EMBO/sd-character-level-ner huggingface dataset:
Reduced fibrotic remodeling in losartan-treated C7-deficient forepaws.Figure 4C7-hypomorphic mice were treated with losartan for 7 weeks, and the forepaws of age-matched untreated, losartan-treated, and wild-type mice were subjected to immunofluorescence staining with antibodies to fibrosis markers (A, B) and to picrosirius red staining (C).A, B Tenascin-C (red) (A), fibronectin (green) (B). The nuclei were visualized with DAPI (blue). Images acquired with a 20× objective, scale bar = 100 μm. Note that losartan did not completely abolish the staining, but effectively limited fibrosis to the site of initial tissue damage, that is, adjacent to the dermal-epidermal blistering (denoted by white asterisks) in C7-hypomorphic skin. C7-hypomorphic mice were treated with losartan for 7 weeks, and the forepaws of age-matched untreated, losartan-treated, and wild-type mice were subjected to immunofluorescence staining with antibodies to fibrosis markers (A, B) and to picrosirius red staining (C). C Picrosirius red staining and visualization of the collagen fibers under cross polarizing light. Under this light, thin fibers appear green and thick rigid collagen bundles orange-red. The staining revealed significantly reduced collagen fiber size in losartan-treated skin, indicating softer tissue similar to wild-type skin. Below, the bar graph shows quantification of picrosirius red-positive areas, n ≥ 19 areas quantified, values represent mean ± S.E.M. Unpaired t-test with Welch's correction, ***P-value wild-type vs. C7-hypomorph < 0.0001; ***P-value C7-hypomorph + losartan vs. C7-hypomorph = 0.0004; *P-value wild-type vs. C7-hypomorph + losartan = 0.0189. Images acquired with a 20× objective, scale bar = 50 μm. C7-hypomorphic mice were treated with losartan for 7 weeks, and the forepaws of age-matched untreated, losartan-treated, and wild-type mice were subjected to immunofluorescence staining with antibodies to fibrosis markers (A, B) and to picrosirius red staining (C). D Immunofluorescence staining of forepaws as above with an antibody to αSma (red). αSma is present both around blood vessels and in myofibroblasts. Note the increase of αSma+ myofibroblasts in C7-hypomorphic paws and reduced number of αSma+ cells in losartan-treated C7-hypomorphic forepaws. Images acquired with a 20× objective, scale bar = 100 μm.
Figure 3A Immunoblotting of subcellular fractions from serum-starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria associated-ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker. B Immunoblotting of subcellular fractions from rat heart. C Immunoblotting of subcellular fractions from rat kidney. D Electron micrographs showing subcellular localization of Nox4 in wild-type (WT) and Nox4KO mouse hearts, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). hiPSC-CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrow heads show peri-mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M=mitochondria, ER=endoplasmic reticulum, T=T-tubule. Scale bars: 100 nm.
Figure 2A-P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines. Q Tabular summary of binding affinity (KD), association (kon) and dissociation constant (koff) determined for the individual RBD variants.
Figure 1Diagram depicting CRISPR/cas9-mediated deletion of the full coding sequence of ap2-o3 in the 17XNL and ap2-o3::6HA strains respectively, generating the Δap2-o3 and ap2-o3::6HA;Δap2-o3 mutants. Immunoblot analyses the AP2-O3 expression in gametocytes of the ap2-o3::6HA and ap2-o3::6HA;Δap2-o3 strains. BiP was used as the loading control. Female and male gametocytes formation in mouse. Gametocytes were counted via Giemsa staining of thin blood smears. Gametocytemia was calculated as the ratio of male or female gametocytes over parasitized erythrocytes. Representative images of purified female and male gametocytes after Giemsa staining. Ookinete maturation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes. Number of ookinetes in the mosquito midguts. The midguts were dissected at 8 hour post blood-feeding and stained with P28 antibody to visualize the crescent-shaped mature ookinete. n represents the number of mosquitos dissected. Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence. Right panels are the stained midgut oocysts. Salivary gland sporozoite counts in the mosquitoes at day 14 post blood-feeding. Diagram of CRISPR/cas9-mediated gene complementation in the Δap2-o3 mutant. The coding sequence of ap2-o3 from P. yoelii and P. falciparum were tagged with a quadruple Myc epitope (4Myc) and introduced back to the ap2-o3 locus, generating the Com1 and Com2 strains. Western blot of the AP2-O3 expression in gametocytes of the Δap2-o3, Com1 and Com2. Protein bands with expected molecular weight (arrows) and an unspecific band (star) were shown. Co-staining of AP2-O3 and α-Tubulin (male gametocyte specific) in gametocytes of Com1 and Com2 strains. Scale bars = 5 μm. Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes. Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.
