EMBO/sd-character-level-ner huggingface dataset:
MITOL∆C8 ubiquitylates expanded peroxisomes following CCCP treatment (A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies.Figure 13(A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (C) The schematic model of PINK1/Parkin-mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin-catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP-dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of NMS-873, MITOL inserted into the peroxisome membrane is shown here in the non-ubiquitylated form.
uORFs are widespread and translated during zebrafish development.Figure 1(A) Classification of the protein-coding transcriptome in zebrafish, human, and mouse reveals that uORFs are widespread and translated. Transcripts containing at least one uORF are marked in purple, transcripts containing no uORFs but at least one oORF are marked in orange, and transcripts lacking both are grey. In zebrafish, 3 different translation thresholds were applied to classify translated uORFs, and each transcript then classified by its highest confidence uORF: Low confidence (dark pink): RPF RPKM >0; Medium confidence (light purple): ORFScore > 0; High confidence (dark purple): ORFScore > 6.044. (B) uORFs and oORFs are widespread throughout the embryonic transcriptome, with a majority of oORF-containing transcripts also containing at least one uORF. uORF-containing (purple) and oORF-containing (orange) transcripts were counted in mouse, human, and zebrafish, and the overlap is shown by venn diagrams. (C) Metagene analysis reveals features of active translation in uORFs classified as translated. Metagene plots display normalized ribosome protected fragment density surrounding uORF start and stop codons, colored according to frame relative to the ORF being translated. CDS regions with and without uORFs are also shown for comparison. Note the clear phasing of ribosome-protected fragments within high and medium confidence uORFs, and the characteristic start and stop RPF peaks across all classes of uORFs. (D-F) Ribosome profiling reveals in-frame translation of uORFs/oORFs in key developmental regulators. RPF-line plots show the positional distribution of 28 29 nt RPFs (above axes) and mRNA-seq reads (below axes) in the whole gene (below) and first 300nt (inset above) of Nanog (D), POU5F3 (E), and Smad7 (F). All putative ORFs (Distal AUG-Stop) are colored by respective frame (blue, pink and green boxes), as are reads according to their P-site. Note the agreement between ORF color and RPF color, consistent with a strong in-frame distribution of reads within individual transcripts.
Model of the Hsp70/Hsc70-mediated regulation of the heat shock response.Figure 8A Illustration of the stepwise disassembly of Hsf1 trimers by the entropic pulling action of Hsp70 and DnaJB1. Homology model of trimeric DNA bound human Hsf1. Red, accessible Hsp70 binding sites; orange, Hsp70 binding sites not accessible in the Hsf1 trimer, only accessible after unfolding. Unzipping of the Hsf1 trimer may occur simultaneously on all three protomers. B Hsf1 activation-attenuation cycle. In unstressed cells Hsf1 is in monomer-dimer equilibrium, occasionally trimerizing depending on the local concentration. Trimeric Hsf1 either binds to HSE-promoter DNA driving heat shock gene transcription by releasing paused RNA polymerase or is disassembled immediately by Hsp70/DnaJB1 action (not shown for clarity). Hsp70 binds dynamically to the TAD of DNA-bound Hsf1 trimers attenuating transcriptional activity and bind to its HR-B proximal binding site, disassembling Hsf1 trimers and thus removing it from heat shock promoters. Release of Hsf1 from Hsp70 restarts this cycle. Upon heat shock Hsf1 trimerizes at elevated rates due to its thermosensory function, integrating over temperature and time at elevated temperatures. Simultaneously, Hsp70/DnaJB1-mediated attenuation and disassembly is slowed down, due to binding of Hsp70 to misfolded and aggregated proteins. Both parts of the cycle shift the Hsf1 pool rapidly to the DNA bound active state, accelerating heat shock gene transcription. Elevating Hsp70 concentrations successively attenuate transcriptional activity of Hsf1 and disassemble Hsf1 trimers.
