EMBO/sd-character-level-ner huggingface dataset:
Figure 1Schematic of model substrates and translocation assay. Polypeptide substrates consist of truncated variants of proOmpA (orange), including the signal sequence (SS) (black). Methionines and cysteines are indicated in green and purple, respectively. Substrate was incubated with SecA (tan), proteoliposomes containing SecYEG (blue), and ATP. When the cysteines are oxidized, they form a disulfide, which is too large to allow complete translocation of the substrate, generating a translocation intermediate (IM) (bottom left). Depending on the added nucleotide, the polypeptide can slide back and forth in the channel or make a single step into the channel. When the cysteines are reduced, translocation of the full-length (FL) substrate can be completed with hydrolysable nucleotides (bottom right). Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel. Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.
HIV-mediated autophagy induction and nuclear translocation of TFEB is dependent upon BECN1.Fig 7(A) Macrophages transduced with non-specific scrambled shRNA (shNS), or BECN1 shRNA (shBECN1) were infected with HIV for 10 days. Cells were lysed and analyzed for BECN1 and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (B) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.
Figure 4(A) GST-RhpR or His6-RhpR was phosphorylated by RhpSc, PSPPH_3550c, PSPPH_3736c or PSPPH_5115c proteins. 10-μCi [γ-32P]-ATP was mixed with or without GST/His6-RhpR and incubated with reaction components for 20 minutes. The reaction was stopped by adding 5 × loading buffer before SDS-PAGE. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. (B) The results from (A) were quantified by Image J. The autokinase and kinase activities of HKs were compared with that of RhpS. The individual data points were shown (n=2 biological replicates). (C and D) The rhpS/PSPPH_3550/PSPPH_3736/PSPPH_5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain. The wild type, rhpS deletion and quadruple deletion strains were cultured in MM (C) for 6 hours or KB (D) to OD600=0.6. Transcripts of the indicated genes were determined by RT-qPCR. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001. (E) The activity of rhpR-lux reporter in the wild-type, rhpS deletion and quadruple deletion strains. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001. (F) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain. Bacterial virulence levels were estimated by inoculating wild type, rhpS deletion and quadruple deletion strains onto host bean plants. Disease symptoms were recorded 5 days after inoculation. The experiment was repeated three times. (G) Leaf disks (1 cm2) from (G) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.
Lysine 437 ubiquitination of Beclin 1 is required for autophagy induction.Figure 6(A) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy. EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with antibody specific for lysine 63‐linked poly‐ubiquitin chains (anti‐K63‐Ub). ( (B) The autophagy‐specific Beclin 1 is ubiquitinated through K63 linkage. After starved in EBSS for 1 h, HeLa cells were lysed and HeLa with anti‐Atg14L antibody before dissociated with 1% SDS. Immunoprecipitates were further precipitated with anti‐Beclin 1 antibody, followed by immunoblotting with anti‐K63‐Ub antibody. The left panel denotes the scheme for separation of autophagy‐specific Beclin 1. The right panel indicates ubiquitination state of autophagy‐specific Beclin 1. (C) Lysine 437 mutation to arginine (K437R) abrogates ubiquitination of Beclin 1. Different constructs of Flag‐tagged Beclin 1 mutants were co‐transfected with HA‐tagged ubiquitin (HA-Ub) into HEK293T cells. (D) Beclin 1 (WT) or K437R‐Beclin 1 (K437R) mutant was stably expressed in Beclin 1‐silenced HeLa cells. Beclin 1‐knockdown HeLa cells were infected with lentivirus encoding 3XFlag‐tagged WT Beclin 1 (WT) or K437R‐Beclin 1 (K437R). (E) K437R‐Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy. HeLa cells expressing 3XFlag-Beclin 1 or 3XFlag-K437R‐Beclin 1 were treated with EBSS for 1 h, and harvested for immunoprecipitation. (F) Vps34 kinase activity is impaired by K437R‐Beclin 1 mutant. HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti‐HA antibody followed by a Vps34 kinase assay. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection. (G) K437R‐Beclin 1 reduces the activation of Vps34. Beclin 1‐silenced HeLa cells stably expressing GFP‐HeLa were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with 1 h for 1 h. GFP‐WIPI1 dots were visualized by confocal microscopy. (H) K437R‐Beclin 1 mutant fails to induce autophagy after starvation. Beclin 1‐knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 (K437R) were starved with EBSS for the indicated times. (I) K437R‐Beclin 1 mutant suppresses autophagosome formation. Beclin 1‐knockdown HeLa cells expressing WT‐Beclin 1 or K437R‐Beclin 1 were infected with lentivirus encoding GFP‐LC3, and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1. GFP‐LC3 dots were visualized by confocal microscopy. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.
