EMBO/sd-character-level-ner huggingface dataset:
Figure 2Schematic representation of immunoprecipitation of protein-RNA complexes engaged to RNAPII shown in (B). With UV-crosslinking of protein-RNA interactions (UV+) or without (UV-), the chromatin fraction is isolated from the cells and treated with (Pre-RNase+) or without RNase A (Pre-RNase-). RNAPII, shown in gray, is 1st immunoprecipitated. The immunoprecipitants are treated with RNase A to release a protein, shown in green, from the protein-RNA-RNAPII complex. RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in the immunoprecipitation experiments. Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking, protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex (left panel) and RNAPII-U1C-RNA complex (right panel). Read distributions of Chr-FUS-tRIP, U1C-FUS-tRIP, and RNAPII-FUS-tRIP mapped to the relative positions of all coding genes in mouse. The ngs.plot tool [58] was used to calculate the average RPM for a gene structure. The average RPM at each position was normalized for the total RPM mapped to each gene. The standard error of normalized RPM is shown as a semi-transparent shade around the average curve. Arrowhead indicates a peak before the transcription termination site (TTS) in RNAPII-FUS-tRIP. Read distributions of RNAPII-FUS-tRIP (purple lines) and Chr-FUS-tRIP (black lines) around the APA sites repressed (bold lines) or activated (thin lines) by FUS. The p-values for the differences between RNAPII-FUS-tRIP and Chr-FUS-tRIP around FUS-repressed APA sites are indicated by circles. An arrowhead indicates a peak upstream to APA sites in RNAPII-FUS-tRIP. Read distributions of RNAPII-CPSF160-tRIPs generated from Fus-silenced cells (siFus, pink line) and those of control siRNA-treated cells (siCont, green line). The p-values for the differences between siFus and siCont are indicated by circles. An arrowhead indicates a peak before APA sites in RNAPII-CPSF160-tRIP of siFus-treated cells.
a) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK, phospho-JNK, total IRS-1 and phospho-IRS-1 S307.f3(a) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK, phospho-JNK, total IRS-1 and phospho-IRS-1 S307. (b) Liver sections from each type of mouse were subjected to immunohistochemistry using anti-nitrotyrosine antibody. Scale bars, 100 μm. (c) Tissue lysates were prepared from 18-week-old mice of each genotype, and subjected to immunoblot analysis using a kit detecting carbonylated proteins. (d) Immunoblot analysis was performed using anti-phospho-p53 and -p53 antibodies. p21 and β-actin mRNA expression was evaluated by RT-PCR. Numbers below immunoblot bands indicate the fold changes normalized to control bands.
a) Rate of mitochondrial O2·– detection in WT and Pink1 KO mice primary neurons.f6a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons. (b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons. (c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).
Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change). E) Heatmaps show the correlations between the plasma levels of metabolites (other than lipids) and the abundance of the species in the gut microbiome. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient
Figure 4Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl) phosphine. Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide. Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin. MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion. The table shows parameters of mass spectrometry identification of CtUba4C202K and CtUba4C202S with lists of detected peptides containing the K-ε-GGH remnant motif of Urm1. In CtUba4C202K, the conjugation site on K202 was confirmed. In the TBH treated samples of CtUba4C202S several potential conjugation sites were found. We present only high confidence peptides reaching a Mascot score higher than 40, which were detected in all analyzed samples. Asterisks indicate ambiguous site localization of K-ε-GGH. #PSMs: number of peptide-to-spectrum matches; m/z: mass to charge ratio of precursor ion. Localization of all lysine residues (shown as green sticks) on the structure of CtUba4C202K-Urm1. CtUrm1 C-terminus and the lysine residues of CtUba4C202S that become covalently linked to Urm1-COSH in oxidizing conditions are presented in red. Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.
H+ and transmitter bindingA.Figure 3A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition. B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines. C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM.. D. Potential H+-bond network between protonated E406386 (TM7b) and residues in HP2b in EAAT1CRYST transmitter-bound structure. HP2 is depicted in yellow and the 398NMDG motif in green. Residue numbering corresponds to EAAT1WT, and the interatomic distance (angstrom) is parenthesis.
Figure 5A, immunoblotting of NRF2 and Keap1 in MCs and MPCs of Du145TXR or MCF-7ADR cells. Data information: Results are representative of three independent experiments. B, immunofluorescence analysis of NRF2 in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 hours. Scale bar, 10 μm. Data information: Results are representative of three independent experiments. C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments. D, the profiles of predicted binding sequence of NRF2 in NPC1L1 promotor by JASPAR. Data information: Results are representative of three independent experiments. E, ChIP-qPCR analysis of NRF2 binding on the NPC1L1 proximal promoter in MCs and MPCs of Du145TXR or MCF-7ADR cells. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. H-I, cell viability of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments. J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.
Figure 6(A) Immunoblots of cell lysates were performed to analyze galectin-9 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice, and GADPH as a loading control. qPCR analysis of Mmps including Mmp9 (B), Mmp10 (C), Mmp12 (D), from mouse peritoneal macrophages isolated from either wild-type or Galectin-9 KO mice stimulated with AG (1 μg/ml) for 24h. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001. qPCR analysis of Mmp13 (E) from mouse peritoneal macrophages isolated from either wild-type or Galectin-9 KO mice stimulated with AG (1 μg/ml) for 24h. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001. (F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control. (G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice infected with H37Rv for indicated times (MOI=5); GADPH of cell lysates served as the loading control. (H) Immunoblot of lysates of peritoneal macrophages isolated from wild-type and Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments. (I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments. (J) qRT-PCR detection of Mmp transcripts including Mmp9, Mmp10, and Mmp12 in wild-type and Galectin-9 KO peritoneal macrophages stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001. (K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.
| Characterization of EV-vsiRNAs with distinct virus infections.Figure 3A-D, Properties of total small RNA reads and vsiRNAs sequenced from EVs in C57BL/6 suckling mice infected with NoVΔB2 at 3 dpi (A) and 9 dpi (B), SINV at 3 dpi (C) and Ifnar-/- mice infected with ZIKV at 4 dpi (D). Length distribution of total 18- to 35-nt reads in each library, size distribution of 18- to 28-nt virus-derived siRNAs, and viral genomic coverage depth of each nucleotide position by 21- to 23-nt vsiRNAs are presented as described in previous figures. Reads are shown as per million total mature miRNAs. 5' terminal nucleotide and the percentage of 1U vsiRNAs and duplex pattern of the 22-nt vsiRNAs are indicated.
