EMBO/sd-character-level-ner huggingface dataset:
Single cell durotaxis is under the regulation by Golgi-microtubules Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs.Figure 2Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs. Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels. Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH was used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels. Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition. Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels. Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity. Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.
Figure 6(A) Percentage of apoptotic cells in WT and CDC25aKO mESC after treatment with DMSO (CONT) and 25 μM P22077 (USP7i) for 16 hrs. This experiment was repeated 4 times using 3 different CDC25A clones. (* p<0.05, t-test). Bars represent mean values ± SEM. (B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM. (C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).
Transcriptomic changes are associate with innate immune responses and redox-status of stimulated macrophages.Figure 5Heat-map indicating DEG patterns comparing SC-conv and SC-naïve macrophages stimulated with the S-protein. Z-score is indicated in a color score. BP enrichment analysis, based on RNA-Seq data, showing activated signaling pathways upon S-protein stimulation in SC-conv macrophages compared to SC-naïve macrophages. Padj values are indicated (color code) and circle size corresponds to the number of DEGs contributing to the pathways (count). Heat-map indicating DEG patterns comparing SC-conv and SC-naïve macrophages without any stimulation. Z-score is indicated in a color score. Volcano plot showing DEGs in unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to macrophages from SARS-CoV-2 naïve individuals (SC-naïve). Negative log10 padj values are plotted against the log2 fold-change. Selected genes of interest are labeled. IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.
Organization of the SARS-CoV-2 S glycan shield.Figure 4. Organization of the SARS-CoV-2 S glycan shield.A-B. Two orthogonal views of the SARS-CoV-2 S structure rendered as a surface with glycans rendered as dark blue spheres.
Figure 7A schematic model of epigenetic changes during TNF-α-induced EC-activation.
Figure 2(A) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type, 5xFAD and p75NTR knock-out (KO) mice. Scale bar, 20μm. Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading. (C) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH. (D) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice of the indicated ages. Scale bar, 400μm. (E) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N=5 mice per group). *, P<0.05 and **, P<0.01 vs 5XFAD. #, P<0.05 vs 5XFAD/KO (one-way ANOVA followed by post hoc test). (F) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N=4 mice per group; *, P<0.05 KO vs. knock-in genotypes. ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extraction ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet, ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.
Figure 1(A) Three main splice variants a, b3 and c have been confirmed for human USH1C. According to an Entropy plot (upper panel), the coding regions appear highly conserved among mammalian species with some variability in inter-domain segments, but also within the b3-specific PST domain. (B) Homology: A high degree of conservation occurs at the nucleotide level, including exons but also intronic elements. Regulation: High degrees of conservation often correlate with strong indication for transcriptional regulation properties. Thickness and color intensity are according to matches to 6 prediction tools (DNase sensitivity, ChIP-Chip, FANTOM, GeneHancer, FAIRE, PreMod). Coverage: Most of the USH1C gene and its surrounding regions are consistently abundant in the examined species (grey). Gaps (white) appear in all species, but mostly remain small (<500bp). Few intronic areas are consistently affected by the introduction of repetitive elements (triangles, with approximate length in kb). Rodents show a high evolutionary turnover, with large gaps or inserts >1kb in extended introns and adjacent intergenic regions of USH1C. Significant proportions of USH1C are indicated as CTCF/cohesion-binding regions that appear relevant for chromosomal interactions (orange blocks). (C) Exon 2 and its surrounding intronic sequences constitute a consistent block in the alignment with high degree of sequence conservation, strong evidence for regulatory purposes and a lack of repetitive element insertion. For this reason the entire segment was humanized (magenta), including the causative c.C91T / p.R31X mutation as well as three patient specific SNPs (pins). (D) All examined species show identical amino acid sequence on the protein level and high degrees of conservation at the intronic borders on the nucleotide level. The yellow box indicates R31, the amino acid that is mutated in R31X patients.
Generation and characterization of chemotherapy-naïve and -exposed patient-derived models to investigate treatment-imposed reprogramming.Figure 1A Clinical history of a PDAC patient receiving perioperative FOLFIRINOX including follow-up. B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX. C H&E staining of FNB and the resection specimen. Scale bars represent 50 µm. D Phase-contrast images of organoids (upper panel) and respective 2D cultures (isolated from the biopsy before FOLFIRINOX (ID188) or after the neoadjuvant therapy from the resected cancer (ID211). Scale bar represents 200µm. E IF staining of the actin filaments (red) and nuclei (blue) of organoids in the left panel and H&E staining of embedded and sectioned organoids in the right panel (ID188 p30, ID211 p30). F Normalized expression (log2 scale) for gene pairs evaluated during Purity independent subtyping of tumors (PurIST). Normalized expression (log2 scale), gene 1 = basal-like, gene 2 = classical. G Gene Set Enrichment Analysis (GSEA) of pre-(ID188, p7) and post-treatment (ID211, p6) PDO lines. Normalized enrichment score (NES) and the adjusted p-value is depicted. H Whole exome sequencing-based copy number profiles for ID188 (p7) and ID211 (p6) organoids. I Allele frequencies of miss- and nonsense SNVs and Indels which are shared between both organoid lines (purple), private to ID188 (red) or private to ID211 (blue).