Figure 6A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group. B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group. C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10). D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm.G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME H. Immunofluorescence images for MEF2C of mice in (G); n = 9 per group. Scale bars, 50 µm. I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Figure 6(A-B) Wild-type and CD45-deficient primary B cells were stained with Alexa647conjugated antibody against CD22 and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(B) Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from three experiments. (C-E) Wild-type and CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips and imaged. Single particle trajectories were then analysed.(C) Trajectories of CD22 in wild-type (left) and CD45-deficient cells (right) showing diffusion of single particles over 6 s.(D) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median. Data are pooled from three experiments.(E) Confinement analysis of CD22 and IgM. Bars and numbers indicate the median. (F) Two-population analysis of CD22 diffusion. Diffusion coefficient were plotted on a logarithmic scale and fitted to two Gaussian-shaped curves to account for slower (light grey) and faster (dark grey) diffusing populations. Wild-type (blue line and circles, left) and CD45-deficient (blue line and squares, left middle) primary B cells were compared in an overlay (right middle) and their proportions plotted (right). (G and H) CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips, treated with vehicle control (DMSO) or 1µM LatA and imaged. Single particle trajectories were then analysed and diffusion coefficients calculated.(G) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(H) Confinement analysis. Bars and numbers indicate the median. (I and J) CD45-deficient primary B cells were stained with Alexa 647-conjugated antibody against CD22, settled onto nonstimulatory coverslips and treated with vehicle control or sialidase. Cells were then fixed, imaged with dSTORM and analysed.(I) 2D (top) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in pseudo 3D (bottom).(J) Quantification of the distribution of CD22 with Hopkins index and H function. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM.Data are pooled from two experiments.*P<0.05, **P<0.001, ***P<0.0001 (Student's t-test). (K and L) Wild-type and CD45-deficient primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated times. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (p-CD22) and total CD22 as loading control. The intensity of p-CD22, normalized to CD22, was referred to the unstimulated sample of the wild-type cells, set as 1.
Figure 5A Representative image of a spheroid stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). A similarly stained section of a mouse villus is shown for comparison. Arrowheads indicate the apical cell membrane. Scale bars, 50 µm. B, C Jejunal spheroids were cultured as indicated. (B) Quantification of the average expression ± s.e.m. of Fabp1, Ace2, and Maoa mRNAs (n = 3 independent experiments). *p<0.05, **p<0.01 by one-way ANOVA and Tukey's post-test. B, C Jejunal spheroids were cultured as indicated. (C) Representative images of spheroids stained for Ace2 (green) and β-catenin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. D, E Graphs showing the top five most significant pathways (D) and gene ontology cellular component terms (E) associated with Cluster 3. F, G Representative TEM images of EP4i-treated spheroids. (F) The basal plasma membranes are outlined in orange solid lines, lateral plasma membranes are indicated with orange arrowheads, and nuclei are outlined with wide yellow dashed lines. Insets show a magnified view of the apical cell surface. (G) Higher power image of the cytoplasm. Single mitochondria are outlined with a narrow blue dashed line, vacuole structures are indicated with red asterisks. Scale bars, 1 µm. H Graph of spheroid oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio expressed as mean ± s.e.m. (n = 3 independent experiments). **p<0.01by 1-way ANOVA and Tukey's post-test.
Figure 3A Scheme of investigated marker proteins, subjected to FRAP, located at different mitochondrial subcompartments. FRAP curves (curve fitted) of WT Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown. C FRAP curves (curve fitted) of MIC13 KO Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown. D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment). E Diffusion coefficients of the above-mentioned proteins are shown in the table.
The STING-mediated NF-κB response is activated from the ER and specifically pro-viral for MCMV transcription (A) 293T cells were co-transfected with Cherry-STING, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either the pNF-κB, p55-CIB, p125 or p125AA luciferase reporter.Figure 8(A) 293T cells were co-transfected with Cherry-STING, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either the pNF-κB, p55-CIB, p125 or p125AA luciferase reporter. Data is representative of two independent experiments. (B) 293T cells were co-transfected with expression plasmids for cGAS-GFP (stimulated) or ev (unstimulated) together with either cherry-tagged WT STING or cherry-tagged K288R STING. Cells were fixed for imaging 24 hours post transfection. Scale bar represents 10 µm. (C-D) iMEFgt/gt stably expressing cherry-tagged WT STING or K288R STING and either ev or V5-tagged m152 were stimulated with ISD (10 µg/ml) or mock stimulated. At 4 hpi total RNA was extracted to determine IFNb1 (C) and IL6 (D) transcripts by qRT-PCR. Data shown is combined from three (C) or two (D) out of three independent experiments. (E) iMEFgt/gt (-) and iMEFgt/gt stably expressing either cherry-tagged WT STING or K288R STING were infected by centrifugal enhancement with parental MCMV or MCMV m152stop (MOI 0.01). 6 hpi total RNA was extracted to determine MCMV E1 transcripts by qRT-PCR. Data shown is combined from three independent experiments.
