EMBO/sd-character-level-ner huggingface dataset:
A role for Dnmt2 in the regulation of codon fidelity.Figure 6Boxplots with outlier calling showing the significant codon biases in Dnmt2 down-regulated and up-regulated proteins in the DML and dSILAC datasets. For the dSILAC (S-All) dataset, we display the plot for all identified proteins or stromal (S-St) and suspension (S-Ss) cells. A significant increase or decrease of a codon frequency in up- or down-regulated proteins relative to the average frequency of that codon in all identified proteins is displayed as a colored dot. Codons showing no deviation from average values are grouped together in the box. Frequency of amino acid substitutions in the dSILAC dataset. Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant (P < 0.01, Fisher's exact test). Changes in bulk codon occupancy in Dnmt2−/− bone marrow primary stromal cells, compared to wild-type cells. Dnmt2 target codons and Glu codons are highlighted with the indicated colors. The two biological replicates of each cell type were pooled. A: ribosomal A-site; P: ribosomal P-site. Changes in bulk codon occupancy in Dnmt2−/− bone marrow primary stromal cells compared to wild-type cells in the A-site of ribosome were plotted against the P-values obtained in the Kolmogorov-Smirnov test. Dnmt2 target codons are highlighted with the indicated colors.
Domain organization in the C3b-regulator complexes and molecular interactions.Figure 2A. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area. B. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).
Amounts of ATG16L1 protein are influenced neither by the rs2241880 genotype nor by intestinal inflammation.figf1(a) Expression of ATG16L1 in a set of different tissues as detected by RT-PCR (IEC, intestinal epithelial cells). The corresponding β-actin control (with a 518-bp amplicon size) is given below. b) Amounts of ATG16L1 in colonic mucosal biopsy specimens. Proteins (15 μg) from rectal mucosal biopsies of Crohn disease patients and normal controls (N) were separated by denaturing SDS-PAGE, transferred onto polyvinylidine fluoride (PVDF) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase-coupled secondary antibody. The abundant 68.2-kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 (UniProt accession number Q676U5). This major protein form was used for three-dimensional modeling of the ATG16L1 protein (Fig. 2). The 48.5-kDa band corresponds to GenBank EF079890, which contains exon 9 (harboring rs2241880) in the same reading frame as the major form. (c-i) Representative immunohistochemistry results (out of six normal controls and six individuals with Crohn disease) from colonic mucosal tissue samples from normal controls for genotype AA (c) and GG (f) and individuals with Crohn disease for genotype AA (d) and GG (g) are shown to demonstrate the cellular localization of the ATG16L1 protein. Cellular expression patterns in mononuclear cells (h) and intestinal epithelial cells (i) of a Crohn disease sample (rs2241880AA) are shown at higher magnification. e shows a Crohn disease sample without the primary antibody, used as a control. Bar represents 10 μm in c-g.
Figure 2(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody. (B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants. (C) Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5'UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01. (D) EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means±SD (n=3). * P<0.01. (E) C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls. (F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins. (G) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins.
Figure 2Figure 2. Cones are associated with chemosensory arrays, ribosome-excluding zones and filament bundles. Tomographic slices show side (A) and top (B, C) views of cones (c) in three cells, highlighting associated chemosensory arrays (ca), ribosome-excluding zones (rez) and filament bundles (f). For a slice-by-slice view through the tomogram shown in (A), see Movie S1. (D) and (E) show different views of a 3D segmentation of the structures shown in (C), with the conical structure in blue and the filament bundle in red. s, S-layer; m, membrane; a, archaellum; o, other filaments. Scale bar 100 nm (applies to A-C); segmentation not to scale.
JQ1 treatment inhibits retinal microglial activation in the rd10 retina.Fig. 3 JQ1 treatment inhibits retinal microglial activation in the rd10 retina. Intravitreal injection of JQ1 (or vehicle) was performed at PN14, as described in Fig. 1. Eyeballs were collected at PN18, PN24, and PN30. Cryosections were used for immunostaining and fluorescence microscopy. a-c Representative immunostaining images of microglial markers. Scale bar 50 μm. Blue DAPI staining of nuclei. d Quantification: IBA1, CD68, or TSPO positive cells per 500 μm ONL length; mean ± SEM, n = 6 mice. Quantification of IBA1 staining at all five time points (PN18-PN30) is presented in Additional file 1: Figure S3
Figure 3(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm. (C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm. (E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm. (G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.
Increased ROS generation plays a critical role in autophagy and proinflammatory cytokine production in Mtb-Δeis-infected macrophages.Figure 3(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom) (B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane. (C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments. (D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well). (E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown. (F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).
Figure 10A. The cytochrome c release by the indicated Bax mutants was measured in two independent replicates, corrected and shown as in Figure 9. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1. B. Purified wild-type (WT) or mutant (G179I or T182I) Bax protein of the indicated concentration was incubated with the Bax-/-/Bak-/- mitochondria in the absence or presence of tBid protein. The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS-PAGE and immunoblotting with antibody specific to either cytochrome c or Smac. n = 2. C. Purified wild-type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax-/-/Bak-/- mitochondria containing Smac-mCherry protein. Release of Smac-mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation. The dots are the fractions of Smac-mCherry release from three independent replicates, and the lines are the means. D. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.
Figure 7(A-G) Lateral (A-B), ventrolateral (C-F) and ventral (G) images of the cranial vessels in an etv2:EGFP;kdrl:nlsmCherry compound transgenic embryo, which has had the etv2-expressing angioblast population (early VA-L) on the left lateral side of the yolk sac ablated. Images were taken at 30 hpf before (A) and after (B) ablation of the early VA-L, with the site of ablation indicated (B; blue dotted circle). Note that the LDA and VA adjacent to the VA-L become photobleached during the ablation process (B), however these were not ablated as they can be seen developing normally at 54 hpf (D). Follow-up images were taken of both the contralateral (C, E) and ablated (D, F) sides of the embryo's head at 54 hpf (C-D) and 78 hpf (E-G). Although the VA-L (light blue dotted line) is seen migrating towards the FLS (white dotted lined) on the contralateral side at 54 hpf (C), it is missing from the ablated side (D). Subsequently, the LFL (white dotted line) of the ­ablated side is short (F) compared to that of the contralateral side (E). In addition, the HA (pink dotted line) is underdeveloped on the ablated side compared to the contralateral side, as it no longer extends towards nor connects to the left ­AA1 (G). (H) Quantitation of the length of the LFL (µm) on the contralateral (control) side and on the ablated side at 78 hpf (n = 16). (I) Quantitation of the total number of cells within the LFL on the contralateral (control) side and from the ablated side at 78 hpf (n = 16).
Figure 5A. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2. B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing. C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing. In vivo bioluminescent images of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth. E. In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth. F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs. G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.
In vitro characterization of mutated PAPP-A2.Figure 3• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS-PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated. B Western blots following nonreducing or reducing SDS-PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection. •C Comparison of proteolytic activity similar to the experiment of panel A, except that equimolar concentrations of PAPP-A2 and PAPP-A2 (A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments.
In vitro generated OxPAPC preparations represent complex mixtures of OxPL species with distinct bioactivities.Figure 2Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown. Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.
Figure 5(A) SARS-CoV-2 pseudovirus was pre-treated with LMWH enoxaparin (250 IU/mL), or different neutralizing antibodies against SARS-CoV-2 (COVA1-18, COVA1-21 and COVA2-15) and a humanIgG1 isotype control at concentrations of 100 pg/mL, 500 pg/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL and 50 ng/mL for 30 min at 37°C. Binding of pseudovirus to Syndecan 1 expressing cells in absence or presence of LMWH enoxaparin or antibodies was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one-way with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3) (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated for 30 min at 37°C with either LMWH enoxaparin (250 IU/mL) or one of the following neutralizing antibodies (COVA1-18, 1-21 and 2-15), a non-neutralizing antibody (COVA1-27) or a human IgG1 isotype control, all at the concentration of 1 pg/mL. SARS-CoV-2 isolate alone or with blocks was added at a concentration of 100 TICD/mL. Detection of virus binding to Syndecan 1 expressing Namalwa was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01 (n=2 measured in duplicates).
Figure 3 A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth .C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals. D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm.
Figure 5(A) Principal components analysis of 16S rRNA gene-sequencing analysis of gut microbes obtained from Villin-Cre and COPS8-lox alleles expressing (COPS8fl/f ) and COPS8 knockout (KO) (COPS8ΔIEC) mice. PC1 and PC2 explain the 64.47% and 27.95% variation, respectively. Representative data from five biological replicates per genotype. (B) Heatmap of differentially represented bacterial species in feces between COPS8fl/fl and COPS8ΔIEC mice. The top 30 genera that are shared by all of the samples shown. The scale was the percentage composition (log2) based on the 16S rRNA gene sequences analyzed using the Greengene database (gg_otus2013). The hierarchical clustering based on Spearman Rank correlation (average linkage method) was performed using Gene Cluster 3.0 and images generated on Java Treeview. Representative data from five biological replicates per genotype. (C) The relative abundance of bacteria phylum in the feces of COPS8fl/fl and COPS8ΔIEC mice. Representative data from five biological replicates per genotype. (D) LEfSe analysis was applied to identify high-dimensional biomarkers that discriminate between feces from COPS8fl/fl and COPS8ΔIEC mice. Representative data from five biological replicates per genotype. (E) Histologic severity scores of COPS8fl/fl mice and COPS8ΔIEC after fecal transplantation. Data represents the histological score of mice analyzed on day 160. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01 using one-way ANOVA. (F) Inflammation scores in the colon of 5-month-old COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA. (G) Percentage of rectal prolapse of 5-month-old mice COPS8fl/fl and COPS8ΔIEC. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA. (H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA. (I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.
Figure 2A Experimental design. Laser coagulation was performed in all C57BL6/J mice that were either untreated or received 10,000 units IFN-b i.p. every second day until animals were analyzed 3, 7 and 14 days after laser treatment, respectively. B, C Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in control mice (B) or IFN-b treated animals (C).Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 7-10 retinas; unpaired Student's t-test: ***p < 0.0001).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 41-62 cells; unpaired Student's t-test: ***p < 0.0001). F-K Representative fundus fluorescein angiography images of control mice (F-H) or IFN-b treated animals (I-K) 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in control mice and IFN-b treated animals. Values show mean ± SD. (n = 15-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p = 0.0004). M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice (M, N) and IFN-b treated animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µmQ Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 7-12 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: **p = 0.0038).