for low enrichment, and sizes of the dots representing the significance score of each subset (the bigger the dot, the higher its significance). Visualization of the H3K27ac, Lsd1 and H3K4me2 CUT & RUN and of RNA-seq tracks of the TGFB2 promoter region in Epi-CTR, -HOT, -HOTΔP and -HOTΔL cells.
Identification and prevalence of human gut microbial subspecies.Figure 1Human Gut microbial species explored for the existence of subspecies show wide phylogenetic spread according to NCBI taxonomy (A) and include Methanobrevibacter smithii, the main archaeal member of the human gut microbiome, as well as representatives of all abundant phyla. Species names are according to NCBI taxonomy, with species cluster (specI) identifiers according to Mende et al., which splits some named species into multiple specI clusters (Mende et al, 2013). Of 71 investigated species, 44 stratify into subspecies (highlighted in blue). Each species' average abundance across 2,144 human gut metagenomes is proportional to the size of the circles on the cladogram. Bars represent the number of subspecies identified in each, with '1' indicating no subdivision. The black portion of the bar corresponds to subspecies for which no representative genome sequence is available from NCBI. Geographic enrichments of subspecies are displayed as a heat map (showing only significant enrichment, FDR corrected Fisher test p-value < 0.05, per country as maximum log-odds ratio across con-specific subspecies). Subspecies with a restricted geographic range are predominantl­y found in the Chinese and Kazakh population. The 71 investigated species captured an average of 95.5% of sequencing reads that were assigned to any reference genome. The subset of 44 species with identified subspecies accounted for the majority of this abundance (B).
Pedigrees of the families with N-terminal IGSF10 mutations with typical growth charts.Figure 2A: Squares indicate male family members, circles female family members. Black symbols represent clinically affected, gray represent unknown phenotype, clear symbols represent unaffected individuals. The arrow with 'P' indicates the proband in each family and 'us' indicates un-sequenced due to lack of DNA from that individual. The mutation in each family is given next to the family number; a horizontal black line above an individual's symbol indicates they are heterozygous for that mutation as identified by either whole exome sequencing (family 3 and 4) or Fluidigm array (family 1, 2, 5 and 6), and verified by Sanger sequencing. B: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.
Figure 4A. UMAP embedding of human ileum-derived organoids colored by the expression of HAstV1. B. Proportion of cells infected with astrovirus detected in each cell lineage and type. C-D. Volcano plots of genes that are differentially expressed in mock relative to 16hpi, showing the statistical significance (-log10 adjusted p-value) vs log2 fold change (mock/16hpi) for the stem cells (C) and enterocyte lineage (D). E. Dot plot of top lineage-specific expression changes upon HAstV1 infection for mock, 4hpi and 16hpi organoids. F. Same as in E but for ISG. The dot size represents the percentage of cells expressing the gene; the color represents the average expression across the lineage. Triangle and stars represent significantly changing genes (FDR < 0.05) in mock-infected samples vs. 4 hpi as well as and mock-infected samples vs 16 hpi, respectively. G G. UMAP embedding of scRNA-Seq data from human ileum-derived organoids based on the significantly changing ISGs for cells at 16 hpi. H. Unsupervised clustering of the UMAP data from G. I. Distribution of cell lineages and types in the clusters from H. J. Heatmap of differentially expressed ISGs across the clusters from H.
Figure 2A Schematic of tFT-Bcd reporter with mCherry and sfGFP fluorophores. B Cartoon of the fluorescent protein maturation states in the tFT-Bcd reporter. mg and mr represent the sfGFP and mCherry maturation rates respectively (note, mr is an effective rate as mCherry has a two-step maturation process). C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).
Figure 2A. The total observed and predicted metabolome of uRBC and iRBC. The total number of metabolites and those with a unique KEGG identification number are reported. Numbers in parenthesis refer to unmatched metabolites that were predicted from the genomic reconstruction but not observed, or observed metabolites that were not predicted. The total number of metabolites common to both predicted and observed lists (percentage in parenthesis) are referred to as 'matched'. An UpSet plot summarising the overlap of metabolites present in the predicted and observable metabolomes across uRBCs and iRBCs, with the inset depicting the metabolite size of each cell type. B. Metabolic pathway enrichment analysis for the observed metabolome of uRBCs and iRBCs. Unique KEGG ID metabolites were queried against the Small Molecular Pathway Database with -log10 (Holm P) reported for the number of metabolite hits reported for each pathway. C. Extracted ion chromatogram of pyridoxine-P, one of five metabolites detected exclusively in uRBCs. X-axis corresponds to retention time (RT) and y-axis corresponds to arbitrary ion intensity of the extracted peak .The inset indicates the integrated peak area for each biological replicate. ND indicates 'not detected'. D. Extracted ion chromatogram of glycero-P-glycerol, an observed metabolite that was not predicted from genomic reconstructions and is significantly elevated in iRBC compared to uRBC. X-axis corresponds to retention time (RT) and y-axis corresponds to arbitrary ion intensity of the extracted peak. Inset indicates the integrated peak area across each cell type and biological replicate. E. The abundance of all detected polar metabolites was compared between iRBCs and uRBCs and presented as the log2 fold-change (iRBC/uRBC) with respect to the -log10P. Significance cut-off was set with −log10(0.05) + c/(x − x0). Each data point represents a single metabolite and the insets depict the extracted ion chromatograms for two metabolites that were either elevated (acetyl-CoA) or decreased (fructose-1,6-bisphosphate) in iRBC relative to uRBC.
