EMBO/sd-character-level-ner huggingface dataset:
Aged lungs display non-resolving pulmonary immunopathology following influenza infection.Figure 1.infection.Young (2 month) or aged (22 month) C57BL/6 mice were infected or not with PR8 virus. F-H, anti-CD45 was injected intravenously (i.v.) to label circulating white blood cells prior to sacrifice. A. Schematics of experimental procedure. B. Percent of host survival following influenza infection in young or aged mice. C. Percent of pre-infection body weight following influenza infection in young or aged mice. D. Airway viral titers were determined through plaque forming unit (pfu) assay of BAL fluid at 9, 15, and 30 d.p.i. E. Lung histopathology by H & E staining at 0, 9, 30, and 60 d.p.i. F. Percent of left lung lobe parenchyma infiltrated by white blood cells was quantitated by Image J analysis. G, H. Lung cells were stained for monocytes (MNC: CD64- Siglec-F- CD11c- MHCII- CD11bhi) and neutrophils (Neu: Ly6G+) which were enumerated in the lung and divided into circulating (Cir) or parenchymal (Par) following intravenous labeling of immune cells at 9 and 60 d.p.i. G. Representative plots. H. Cell numbers of circulating and parenchymal monocytes and neutrophils at 9 and 60 d.p.i. d.p.i. I. 530 transcripts were examined at the endogenous mRNA levels by nanostring from young or aged lungs prior to (day 0), or 60 d.p.i. and displayed as a heatmap (purple = relatively low expression, orange = relatively high expression) following log2 transformation of raw expression data above detection threshold. Venn diagrams displaying data in both number of genes and % of total genes examined for the ratios infected versus not by age (top right panel) or aged to young by day post-infection (bottom right panel) from nanostring data. Groups from B & C are n=10 young and n=14 aged mice (2 experiments pooled). D is 6 BAL samples per group (2 experiments pooled) for days 9 & 15 and 3 each for day 30. D, E, G, and I are representatives of 2-4 experiments each; H is 1-2 independently significant replicates pooled. Scale bars on histology figures are 600 μm. * p<0.05 Student's two-tailed t-test with unequal variance.
Dynamics of kinase activities.Figure 4a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1). b t-SNE plot displaying local enrichment of phospho-sites corresponding to known substrates associated with the specified kinase. Dot transparency is scaled according to the P-value corresponding to the local enrichment of the annotation term c Illustration of the kinase-substrate interconnections characterizing the AMPK, AKT and ERK signaling pathways. Substrate phospho-sites dynamics are displayed and color-coded according to their clusters, and the corresponding scaled kinetics are presented on the right. d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control. e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.
Figure 1A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001. Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported. Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.
Figure 7(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μm IF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm. IF analysis of P-IκBα and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm. (D) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed. (E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm
FKK6 abrogates Dextran Sulfate Sodium induced colitis in mice expressing the human PXR gene.Figure 5After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. A, % weight loss from baseline (day 1 vs day 10) After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. B, colon length (cm) After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation score After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. D, Fecal lipocalin 2 (pg/ml) After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. E, serum FITC-dextran (μg/ml). After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate).
Mec1-S1991 phosphorylation limits RNA polymerase transcription during replication stress.Figure 9Upon HU-induced replication stress, Mec1ATR and Rad53 are activated through phosphorylation, including phosphorylation at Mec1-S1991. Mec1-S1991 phosphorylation is not required to activate the downstream checkpoint effector kinase Rad53, yet it promotes the attenuation of RNAPII and RNAPIII-mediated transcription during replication stress, acting on a variety of transcription controlling factors. Proteasome-mediated RNAPII degradation during replication stress requires a functional Cul3-Elc1 ubiquitin ligase. RNAPIII is evicted in a Mec1-dependent manner but not degraded. Mec1-induced RNAPII removal from chromatin allows the release of highly transcribed genes from nuclear pore under HU stress, while Rad53 is thought to act directly on the nuclear pores complex (NPC).
Figure 6A Typical growth curves of control and ad-FADDmice maintained on a SD (left panel) or a HFD (right panel) (n = 8 for each genotype). Data are expressed as mean ± SEM. *P = 0.0435, **P =0.0371, ^P = 0.0217, #P = 0.0236, ##P = 0.0158, ###P = 0.0113 (Student's t-test). B Body composition by NMR to show the fat mass and fat mass ratio in control and ad-FADDmice after 10 weeks of HFD (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.0027, **P = 0.0012 (Student's t-test). C Food intake in 15-week-old male control and ad-FADDmice fed a HFD (n = 8 for each genotype). D and E Representative pictures of hematoxylin and eosin-stained sections of epididymalWAT from control and ad-FADDmice at 10 weeks (D) and after 10 weeks of an HFD (E) are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.F The average cross-sectional area of adipocytes in epididymalWAT is shown in the bar graph (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.002 (Student's t-test). NS, not statistically significant.G The distribution of adipocyte cross-sectional area in epididymalWAT of HFD-fed control or ad-FADDmice is shown in the bar graph (n = 6 for each genotype). H Representative pictures of hematoxylin and eosin-stained sections of brown adipose tissue from HFD-fed control and ad-FADDmice are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.