Figure 6(A-D) 2D 1H-15N-HSQC spectra of the TRIM25 RING domain at a concentration of 125 µM (A) and 570 µM (B), the monomeric TRIM32 RINGcore (C) and TRIM32-RING dimer (D). (E) Isotropic rotational correlation times of the different TRIM25 and TRIM32 RING constructs, obtained from the relaxation analysis of resonances in the corresponding 2D 1H-15N HSQC spectra (dark grey) or calculated from available structures by HYDRONMR (light grey). (F) Scattering profiles of RING constructs (left) and their normalised pair-distribution functions P(r) (middle). The right hand panel shows low-resolution ab initio models derived from the SAXS data analysis. (G) Scattering profiles, their normalised pair-distribution functions P(r) and low-resolution ab initio models for RING and B-box containing constructs. (H) Scattering profiles, their normalised pair-distribution functions P(r) and low-resolution ab initio models for the RBCC domains of TRIM25 and TRIM32. The curves and envelopes are reported using the same colour scheme in all respective panels and the envelopes for the constructs are drawn to scale.
FKK6 does not abrogate Dextran Sulfate Sodium induced colitis in pxr -/- mice.Figure 6As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown. As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown. As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown. As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown. As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.
CatD−/−hippocampal neurons as a model system for autophagic vacuole accumulation.Figure 8.(A) Intracellular sorting and maturation of catD. Wild-type and PS1−/− neurons were metabolically labeled and chased for the indicated time periods. Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46-kD intermediate vs. 52-kD precursor (mean ± SEM, n = 3). (B-D) TLN localizes to autophagic vacuoles of catD−/−hippocampal neurons. Lysotracker staining revealed sparsely distributed small-sized organelles in wild-type and PS1−/− neurons in contrast to catD−/−neurons where high numbers of large Lysotracker-positive organelles were found (B, arrowheads). Although TLN accumulations in PS1−/− were fully negative for Lysotracker, some acidic organelles tended to closely associate (C, arrowheads, middle). In catD−/− neurons, TLN was clearly detected in the large acidic organelles (C, bottom). Bars, 20 μm. (D) At the immuno-EM level, the large acidic organelles seen in catD−/−neurons represent dense autophagic vacuole-like structures (star) that label positive for TLN (10-nm gold). M, mitochondrion. Bars, 200 nm.
Figure 5Side-by-side comparison of CNFY and nigritoxin. Nigritoxin is a toxin of crustaceans and insects. The translocation domain of nigritoxin (PDB entry 5M41, (Labreuche et al, 2017)) and domain D1 of CNFY show partial structural similarity (highlighted areas). This similarity was identified with DALI (Holm & Rosenström, 2010) which was also used to align both structures. The ART-like domain D4 of CNFY. Essential residues of canonical ARTs are not conserved in CNFY (RSE-ARTs exemplified by C. perfringens iota toxin, PDB entry 4H03 (Tsurumura et al, 2013); HYE-ARTs exemplified by P. aeruginosa ExoA, PDB entry 2ZIT (Jørgensen et al, 2008); carbon atoms of NAD+ shown in black). The deamidase domain D5 of CNFY. C866 and H881 form a conserved catalytic dyad also found in the deamidase domain of E. coli CNF1 (PDB entry 1HQ0, (Buetow et al, 2001)) and Burholderia pseudomallei lethal factor BLF1 (PDB entry 3TU8, (Cruz-Migoni et al, 2011)).