Figure 2(A) Measurement of PMO uptake in dystrophic muscle cells (H2K mdx cells) in the presence of MOTS-c (scale bar: 10 μm) (n=6; **p<0.001). PMO-M refers to PMO and MOTS-c (the same is for the rest unless otherwise specified). (B) Tissue distribution of FITC-labeled PMO (50 mg/kg) mixed with MOTS-c (500 μg) in mdx mice 48 h after single intravenous injection. NC represents untreated mdx control. The abbreviation is the same as Figure 1D. Color scale represents the fluorescence intensity. (C) Quantification of fluorescence intensity in body-wide tissues of mdx mice treated with PMO-M or PMO alone (n=3; *p<0.05). The comparison was conducted between PMO-M and PMO. (D, E) Immunohistochemistry (D) and quantitative analysis (E) of dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 0.5 μg PMO mixed with MOTS-c (20 μg) (PMO-M) or PMO alone (scale bar: 100 μm) (n=3; **p<0.001). C57 means wild-type control C57BL/6. (F) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO-M or PMO alone (n=3; *p<0.05). 0.5 µg and 2.5 µg total protein from C57BL/6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA muscles from C57BL/6 were used as normal controls (the same is for all Western blots unless otherwise specified). (G) Systemic evaluation of MOTS-c (500 μg) and PMO at the dose of 50 mg/kg/week for 3 weeks in adult mdx mice intravenously. Tissues were examined two weeks after last injection. Immunohistochemistry for dystrophin- positive fibers in body-wide muscles of treated mdx mice (scale bar: 100 μm). C57 means wild-type control C57BL/6. (H, I) Western blot (H) and quantitative analysis (I) for dystrophin expression in body-wide muscles from treated mdx mice (n=3; *p<0.05). 0.5 μg, 2.5 μg, 5 μg and 10 μg total protein from C57BL/6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control.
Figure 2(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test. Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm. Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm. (H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3. (I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.
a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3).f1a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3). c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr;IEC hypomorphic Paneth cells. d, e, Crypt showing GFP-LC3 punctae (d), quantified in e (n = 10; unpaired Student's t-test; mean ± s.e.m.). Scale bar, 5 µm. f, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK-eIF2α branch (n = 3). j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.). k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.
p62 is found both in autophagosomes and in cytosolic aggregates/sequestosomes.Figure 4.(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo). (B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1. (C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.
Figure 4(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL). (B) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T- from A-T-. Both CSF p-tau217 and p-tau231 a showed a statically higher accuracy discriminating A-T- from A+T- than CSF p-tau235 (C) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T+ from A-T-. However, all three assays had statistically the same accuracy when distinguishing A-T- from A+T+. (D) ROC analysis comparing AUC values of CSF p-tau235, p-tau231 and p-tau217 discriminating A+T+ from A+T-. CSF p-tau235 demonstrated statistically superior diagnostic accuracy than CSF p-tau217 discriminating the two preclinical groups (A+T- and A+T+). In this scenario, no differences in diagnostic performance between CSF p-tau235 and p-tau231 were observed. Data information: Box-and-whisker plots show the median and the 25th and 75th percentiles.
Figure 2(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments. (B) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments. (D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated. (E) DNA-RNA hybrid metaplot distribution over γH2AX ChIP-seq peaks. Peak metaplot shows the mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC). (F) Venn diagram representing the overlap between γH2AX-positive genes in K562 cells (γH2AX ChIP-seq) and genes that specifically accumulate hybrids in control cells (top) or in DDX5-depleted cells (bottom).
Overview of Oxford and Nepal comparison.Figure 1(a) Overview of enteric fever cohorts used in this study (T1: Typhoid CHIM study 1; T2: Typhoid CHIM study 2; P1: Paratyphoid CHIM; 03NP: Nepali cohort. ST: S. Typhi; SPT: S. Paratyphi A; sEF: suspected Enteric Fever; D0: day of challenge which represents the control samples in the Oxford CHIM; CTRL: endemic community controls; nD7: day 7 after challenge in participants who stayed well in the CHIM; BC+: Blood-culture positive; BC-: Blood-culture negative; Dx: Diagnosis). (b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls. (c) Circular plot depicting the overlap of BTMs between enteric fever and nD7 samples from Oxford and Nepal. Tracks (from outer to inner): cohort and samples; BTM labels; direction of enrichment (blue: down; red: up; compared to healthy controls). Cords represent overlap of enrichment between given cohorts (red: overlap between P1-SPT and T1-ST; green: overlap between T1-nD7 and P1-nD7; blue: overlap of 03NP-ST with P1-SPT and T1-ST; purple: overlap of 03NP-SPT with P1-SPT and T1-ST; yellow: overlap between 03NP-SPT and 03NP-ST). (d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).