Phenotype of atg4da morphant zebrafish embryos.Fig 5(A-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye. (G) RT-PCR assay showing the efficiency of the atg4daSMO.
Figure 3(A) A schematic shows 10 µm cryosections of unfixed brain cryosections from 6-months old APP/PS1mice, which show a high amyloid plaque burden (right panel with methoxy-X04 staining), were incubated with antibody for 1 h, followed by adding BMDM or primary microglia on top of the sections. After 24 h incubation, sections were analyzed by immunostaining or immunobloting. (B) mAb11 but not the IC co-localized with methoxy-X04. BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.
Figure 2A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated. B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.
Figure 1(A) Schematic representation of mouse immunization and challenge schedule. Female BALB/c mice (n = 5 per group; 4-6 weeks old) were immunized with single dose of live RSV A2 virus (104 pfu dose) intranasally, or two doses given i.m. with 20 μg of RSV pre-fusion or post-fusion form of F protein or F peptides from RSV strain A2 with Emulsigen adjuvant, or with KLH or with PBS as a control. After the second immunization, blood was collected from the tail veins on day 35. On day 42, mice were challenged intranasally with 106 PFU/10μL of RSV rA2-Line-19F-FFL. Mice were sacrificed on day 5 post-challenge, when lungs and blood were collected. (B) Alignment of antigenic site peptides within the RSV F protein used for mice vaccination in the study. Previously described antigenic sites (sites φ, I, II and IV) are shown above the F protein schematic, and the antigenic site peptides used in this study are depicted below.
Figure 2(A) Experimental scheme of dual labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin.(B) Representative IdU tract length distributions in shWRNIP1WT (left graph) or shWRNIP1 cells (right graph) after treatment. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.
Figure 8(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II). (B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II). (C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83). (D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.
Human liver interactome delineates tumor-stroma cross talk (A) Summary of the total number of ligand-receptor interactions among clusters with at least 20% tumor cells.Figure 4Summary of the total number of ligand-receptor interactions among clusters with at least 20% tumor cells. Interactions of tumor TME cell types with the carcinoma cluster marked by red box. Network of recurring ligand-receptor interactions between carcinoma cells and stromal cells from the malignant sites. Node colors denote the cell type cluster in which the ligands/receptors are enriched. Gray arrows color indicates the interaction Zscore (Methods). Recurring modules are shaded. Included are all recurring interactions that significantly appeared in at least three patients. Dot-plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in (A). For each gene, dot size represents the fraction of positive cells for each cluster. Recurring interaction motifs between CAFs and tumor immune cells. Top - CAFs and SAMs co-modulate carcinoma -stroma interaction. CAFs produce DCN that modulates the interaction between the CAF-SAMs-expressed ligand HGF and the carcinoma-expressed receptor MET. Bottom - CAFs produce CTHRC1 that modulates the interaction between the WNT5A ligand, expressed by CAFs, SAMS and TM1 and the carcinoma-expressed receptor FZD5. Strategy for computing interaction scores for each tumor. A score is computed as the sum of the products of all ligands and matching receptors of the recurring interaction network. These are compared to the average score obtained when randomizing the real interaction network in a manner that preserves the number of outgoing and incoming interactions of each ligand and receptor (Scorerand). The ratio of the real and randomized scores constitutes a network score. Network score increases with increasing tumor stage. Analysis for 383 TCGA sample of Liver Hepatocellular Carcinoma (LIHC) and Cholangiocarcinoma (CHOL). p-value determined by Wilcoxon rank-sum test. Empty circles are the medians over all repeats. Dashed lines are the median over the tumor stage.
Comparative transcriptome analysis reveals positive and negative lncRNA regulators in brite adipogenesis.Figure 1(A) Experimental design of transcriptome analysis. Primary preadipocytes and differentiated adipocytes ((w/o) rosiglitazone), isolated from inguinal white adipose tissue (iWAT) of five inbred mouse strains of varying browning propensity (evaluated based on Ucp1 transcript levels in RPKM (reads per kilobase per million mapped reads)). Mean values ± SD, n=3 (biological replicates) (Data ref: Array Express E-MTAB-8344, 2019) (Li et al., 2019) were subjected to RNA sequencing to identify regulated long noncoding (lnc) RNAs. (B) Principal component analysis (PCA) of undifferentiated and differentiated adipocyte samples, n=3 (biological replicates). (C) hierarchical clustering of the long noncoding transcriptome of undifferentiated and differentiated adipocyte samples, n=3 (biological replicates), euclidean distance. (D) Heatmap, displaying row-wise centered log2 normalized counts. (E) (F) Venn diagrams of positively and negatively regulated lncRNAs selected according to correlation with Ucp1, regulation during differentiation and regulation by rosiglitazone treatment, n=3 (biological replicates). (G) GO term analysis of different mRNA clusters derived from lncRNA-mRNA coexpression network (according to Bai et al. 2017) that point towards lncRNA associated cellular processes, hypergeometric test.