Figure 1(A) DNA sequence analysis of SLC25A46 cDNA showing a homozygous c.425C>T mutation in the subjectfibroblasts compared to the control. (B) Schematic representation of SLC25A46 protein and position of the mutated p.Thr142Ile amino acid. (C) BN-PAGE analyses of OXPHOS mitochondrial complexes in fibroblasts from control and subject, overexpressing an HA-tagged version of wild-type SLC25A46. Each of the five OXPHOS complexes (I-V) was visualized with a subunit-specific antibody that recognizes the native complex as follows: COI (NDUFA9), COII (SDHA), COIII (UQCRC1), COIV (COX4), COV (ATP5A1). Overexposure of the blot shows a defect in assembled complex IV (asterisk) and some free unassembled COX4 subunit in the subject (arrowhead). Complex II was used as a loading control. (D) Immunoblot analysis of the same samples as in (B) for expression of SLC25A46 and individual structural subunits of the five OXPHOS complexes. The 70 kDa subunit of complex II (SDHA) was used as a loading control. (E) Seahorse analysis of oxygen consumption rate in fibroblasts from control compared to subject fibroblasts with or without expression of an EGFP-tagged version of wild-type SLC25A46 (top panel), and in fibroblasts from control compared to those in which SLC25A46 is suppressed by a specific siRNA (bottom panel). n=3, error bars represent mean +/- SEM and p-values were calculated using a t-test. (F) Polarographic analysis of oxygen consumption in permeabilized fibroblasts from control and subject. Left panel. State 1: no exogenous substrates; state 2: addition of glutamate and malate (GM); state 3 (GM): addition of ADP; state 3 (Succ): addition of succinate; Oligo: inhibition of ATP synthase by oligomycin. Serial addition of 1 to 4 µM CCCP measuring maximal uncoupled respiration. (control n=4 vs subject n=5). Right panel: Complex IV driven O2 consumption was measured in presence of antimycin A, TMPD and ascorbate. (control n=4 vs subjectn=5).
Fig.Figure 2(A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented. (B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.
Figure 4Schematic of inferred topology of neuropeptidergic signalling axes between SCN neuronal sub-populations in circadian day. Top: clusters are annotated in the overview t-SNE for circadian day (A.) and night (B.), respectively. Below: the identified SCN subpopulations are depicted as nodes of each neuropeptide-receptor network, connected by edges that are weighted (shown as differential line thickness) based on the average count of the respective cellular gene expression for each ligand and receptor. The nodes of the inferred topology networks are colour-coded to match those of the overview t-SNE plots. Nodes further highlighted in yellow mark ligand-enriched source clusters. The edges are colour-coded for each neuropeptide-receptor signalling pair: Prok2/ProkR2 - purple, Vip/Vipr2 - green, Avp/Avpr1a - blue. The sum of edge weights within each day and night inferred signalling network is presented below each map to provide a quantification of the overall degree of connectivity.
Figure 2"Far Western" blot analyses of BER protein interactions. Recombinant purified human TDG (10 µg), POLß (1 µg), APE1 (1 µg), and LIGIII (1 µg) probed with XRCC1 and XRCC1-SUMO (20 µg) and immune-stained for XRCC1. Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions. 0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins. Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII. 1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated. 0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO. "Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated. Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.
Partial rescue of the IgG response by adoptive transfer of naive functional T cells in Cd40lg-/- mice.Figure 5A Schematics of adoptive transfer of spleen derived CD45.1.2+ CD3+ WT T cells (donor) into CD45.2 Cd40lg-/- mice (recipient) pre-conditioned with 300 mg/kg CPA or not (NC) and immune response studies. B Total counts of recipient CD3+ cells gated within CD45.2+ cells in PB of experimental mice (n=4 CPA 300, 9 NC). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 1 (****p<0.0001). Mean ± SEM. C Total counts of engrafted donor CD45.1.2+ cells in PB of mice from (B). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to plateau (***p=0.0001). Mean ± SEM. D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM. F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR. G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM. H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.
α1-tubulin specific amino acids modulate microtubule length while modulating expression level tunes microtubule numbers A.Figure 5Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter. SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test. qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates. Schematic cartoon of sporozoite indicating the apical, central and nuclear sections as analysed in panels (E-G). TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm. TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line. Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites. Cartoon summarizing the effect of α2-tubulin expression on microtubule numbers and lengths. Numbers indicate the median number of observed microtubules per line. Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).