Figure 1a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min. b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations. c, Coefficient of variation (CV) of phase durations. d, Percentage of the total variation in cell cycle duration contributed by individual phases. e, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.
Figure 2A. Experimental scheme to analyze the effects of LincGET depletion on embryonic development. phCG, post human chorionic gonadotropin injection. LNA, locked nucleic acid. IF, Immunofluorescence. LNA was injected at phCG 25 hours. For BrdU staining, BrdU was added at phCG 30 hours. For EU staining, EU was added at phCG 40 hours. IF, including detection of BrdU and EU, was performed at phCG 48 hours. B. LNA efficiently mediated LincGET knockdown. LNA was injected at phCG 25 hours and embryos were collected at phCG 48 hours at late 2-cell stage for TM-qPCR analysis. The error bars represent s.e.m. About 50 embryos of each stage were used and three experimental replicates were performed. C. LincGET depleted embryos arrest at the 2-cell stage. LNA was injected at phCG 25 hours and photos were taken at phCG 114 hours at the late blastocyst stage. Embryos injected with Control-LNA can develop to the late blastocyst stage, while LincGET depleted embryos arrest at the 2-cell stage. Scale bar, 100 μm. At least three experimental replicates were performed for each LNA injection (Table 1). D. LincGET depletion results in developmental arrest at the G2 phase of 2-cell stage without affecting DNA integrity and replication. We use BrdU to visualize S and G2 phases, CAF-1 to visualize S phase, and PI to visualize the M phase. Cyclin B1 is a marker of G2 stage and H2A.X is a marker of DNA damage. Aphidicolin-treated embryos arrest at the S phase without DNA replication. LNA was injected at phCG 25 hours and embryos were collected at phCG 48 hours at the late 2-cell stage for IF analysis. Scale bar, 50 μm. Three experimental replicates were performed and about 15 embryos were used in each group. E. EU staining indicates the normal major ZGA process after LincGET depletion. EU was added to the culture medium at phCG 40 hours and EU signals were detected at phCG 48 hours. Scale bar, 50 μm. Three experimental replicates were performed and about 15 embryos were used in each group. F. Genes related to major ZGA initiation, like Hsc70, Hsp70, Erv4, Eif1a, and Zscan4, are expressed normally in LincGET depleted L2C embryos compared to that in Control embryos. Embryos injected with LNA were collected at phCG 48 hours at the late 2-cell stage for TM-qPCR analysis. The error bars represent s.e.m. About 100 embryos were used for each group and three experimental replicates were performed. n.s., p > 0.05. G. The transcription of pericentric satellites is normal after LincGET depletion. Embryos injected with LNA were collected at phCG 50 hours at the late 2-cell stage for TM-qPCR analysis. The error bars represent s.e.m. About 50 embryos were used for each group and three experimental replicates were performed. n.s., p > 0.05. H. DNA-FISH analysis of major transcripts shows that the pericentric domain reorganization towards chromocenters is not affected by LincGET depletion. Scale bar, 50 μm. Three experimental replicates were performed and about 15 embryos were used in each group.
Figure 4A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting. B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm. C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments. C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.
Figure 3. Schematic representation of zebrafish Endouc protein and its internal deletion mutants. . Histograms presenting the relative luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop-luc, phRG-TK, and each of the expression plasmids as indicated followed by analysis of luc activity at 96 hpf. Embryos microinjected with pCS2 vector during normal condition served as a control group (non-stress; blank column), while the microinjected embryos treated with 40℃ at 72 hpf for one hour were the heat-shocked group (solid column). The relative luc activity of microinjected embryos shown on the right was normalized by the amount of RNA in the injected embryos shown on the left. The relative luc activity is represented by the fold increase in Fluc/Rluc ratio over that obtained from control group. The luc activity mediated by the huORFchop element was measured by a dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR. . Histograms presented the luc activity obtained from HEK 293T cells co-transfected with puORFchop-luc, phRG-TK, and each one of the plasmids as indicated and treated with either DMSO (control group) or Thapsigargin (TH; stress group) followed by analysis of luc activity. The luc activity of cells transfected with pCS2 vector and kept at normal condition served as a control group. The relative luc activity of transfected cells shown on the right was normalized by the amount of RNA in the transfected cells shown on the left. The relative luc activity is represented by the fold increase of Fluc/Rluc ratio over that obtained from pCS2-transfected control group normalized as 1. The luc activity mediated by the huORFchop transcript was measured by the dual-luciferase assay, and its corresponding huORFchop-luc transcript was measured by RT-qPCR. D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated, followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.