Figure 6A. Representative images for co-localization between PML (green) and telomeres (red FISH probe). White arrows indicate co-localization events, marking APBs. Quantification of images is shown on the right. A total of 85-300 nuclei were scored from four independent experiments. Each dot represents the number of events per nucleus: PML foci or APBs. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution. B. Experimental scheme for ATSA (ALT telomere DNA synthesis in APBs), used to measure G2/M telomeric DNA synthesis, within PML bodies. C. Representative images for co-localization between EdU (green) and telomeres (red FISH probe) within PML bodies (cyan) in U2OS and U2OS-hTR cells. White arrows indicate co-localization events. D, E. Quantification of images shown in (C). Co-localization events between EdU foci and APBs were analyzed (D) and the percentage of APBs that are actively engaged in G2/M telomeric synthesis was calculated (E). A total of at least 50 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.
ATG4C and BECN1 were targets of MIR376A.Figure 4(A and B) MIR376A mature miRNA sequence and MIR376A binding target sequences in the 3' UTR of ATG4C (A) and BECN1(B) mRNAs were depicted. MIR376A seed sequence was shown in bold letters and and complemetary sequences were indicated. (C and D) qPCR analysis of ATG4C and BECN1 mRNA expression in control miRNA (MIR-CONT) or MIR376A overexpressing cells under non-starved (NON STV) or starved (STV) condition (mean ± S.D. of independent experiments, n = 3, *p<0,05). (E and F) Immunoblots showing ATG4C and BECN1 protein levels following control miRNA (MIR-CONT) or MIR376A overexpression. ACTB was used as a loading control. ATG4C/ACTB or BECN1/ACTB densitometric ratios were marked.
Figure 4(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001. (b) Representative spatiotemporal plots of the detrended Venus::HES5 pattern along ventral-dorsal direction in DMSO control (left panel) and DBZ conditions (right panel) obtained by averaging kymographs data in the same region over 2 hr time intervals. (c) Schematic indicating the correspondence between Venus::HES5 spatial oscillator represented as detrended level and phase angle characteristics; the spatial oscillator traverses repeated cycles including start (HES5 low-orange arrowhead), middle (HES5-teal arrowhead) and end (HES5 low-red arrowhead) which in phase space corresponds to phase angles 0, π and 2π respectively. (d) Phase maps corresponding to DMSO (left panel) and DBZ (right panel) detrended Venus::HES5 data shown in (b). (e) Phase synchronisation measure (see Materials and Methods) of the detrended Venus::HES5 spatial oscillator measured over time in E10.5 Venus::HES5 spinal cord periodic slices treated in DMSO vs DBZ conditions up to 10h; dots indicate DMSO (21 kymographs, n=3 experiments) and DBZ (19 kymographs, n=4 experiments); bars indicate mean and SD; 2 tailed Mann-Whitney test with significance ****p<0.0001. (f) Spatial peak: trough fold change in Venus::HES5 intensity profile in the D-V axis measured at 2h and 10h in DMSO and DBZ treated E10.5 Venus::HES5 spinal cord slices; dots indicate average over 3 z-slices from DMSO (n=3) and DBZ ( n=4) experiments; lines indicate median per condition; 1 tailed unpaired t test with significance *p<0.05. (g) Percentage of ex-vivo slices with significant spatial period detected after 10-12hrs of DMSO and DBZ conditions; significant spatial period defined as multiple significant peaks in auto-correlation detected above the 95% confidence bounds; dots indicate % per experiment; bars denote median and inter-quartile range of DMS0 (n=3) and DBZ (n=4) experiments; 1 tailed t test with significance **p=0.0062. (h) Peak:to:peak distance in auto-correlation plots of detrended Venus::HES5 spatial profile in DMSO and DBZ treated E10.5 Venus::HES5 spinal cord slices; gray dots represent significant mean peak-to-peak distance of DMSO (100) and DBZ (105) auto-correlation functions collected from 3 z-stacks per slice and 2 repeats (left and right of ventricle) with multiple timepoints; bars indicate median per experiments from DMSO (n=3) and DBZ (n=4) experiments; error bars indicate SD; 2 tailed Mann-Whitney test ****p<0.0001.
Figure 5(A-J) Isotope-labeled carbon tracing experiment showing glucose and lactate metabolism in HBVPs. HBVPs were treated with isotope-labeled nutrients (­­CON: control, [13C6]-glu : 5mM [13C6]-glucose, [13C3]-Lac : 2mM [13C3]-Lactate, [13C3]-Lac-20 : 20mM [13C3]-Lactate in glucose free media) for 12hr and analyzed by a Q-exactive MS spectrometry. Barplots show relative fraction of unlabeled (M0) and labeled forms (M1~6) in various metabolites including A. Glucose, B. Lactate, C. Citrate, D. Aconitate, E. α-ketoglutarate (αKG), F. Succinate, G. Malate, H. Aspartate, I. Alanine, J. Glutamate. (n=3 from independent experiments.) K. Schematic diagrams showing the glucose or lactate derived carbon flow in pericytes (HBVPs) when [13C6]-glucose or [13C3]-lactate was used as labeling substrate(s). (Unlabeled carbon : black circle, labeled carbon : red circle)