Figure 5A) Expression of PTGES in normal and tumor tissue. B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression. C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma. D) IL15 gene expression analysis in high and low NK gene signature (NK GS). E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression. F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.
a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig.f5(a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig. 4. Endogenous LC3-II and tubulin levels were assessed by western blot. (b) Quantification of the representative experiment in triplicate shown in a, of two independent experiments. *P=0.02 (DMSO) and 0.08 (Baf) by 2-tailed Student's t-test. (c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with pEGFP vector or GFP-FAM21, and subsequently treated with bafilomycin A1, lysed and subjected to western blot as in a. (d) Quantification of the representative experiment in triplicate shown in c, of two independent experiments. **P=0.008 by 2-tailed Student's t-test. (e) Cells were depleted of WASH1, treated with bafilomycin A1 and examined for endogenous LC3-II, tubulin, WASH1 and GAPDH levels. A representative experiment of six independent experiments is shown. (f) Quantification of the representative experiment shown in e. **P=0.002 (DMSO) and P=0.0028 (Baf) by 2-tailed Student's t-test. g) HeLa cells depleted of WASH1 by siRNA were transfected with GFP-LC3 for 24 h. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of two independent experiments is shown, with 757 (control) and 695 (knockdown) cells analysed. ***P0.0001 by 2-tailed Student's t-test. (h) HeLa cells depleted of FKBP15 were transfected with GFP-LC3 and vesicles were quantified as in g. A representative experiment of two independent experiments in triplicate is shown, with 579 (control) and 815 (knockdown) cells analysed in total. (i) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP-Q74 construct for 24 h and fixed in PFA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of two experiments, each in triplicate with min 220 cells counted per replicate (j) FKBP15 was depleted in HeLa cells as in e. A representative blot from three independent experiments in triplicate is shown. Error bars indicate s.e.m.
a,b) RT-qPCR analysis of levels of (a) LINE-1 RNA (5′UTR, ORF1, n=3, *P=0.f2(a,b) RT-qPCR analysis of levels of (a) LINE-1 RNA (5′UTR, ORF1, n=3, *P=0.009 LINE-1 5′UTR, P=0.03 LINE-1ORF1, t-test) and (b) AluYa5 and AluYb8 in cells (n=3, *P=0.0018 AluYa5, P=2 × 10−6 AluYb8, analysis of variance (ANOVA)) treated with siRNA-targeting ATG5 or control (10 nM, 48 h) (c) RT-qPCR analysis of AluYa5 and AluYb8 in 250-350 nt RNA isolated from cells treated with siRNA-targeting ATG5 or control (10 nM, 48 h, n=3, *P=0.046 AluYa5, P=0.043 AluYb8, ANOVA). (d) RT-qPCR analysis in cells treated with dimethylsulphoxide or Bafilomycin (400 nM) and actinomycin D. (5 nM, n=3, *P=0.01 LINE-1 both 4 and 8 h, P=0.04 AluYb8, ANOVA). (e) RT-qPCR analysis of pulse-labelled RNA recovered 0 and 4 h after pulse in cells transfected with siRNA-targeting ATG5 or control (10 nM, n=3, *P=0.01 both LINE-1 and AluYb8). f) Percent of cells expressing GFP when co-transfected with LINE-1-RP GFP reporter of retrotransposition and siRNA-targeting ATG5 or control. Results are normalized to similar experiments with a retrotransposition-incompetent control (n=3, *P=0.045 ATG5-1, P=0.048 ATG5-2, ANOVA). (g) Relative number of cells at experiment's termination if cells were transfected with siRNA-targeting ATG5 or control. (h) Percent of cells expressing GFP when co-transfected with plasmid expressing GFP and siRNA-targeting ATG5 or control (n=3). (i) Representative plates from Alu-retrotransposition assay transfected with control siRNA or siRNA-targeting ATG5. (j) Relative number of G418-resistant colonies in cells transfected with siRNA-targeting ATG5 or control, Alu-retrotransposition reporter and retrotransposition-enhancer LINE-1 ORF2p (n=3, *P=0.01 ATG5-1, P=0.02 ATG5-2, ANOVA) All error bars represent s.e. of the mean. All experiments were performed with a minimum of three biological replicates.
Figure 3(A) Summary of cell types positive for ACE2 using at least one antibody. Illustrations were in part adapted from https://biorender.com/. /. (B) Details of cell type-specific protein expression levels based on immunohistochemistry in tissues showing distinct immunohistochemical staining in at least one cell type using at least one antibody. Left panel: MAB933 (R&D Systems). Right panel: HPA000288 (Atlas Antibodies). Dot size: level of immunohistochemical staining based on staining intensity and fraction of positive cells.
a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and &bgr-oxidation (b) as compared to cells in regular medium alone (*P  0.figf2a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34). c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37). e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m. f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM. g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.