EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) A schematic diagram indicates sequence features of Lilli and the AFF proteins (Nilson et al, 1997; Wittwer et al., 2001). A correspondence is seen between the C-terminal half of Lilli and the C-terminal two-thirds of the AFFs (the Lilli-like segment). Scale bar indicates length in amino acids (a.a.). pSer, poly-serine domain; NLS, nuclear localization sequence; CHD, C-terminal homology domain; POU, POU transactivation domain; AT, AT-hook DNA-binding motif; NHD, N-terminal homology domain; ALF, AF4/LAF4/FMR2 homology domain. AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm. AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.
A cross-tissue survey of ACE2+TMPRSS2+ cells shows enrichment in cells at reported sites of disease transmission or pathogenesis.Figure 1.(a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows). (b) Proportion of ACE2+TMPRSS2+ cells (y axis) within cell clusters (dots) annotated by broad cell-type categories (rows) within each of the top 7 enriched datasets (color legend, inset). (c,d) Significant co-expression of ACE2+TMPRSS2+ or ACE2+CTSL+ highlights cells from tissues implicated in transmission or pathogenesis. Significance of co-expression (dot size - log10(adjusted P-value), Methods; red border: FDR<0.1) of ACE2+TMPRSS2+ (c) or ACE2+CTSL+ (d) and effect size (dot color, color bar) for finely annotated cell classes (columns) from diverse tissues (rows). Only tissues and cells in at least one significant co-expression relationship are shown (Methods). (e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).
Figure 2A, Experimental scheme of flagellar motor tethering. The stub of sheared flagellum is tethered to glass by an anti-flagellin antibody in a flow chamber B, Demonstration of rotation rate measured from a single cell; negative and positive values (below and above the blue bar) are counterclockwise and clockwise, respectively. The switch from negative to positive rotation rate at 3 s is shown in the montage to the right. C, The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. D, negative stimuli. E, Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM. F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate. G, Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation at time segments 0-20 s and 160-180 s. The variants shown are fliM∆N ∆cheZ cells expressing CheY in the absence of presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ∆cheA cells expressing CheY, in the absence of presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ∆cheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used. H, FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ∆cheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ∆cheA cells expressing FliMN-CheY, in the absence of presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ∆cheA cells expressing FliMN-CheY(D13K) (burgundy; EW739).  I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber.
Figure 5(A) Alignment of observed FRET states with the ATP hydrolysis cycle. Two-helix FRET states are depicted in shades of blue and clamp states in shades of red. The red gradient from low to high FRET indicates uncertainty as to the precise point of clamp closure. The subdivisions of ATP hydrolysis (wavy lines) are meant to indicate qualitative, rather than quantitative, durations of the hydrolysis steps. ADP~Pi indicates the transition state of ATP hydrolysis. (B) SecY is shown in blue and SecA domains in pink (THF, two-helix finger). Domain movements are indicated by red arrows. Clamp closure occurs either during ATP binding or hydrolysis. See text for details.
Self-organization map (SOM) analysis based on relative abundances of serum GM3 species.Figure 3(A) Procedure (schematic) for self-organization map (SOM) analysis, a pattern recognition method using neural-network-type artificial intelligence. Complex patterns of multi-dimensional information (in this case, expression patterns of the ten major GM3 species in human subjects) are mapped onto a 2D surface. Subjects having similar GM3 patterns are located proximal to each other and form several clusters (red arrows), whereas subjects having different GM3 patterns are located distal to each other (blue arrows). (B) SOM analysis of control and lipidemia subjects based on expression patterns of ten GM3 species. (C) Mapping of expression levels of ten GM3 species onto SOM in (B). (D) Identification of sub-clusters having different GM3 patterns based on SOM in (B). (E) Metabolic pathways for fatty acids: elongation, desaturation, and α-hydroxylation (α-oxidation) (schematic). Sub-clusters identified by SOM analysis are mapped on metabolic pathways. (F) Heat-map analysis for GM3 species and clinical markers of six clusters. Sample sizes: sub-cluster-1 to -3 (total), n=22; cluster-4, n=7; cluster-5, n=9; cluster-6, n=12. (G) Self-organization map (SOM) based on four GM3 species as indicated at bottom. (H) ROC curve derived from Bayesian regularized neural networks (BRNN) based on four GM3 species in (G).
Isolation of acetylated histone peptides.Figure 1(A) Schematic overview of the experimental set up to isolate and identify acetylated histone peptides following UV irradiation. SILAC labeled cells are mock or UV (16 J/m2) treated one hour before harvesting. Heavy and light labelled cells are mixed in a 1:1 ratio followed by acid extraction to isolate histones. Histones are separated in 3 fractions each digested with either pepsin, trypsin or GluC. The limited digestion with trypsin is performed for 2, 10, 30 and 120 min. The different fractions of digested peptides are combined and followed by acetyl-lysine immunopurification. The acetylated-lysine enriched histone peptides are analyzed by LC-MS/MS (B) Histones were isolated by acid extraction from HeLa cells one hour after UV (16 J/m2) or mock treatment and were loaded on a 4-15% gradient SDS-PAGE gel and stained with Coomassie Brilliant Blue. The different fractions originate from the same amount of cells and contain the non-acid-soluble proteins (pellet), histone H1 and high mobility group proteins (H1 fraction) or the core histones (core histone fraction). * indicates 21 kDa protein band, most likely representing histone H1. ** indicates several protein bands ranging between 10-15 kDa most likely representing core histones (C) Western blot of isolated histone fractions from HeLa cells one hour after UV (16 J/m2) or mock treatment. Western blots were stained with α-histone H1.2 (top panel), α-histone H2B (middle panel) and α-yH2AX (bottom panel) (D) Table listing the coverage of the histone sequences using our peptide digestion procedure by LC-MS/MS analysis (E) Table listing the number of acetylation sites on core histones identified by MS after acetylated peptide enrichment
| Relationship between NANOS and PUMILIO.Figure 2Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) or with FLAG-tagged PUMILIO1 or PUMILIO2, and HA-tagged NANOS1, NANOS2 or NANOS3 with or without MYC-tagged DND1 (B). C, Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag-tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG-tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG-tagged PUMILIO1 while the anti-PUMILIO2 antibody could detect both Flag-tagged PUMILIO1 and PUMILIO2 but less actively. D, FLAG-tagged NANOS2 was immunoprecipitated with an anti-FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG-tagged NANOS2. Precipitates were analyzed by western blotting with the indicated antibodies. Note that co-precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co-precipitated.
Figure 2The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test). Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR. YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells. Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test). RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).
NOD2 activation after M.Figure 4(A) Intracellular bacterial burden was measured by quantifying CFU, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments. The production of (B) TNF‐α, (C) IL‐6 and (D) IL‐1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments. (E) LL37 and (F) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test. (G) LL37 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented. (H) The amount of protein normalized to tubulin relative to infected‐only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.
Figure 6A Schematic of IMC assembly during gametocyte to ookinete. IMC assembly initiates in apical and extends along protrusion of the fertilized zygotes. B Endogenous DHHC2 is palmitoylated in the dhhc2::6HA gametocytes. C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in female gametocytes, female gametes, and zygotes of WT parasites. Left panel shows the expression of HA-tagged DHHC2 in female gametocyte by co-staining with antibodies of HA and α-Tubulin II (male gametocyte specific). Scale bar = 5 μm. D IFA of DHHC2 and P28 in female gametocytes, female gametes, and zygotes of the dhhc2::6HA parasite. Gametocytes were stimulated with XA and 22°C in vitro. P28 is expressed specifically in female gametes and zygotes. Scale bar = 5 μm. E Quantification of parasites with DHHC2 polarization in P28 positive cells during gametocyte to zygote differentiation x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ±SD from three independent tests. F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD. G No change in DHHC2 expression in zygotes of the dhhc2::6HA parasite-derived mutants with individual gene disruption of cdpk4, hap2, and dozi. H Quantification of parasite with DHHC2 polarization in P28 positive cells. Parasites are indicated in (G) or from a genetic cross (dhhc2::6HA/cdpk4 and WT). x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ± SD from three tests, two-tailed t test, ***, P<0.001, ****, P<0.0001, ns, not significant. I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2::6HA parasite. GAP45 is an IMC marker and is only detected in zygote after fertilization, but not gametes. Scale bar = 5 μm.
Expression and localization of Nrg3 in vivo.Figure 1A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3. B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant). C) Schematic display of the structure of Nrg3 before (left) and after cleavage by Bace1 (right). The following domains of Nrg3 are indicated: black, N-terminal intracellular domain and transmembrane domain; blue, extracellular domain; red EGF domain; black, stalk region with Bace1 cleavage site indicated by an arrow and C-terminal transmembrane domain; green, intracellular domain. D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes. E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F). G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization. H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).
a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.figf6a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b). (c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.
Figure 4Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test. Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm. Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining. Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193. Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242. CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001.
Fig.Figure 5(A) Scheme representing the study design. FVIII deficient mice (FVIII -/-, n=6-9/group) were administered with 2 doses (1x1010 or 1x1012 vg/mouse) of AAV vector. Control mice were injected with PBS. Red symbols represent timing of blood collection. (B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker. (C) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group). (D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD. (E) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. (F) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.
Figure 7Hrd1 uses Ubc7 for both priming and elongation and selects Ubc7 over Ubc6 for priming through higher binding affinity and optimal stimulation through its arginine linchpin ("affinity-driven pairing"). Doa10 uses Ubc6 for priming. Although it binds the conjugates of the two E2s with similar affinity, its suboptimal histidine linchpin is ineffective at stimulating Ubc7. In contrast, Doa10 pairing with Ubc6 is highly active due to the high basal activity of Ubc6~Ub that does not require substantial stimulation by a linchpin. We call this mode of E2 selection "rate-driven pairing." E3-mediated E2 stimulation is dispensable during elongation, allowing Doa10 to pair productively with Ubc7. For later elongation steps, Cue1 plays an important role in driving reaction rates although Ub chain elongation is generally not rate-determining for the kinetics of protein degradation. Finally, both RINGs retain the ability to use the non-preferred E2 for priming (dashed arrows), possibly providing a "backup mechanism" for priming.