Fig.Figure 2(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01. (B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.(D) Table representing a manually assembled list of microRNAs revealed by microarray analysis and statistically induced by TSA in FAPs-derived EVs. (E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2). (F) Graph representing miR-206-3p violet area quantification relative to (E). (G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E). (H) Graph showing the percentage of eMyHC positive fibers of condition described in (G). (I) Representative images of Masson's Trichrome staining in conditions described in (E). (J) Graph showing the fibrotic area quantification of conditions described in (I).
Figure 8A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells. C. Chord diagram integrating associations between symptomatic malaria signature genes, immune cell populations and antibody responses. Blue lines within the chord diagram represent positive correlations between two variables, while red lines represent negative correlations. (Benjamini-Hochberg adjusted Spearman's Rho, FDR<5%). D. Chord diagram integrating associations between asymptomatic malaria signature genes, immune cell populations and antibody responses. Blue lines within the chord diagram represent positive correlations between two variables, while red lines represent negative correlations. (Benjamini-Hochberg adjusted Spearman's Rho, FDR<5%).
Figure 6A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf). C. Upon depolarization, endogenous LC3C is localized to mitochondria labeled with mt-mCherry in U2OS cells. An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria (upper panel) and a representative image (lower panel) is given. D. Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy.
SARS-CoV-2 challenge does not affect the IFN response in alveolar macrophages.Figure 3(A) Timeline of the experiment. Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions). Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2. Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 8Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom). (B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right). (C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).
Fig.Figure 5Schematic representation of the scRNA-seq analysis used on a 1:1 mixture of CD45- and CD45+ eWAT cells (excluding F4/80hi resident-macrophages and Ly6C+ monocytes) obtained from lean/obese (HFD 16 weeks) WT and CD169-DTR mice (7 days DT injected, n = 5-8 mice per group). tSNE projection of 18,798 pooled cells (5032 cells from ND WT mice, 3494 cells from ND CD169-DTR mice, 5096 cells from HFD (16 weeks) WT mice, and 5176 cells from HFD (16 weeks) CD169-DTR mice) isolated from eWAT of lean and obese WT/CD169-DTR mice. The projection shows 23 distinct clusters of CD45- and CD45+ cell populations identified via unsupervised clustering. Six specific CD45- cell clusters were identified, including adipocyte stem cells (ASC1 and ASC2), stromal cells, vascular endothelial cells (VEC1 and VEC2), and mesothelial-like cells (MLCs). Seventeen different CD45+ cell clusters were identified, including B and T cells, NK cells, and several myeloid subpopulations. NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T. Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange). Four tSNE plots of 5032, 3494, 5096, and 5176 cells obtained from lean/obese WT and CD169-DTR mice. Orange and blue shaded areas depict the CD45- and CD45+ cell clusters, respectively. Hierarchical heatmap showing the percentage (log2) of all 23 cell clusters identified in lean/obese (HFD 16 weeks) WT and CD169-DTR mice.
Figure 1A. A representation of the primary structure of human CCDC76 (hTrmt13). B. Schematic of the modified CLIP-seq approach employed to profile genome-wide hTrmt13-RNA interactions. C. Autoradiograph of hTrmt13-RNA complexes in the MDA-MB-231 cells immunoprecipitated with an antibody against hTrmt13 (right lane) and IgG as a negative control (left lane). * RNA residing in the regions between marked lines was recovered for sequencing. D. Pie chart showing distinct categories of RNA species bound to hTrmt13 identified by CLIP-seq. E. Pie chart showing the different species tRNAs bound to hTrmt13 from CLIP-seq. F. The capacity of HctRNAs to be methylated by hTrmt13. Error bars represent mean ± SD for at least three independent experiments. G. A representative secondary structure of tRNA with Nm4 modification marked. H. Mass chromatograms of the nucleosides, Um (Q1/Q3 = 259.1/113.1), Cm (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13 KO cells. Um, 2′-O-methyluridine, Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. These endogenous tRNAs were purified separately from total cellular RNAs using sequence-specific probes. I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.