| Characterization of viral siRNAs in murine EVs.Figure 2A, Model of sample preparation for small RNA deep sequencing. B, Size distribution of the purified EVs analyzed by NTA. C, TEM images of purified EVs. Scale bar: 100 nm. D, Western blotting of HSP70, tsg 101, CD81, CD63, and CD9 in EVs, whole blood, and blood clot derived from suckling mice infected with DMEM or NoVΔB2. Staining of β-actin was used as a loading control. E, Size distribution of total sequenced small RNAs in the libraries constructed from EVs of DMEM challenged BALB/c suckling mice, and EVs, whole blood, blood clot of BALB/c suckling mice infected with NoVΔB2 at 3 dpi. F, G, Size distribution of 18- to 28-nt vsiRNAs (F) and genomic coverage depth of each nucleotide position by 21- to 23-nt vsiRNAs (G) were analyzed from BALB/c suckling mice libraries infected by NoVΔB2 at 3 dpi. H, Pie charts of the top ten most abundant miRNAs and vsiRNAs in hindlimb, EVs, whole blood, blood clot from BALB/c suckling mice infected by NoVΔB2 at 3 dpi. Red charts indicate 18- to 28-nt vsiRNAs abundance. The percentage of miRNA reads was shown above. The abundance of total miRNAs and 18- to 28-nt vsiRNAs reads was set as 1.
a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation.figf5(a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021. (b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039. (d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.
Figure 3A. Two pFastBac1 plasmids containing PTENα with a C-terminal His-tag with or without CUG816>CUC mutation was used for in vitro purification and mass spectrometry sequencing. The initiation codon of canonical PTEN (AUG1032) was mutated to AUA and the initiation codons of PTENα (CUG513) and PTENβ (AUU594) were mutated to CUC to avoid co-purification of PTEN, PTENα, and PTENβ with PTENε. B. His-selected affinity purification of PTENε. Sf9-expressed His-PTENε was purified using nickel affinity chromatography. Combined fractions were separated by SDS-PAGE. The protein band ranked third by molecular weight in the M2 lane can be eliminated by the CUG816>CUC mutation (M1 lane vs. M2 lane), indicating it is PTENε. Mass spectrometry analysis of the band of PTENε protein revealed five segments of peptide that match the N-terminal extended sequence of PTENε, including the most proximal N-terminal peptide of PTENε, MAAEEKQAQSLQPSSSR. C. The MS/MS spectrum of the peptide (MAAEEKQAQSLQPSSSR) that matches the N-terminal sequence of PTENε. D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody E. The MS/MS spectrum of the peptide (MAAEEKQAQSLQPSSSR) of endogenous PTENε in mice that matches the N-terminal sequence of PTENε. Protein lysates of PtenFLAG knock-in liver tissues were subjected to immunoprecipitation with anti-FLAG M2 agarose. The bound proteins were separated with SDS-PAGE, and gel slices of the band ranked fifth by molecular weight were analyzed by mass spectrometry.
Multi-PAS genes with low APA diversity have higher gene expression levels.Figure 3Genes with higher dominant PAS usage (DPU) (A) or with lower APA Shannon index (B) express at higher levels (liver shown as example). The number of genes in each group is indicated on top. Mann-Whitney U test significance levels are indicated for neighboring groups: *P < 0.05; **P < 0.01; ***P < 0.001. The boxplots show the Q1 to Q3 quartile values (the box limits), the median (the horizontal green lines) and values within the 1.5 * IQR (the whiskers). Spearman correlations between gene expression level and dominant PAS usage (DPU) in each tissue for all genes (blue), genes with DPU equal to or above 90% and genes with DPU less than 90%. Genes with DPU less than 90% exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue. An empty square indicates that the P value of Spearman's rho is above 0.01. (ESC: embryonic stem cells, CER: cerebellum, COR: cerebral cortex, HEA: heart, KID: kidney, LIV: liver, LUN: lung, MUS: muscle, SPL: spleen.) Positive Spearman correlation between APA Shannon index and replicate variability in liver.
Figure 9A Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.C Average chromatin accessibility (normalized read counts) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=3) and R6/1 (N=3) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. Wilcoxon Mann Whitney test was used for statistical analysis. Exact P values are reported in Appendix Table S3. D Venn diagram showing the total number of genes found in common and wild-type (WT) specific LADs regions (left). Bar graph showing the number of up- and down-regulated genes found in common and WT specific LADs regions filtering only according to adjusted P-value (adjusted P-value <0.001) or additionally with fold change (adjusted P-value <0.001, |FC| > 2) obtained from deseq2 differential expression analysis (see methods). E UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, lamin B1 ChIP-seq (log(Lb1 ChIP/Input)) for WT (N=3) and R6/1 (N=3) mice hippocampus, hippocampal H3K9ac, CTCF and H3K9me3 for WT mice hippocampus in Fos locus (left). Enhancer regions (E1-E5) are indicated with arrows. Boxplot of lamin B1 enrichment (log10(Lb1 ChIP/Input)) for regions with unchanged, decreased (closed in R6/1) and increased (opened in R6/1) chromatin accessibility in R6/1 (N=3) mice hippocampus. * P < 0.05 as compared to WT (N=3) mice. (Wilcoxon Mann Whitney test). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.