Figure 3A Viral bait-bait correlation. B The overlap of proteins in each cluster is shown in a Venn diagram. C A bar diagram of the enriched pathways associated with the clusters shown and ranked by p-value. D Host receptor bait-bait correlation; The N- and C-terminal tags on the same bait are indicated. E The bar chart represents the association of cluster 1with specific pathways.
CACNA1A localizes to the lysosomes.Fig 5A. CACNA1A co-localizes with LAMP1 in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm. C. Quantification of LAMP1 positive organelles that are also CACNA1A positive. Data are presented as means ± SD. (ns: not significant). D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.
Proposed role of IL-1 in neuroinflammation in MPSIIIA.Figure 7 MPSIIIA GAG primes a TLR4 mediated innate inflammatory response, resulting in production of intracellular pro-IL-1β (blue arrows). This step can potentially also be achieved by delivery of cholesterol crystals, amyloid beta deposition or high levels of cellular ATP. Cholesterol, amyloid beta and ATP are each able to activate the formation of the NLRP3 inflammasome, usually via lysosomal destabilisation, inducing cleavage of pro- I-1β by caspase 1 and secretion of IL-1β (red arrows), but only if cells are first primed. Extracellular IL-1β is able to bind IL-1R1 expressed on astrocytes and neurons, leading to astrocyte activation and changes in neuronal activity. Inhibition of IL-1 signalling reduces potentiation of microglial and astrocytic responses, presumably by reducing astrocyte activation, which leads to prevention of behavioural and cognitive changes in MPSIIIA
Figure 2(A) Schematic of the chemical mutagenesis approach. (B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed. (C) Ak2 mutations identified in the resistant clones analyzed in (B). (D) Protein sequence alignment of mouse and human AK2. Black stars indicate residues involved in nucleotide binding. Black boxes mark sites mutated in the identified cell clones with alternative residues indicated above. (E) Structure of human AK2 in complex with bis(adenosine)-5'-tetraphosphate (pdb:2c9y). Ak2 mutations identified in the remdesivir resistance screen are highlighted in magenta. (F) Schematic of remdesivir cellular uptake and intracellular activation. Modified from Eastman et al. (Eastman et al., 2020).
Figure 5A. View of ALK (grey) bound to alectinib (magenta) centred on the active site highlighting the αC-helix (cyan), αAL-helix (orange) and Lys-Glu salt bridge (blue-red), based on the crystal structure (PDB:3AOX). B. Magnified views of ALK, in the vicinity of the Lys-Glu salt-bridge, bound to inhibitors: alectinib (ALE, PDB:3AOX), ceritinib (CER, PDB:4MKC) and lorlatinib (LOR, PDB:4CLI). The cyan wiremesh is a 2Fo-Fc electron density map. The black line marks the interatomic distance between terminal atoms in the Lys-Glu salt-bridge. C. Schematic illustration of the regulatory features of the ALK kinase domain and the effects of mutations. The side chains of key residues are shown as sticks. The αC-helix of inactive ALK interacts with the JM domain through hydrophobic contacts and the αAL-helix through polar contacts. The interactions between αC-helix, the JM domain and the A-loop are destabilised by mutations such as F1174L, R1275Q that drive the activation of ALK. The Lys-Glu salt-bridge can be disrupted by an inactivating mutation (K1150M). D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.
Figure 5A Time course expression profiles of CASPLs in response to Pst DC3000 (AvrRpm1) treatment. Analysis was based on the Genevestigator database (http://www.genevestigator.ethz.ch/). The darker color corresponds to stronger expression. ND, not determined. Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. ata are shown as means ± SD (n = 4; 3-9 leaves each). ata information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); dpi, days post-inoculation. C Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). Data information: AvrRpm1, Pst DC3000 (AvrRpm1) dpi, days post-inoculation. D Quantification of leaves (n ≥ 30) with spreading cell death after Pst DC3000 (AvrRpm1) inoculation. dpi, days post-inoculation. E Measurements of Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) growth. Data are shown as means ± SD (n = 3). Data information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation. F Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in caspl mutant plants. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm.