EMBO/sd-character-level-ner huggingface dataset:
Figure 2Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of microglia homeostatic (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12, Mef2c), phagocytic (Tyrobp, Trem2), and inflammatory (Il1β, Tnf, Ccl2, Mrc1, Arg1) markers were analyzed by qPCR. Bars represent mean of relative expression (% of saline; Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test; n=4). Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains. Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b+CD45int cells from saline or LPS-injected mice. Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia (n=4) and isolated bone marrow monocytes (n=2) by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test). Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).
Figure 3Selected regions from 1H-15N TROSY spectra of 2H,13C,15N-labelled R0RBR parkin bound to unlabelled pUb in the absence (black contours) and presence (blue contours) of one equivalent invisible 2H-labelled UbcH7-Ub. A large number of signals for residues including S145, N235, S255, E399, Q400, W403, E404 and D464 either shift or are absent in spectra with UbcH7-Ub. Details of the re-modelled region in parkin observed upon UbcH7-Ub binding. The figure shows the sidechains of residues in the RING0 (brown), RING1 (blue), tether (purple and RING2(Rcat) (pale green) interface that surround W403 (red) and are most affected by UbcH7-Ub binding.
Fig.Figure 4A) Elevated Plus Maze. Percentage of time spent in open arms and number of entries in open, closed and total (open+closed) arms. (n = 12 for time in open arm, n=24 for arm entries). B) Novel object recognition. Locomotor activity (distance in m) in the whole arena and percentage of time spent in the center during 15 min habituation. Locomotor activity during the acquisition and retention trials. Recognition index. (n = 24). C) Social recognition. Number of entries in the two side compartments during preference session (left), social preference (percentage of exploration of a congener vs an object) (center) and social memory (percentage of exploration of a novel vs familiar congener) (right). (n=24).
Figure 5Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870 Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685 RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h. Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).
Figure 1A-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4). For SPR-based experiments, (F) Gal-3 (density 650 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin. For SPR-based experiments, (G) Gal-3 CRD (density 1180 RU), were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin. For SPR-based experiments, (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin.
hTAPBPL-Ig ameliorates established EAE in mice.Figure 6C57BL/6 mice were immunized with 200 µg MOG35-55 emulsified in CFA and 500 ng of purified Bordetella pertussis toxin. When EAE symptoms occurred, the mice were injected i.p. with 25 µg hTAPBPL-Ig or control Ig protein 3 times per week. (A) Mean clinical scores. Deletion of Tregs partly reverses the effect of TAPBPL on EAE. Groups of EAE mice as in (A) were injected i.p. with anti-CD25 antibody. Mean clinical scores are shown. At the end of the studies, spleens and spinal cords were harvested and analyzed for the (B, D, F, H) percentages and (C, E, G, I) numbers of (B and C) CD4+ T cells and (D and E) CD4+CD25+FoxP3+ Tregs, the expression of CD69 by (F and G) CD4+ and (H and I) CD8+ T cells, the (J, L) percentages and (K, M) number of CD44hi CD62Llo effector memory and CD44lo CD62Lhi naïve (J and K) CD4+ and (L and M) CD8+ T cells in the spleens. The (N, P) percentages and (O, Q) numbers of CNS infiltrating (N, O) CD4+ T cells and (P, Q) CD4+CD25+FoxP3+ Tregs. Splenocytes from (B) were cultured with graded doses of MOG35-55 for 72 hours. [3H] thymidine (1 μCi/well) was added to the cultures 12 hours before harvest. T cell proliferation was measured by [3H] Thymidine incorporation. The data are expressed as mean + SD and representative of 3 independent experiments with n= 8/group. Significance was calculated by two‐tailed Student's t‐test except (R) that was analyzed by two‐way ANOVA with Tukey test. * P<0.05, hTAPBPL-Ig group was compared with control Ig group; ** P<0.05, hTAPBPL-Ig + anti-CD25 antibody group was compared with hTAPBPL-Ig + isotype antibody group.
Figure 3A. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.
Figure 6Figure 6Sedimentation analysis of salt-dependent H1-oligomer assembly.A Representative experiment showing the second moment sedimentation coefficients of the H1-oligomers as a function of MgCl2. The dashed line indicates the upper limit of measurable sedimentation coefficients (~106 S). The white symbol is intended to show that the sedimentation coefficient of the H1-oligomers in 5 mM MgCl2 is beyond the detectable limit.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).C The g(s*) profiles in 2.5 mM, 3 mM, and 3.5 mM from panel (B) are re-plotted on a smaller scale.
Figure 6a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL). b) Residues with CSPs greater than 1 stdv (HHARI residues 333, 337-342, 352-354, 363, 365, 371) are colored red in a cartoon (left) and surface (right) representation of HHARI RING2 (PDB 2m9y). The active site C357 is in shown in yellow. c) Sequence alignment of HHARI and Parkin RING2 domains. HHARI residues perturbed by Ub binding (panel a) are in red. Residues that decrease auto-ubiquitination activity when mutated in HHARIRBR are indicated with black boxes. d) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto-ubiquitination assays. Time points (10min) are visualized by western blotting for the GST-tag on HHARI. Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared. e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).
Figure 2(A) Visualization of the overlap of the top DF reactions between each pair of datasets analyzed using Fisher's exact tests (Methods). The dot size corresponds to the effect size of the overlap as measured by odds ratio, and the color corresponds to the negative log10 adjusted one-sided P value (grey means below 0.05). (B) A summary visualization of the metabolic pathway enrichment result for the top consistent DF reactions across the datasets, with more importance given to the various in vivo patient datasets (Methods). Y-axis represents the odds ratio of enrichment, the dot color corresponds to the adjusted P value from Fisher's exact tests, and dot size corresponds to the number of enriched reactions within each pathway. Half-dots plotted on the top border line correspond to infinity odds ratio values. The pathways on the X-axis are ordered by P value and only those with FDR<0.1 are shown. (C-E) Visualization of the relatively consistent DF patterns in selected enriched pathways. The DF results are based on metabolic modeling using the human GEM Recon 3D (Brunk et al. 2018), but for clear visualization, the metabolic network graphs are based on the human GEM Recon 1 (Duarte et al. 2007) to reduce the number of metabolites and reactions displayed (Methods). Metabolites are represented by nodes, reactions are represented by directed (hyper) edges, with edge direction corresponding to the consensus reaction direction and edge color corresponding to the consensus DF direction across datasets (Methods). Red and blue colors correspond to increased and decreased fluxes, respectively; grey color corresponds to reactions not showing consistent DF changes across datasets, some of such reactions are not shown to increase clarity. (C) Pyrimidine synthesis. (D) Inositol phosphate metabolism. (E) Fatty acid synthesis. Metabolites are labeled by their names in (C) or IDs in (D,E), with suffixes denoting their cellular compartments: [c] cytosol; [m] mitochondria. The mapping between the IDs and metabolite names in (D,E) is given in Table EV5C.
Brain histopathological analysis in the brain of representative mice from different experimental groups.Figure 3A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C (N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.
Skin fibroblasts derived from HGPS patients display deficiency in ciliogenesis.Figure 1A Normal human fibroblasts and HGPS fibroblasts were serum-starved for 48 h and stained for progerin (red), acetylated tubulin (green) and DNA (blue). The representative images show that the cell with high progerin (inset 2), but not low progerin (inset 1), displays defective cilia formation. Scale bars, 20 μm or 2 μm (in the magnified images). B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software (n ≥ 148). The dashed line indicates the background level of progerin. A.U., arbitrary units. The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 m. D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets). E,F The cells were subjected to immunofluorescence stain as in (D). The percentage of the cells with cilia in the total counted cells (E, n ≥ 50) and the cilia length (F, n ≥ 50) were measured.
Gemc1 deficient animals are growth impaired and develop hydrocephaly.Figure 1A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2. B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test. C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm. D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2). E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted. F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.
Figure 7Under normal conditions, TCF11/Nrf1 is a short-lived ER-membrane (endoplasmic reticulum) resident protein, which is rapidly subjected to proteasome-mediated degradation following retro-translocation to the cytosol by ER-associated degradation machinery (ERAD). In case of proteasome dysfunction (i.e. proteotoxic stress), the half-life of TCF11/Nrf1 is prolonged and become then a substrate for the NGLY1 and DDI2, thereby giving rise to a C-terminal cleaved fragment that enters into the nucleus. After nuclear translocation, cleaved TCF11/Nrf1 associates with Maf and promotes the expression of proteasomes genes, so that protein homeostasis can be restored. In patients carrying the c.1127+337A>G homozygous PSMC3 variation, defective proteasomes promote a constitutive activation of TCF11/Nrf1 thereby resulting in increased assembly of newly synthetized non-functional proteasomes, which in turn activate TCF11/Nrf1 again. Over-activation of TCF11/Nrf1 results in pathway exhaustion thus rendering patient cells incapable of responding to further proteotoxic stress.
Figure 5(A) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.
Figure 2A) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01) B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels. C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted. D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated. E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001). F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated. G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).
Figure 6A Schematic representation of the DNA strand exchange assay with ssDNA. B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM. E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM. F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM. G A scheme of streptavidin pulldown assay performed in (H, I). H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM). I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.
Figure 2SILAC analysis of mitochondrial proteome of ∆IV and ρ0 cells. Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV2). 568 high fidelity mitochondrial proteins (gene/protein list in Table EV1A) in ∆IV cells and 572 such proteins in ρ0 cells were quantitatively measured. Significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated. Western blot analysis of representative mitochondrial proteins endogenously tagged with HA. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Polysome fractionation analysis of OXPHOS mutants. Polysomes were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.