Figure 2(A) Anti-CMS dot blot assay assessing the global amount of 5hmC levels in CD4+ naïve T cells and WT Treg cells. Data are representative of three biological replicates. (B) Heatmpas of differentially hydroxymethylated regions (DhmRs) between CD4+ naïve T cells and WT Treg cells. (C) Scatter plot of 5hmC changes (Y-axis) and gene expression changes (X-axis) between CD4+ naïve T and WT Treg cells. DhmRs associated with a differentially expressed gene are denoted in color (blue: WT Treg cells; black: CD4+ naïve T cells). (D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom. (E) The graphs show CMS (5hmC) signal (RPM) in DhmRs denoted by the shaded region in D for IP and Input in CD4+ naïve T cells and WT Treg cells. FDRs are calculated based on the Benjamini-Hochberg correction of P-values determined using a negative binomial distribution (see methods). Error bars show mean ± S.D. from two independent biological replicates. (F) The graphs show the expression levels of Il2ra (upper panel) and Foxp3 (lower panel) in CD4+ naïve T cells and WT Treg cells. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT Treg) independent biological replicates.
Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.Figure 11Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.
Figure 8A The relationships between heme, Bach1, FOG-1, TAL1, Lmo2, and Mi2β regulation of GATA-1 target genes in G1E-ER-GATA-1 cells. "Sensitive" implies significant deregulation upon heme deficiency, FOG-1-binding deficiency, or knockdown of the respective factor. TAL1, Lmo2, and Mi2β knockdowns were reported previously [76, 77]. Expression of TAL1, Lmo2, and Mi2β decreased 70%, 75%, and 50%, respectively, by the knockdown. FOG-1 sensitivity was determined using G1E-ER-GATA-1 (V205G) cells expressing mutated GATA-1 defective in FOG-1 binding [87]. B Cellular and molecular consequences of heme deficiency on GATA-1 function. Type I incoherent feed-forward loop [88] under normal heme conditions that controls heme biosynthesis and globin chain production. This loop also activates a p62-dependent pathway that can induce Nrf2 and confer cytoprotection [70], and promote autophagy, a process vital for erythroid cell maturation [89-92]. In heme deficiency, Bach1-sensitive and -insensitive (X?) mechanisms disrupt multiple mechanistic steps, thus impairing hemoglobin biosynthesis, cytoprotection, and autophagy. As impaired autophagy and associated molecular defects would compromise cell survival and/or maturation, this underscores the critical implications of the heme mechanism to amplify GATA-1 activity described herein. The red arrows indicate relationships derived from this study. The black arrows indicate relationships that were either known or predicted from existing knowledge.
Comparison of the single-cell transcriptomes of normal tissue, BRCA1+/- preneoplastic tissue and BRCA1-associated tumors.Figure 4A t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples. B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12). C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genes D t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs). E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15). F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells. G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line. H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow). I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.
Figure 1A Yeast two-hybrid assay for the interaction of PknG with UbcH7. Yeast strains were transformed with the indicated plasmids, wherethe TAK1-TAB2 interaction served as a positive control. Left, low stringency. Right, high stringency. B Immunoprecipitation (IP) of UbcH7 by Mtb PknG in U937 cells. Cells were infected with wild-type (WT) Mtb or Mtb ΔpknG at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. IP products were immunoblotted with the antibody against UbcH7. C Pull-down of His-tagged E2s (5 μg each) by GST-PknG (10 μg each). Pull-down products were immunoblotted using the antibody against the His-tag. D Schematic diagram of the Mtb PknG domains. E, F Topology diagrams of the β-grasp folds of ubiquitin (E, left), the ubiquitin-fold domain (UFD) of the E1 enzyme (E, right), and the putative Ubl domain in PknG (F, left) are shown, and a cartoon depiction of the β-grasp fold of PknG is included (F, right). Strands in the topology diagrams are illustrated as cyan arrows, and the helices are highlighted in magenta. G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B. H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.