EMBO/sd-character-level-ner huggingface dataset:
Mitochondrial AcCoA Production by Ach1p- or Mpc1p-Associated Pathways Is Required for Autophagy during Aging (A) Fluorescence microscopy of GFP-Atg8p expressing wild-type (WT) and ACH1-deleted (Δach1) yeast grown to day 1 (young) and aged for 3 days in SC 2% glucose medium; propidium iodide (PI) counterstaining served to visualize dead cells.Figure 2(A) Fluorescence microscopy of GFP-Atg8p expressing wild-type (WT) and ACH1-deleted (Δach1) yeast grown to day 1 (young) and aged for 3 days in SC 2% glucose medium; propidium iodide (PI) counterstaining served to visualize dead cells. Scale bars represent 5 μm.(B) Quantification of cells depicted in (A) with 150-300 counts (blinded) for each replicate. Autophagic cells were defined as cells with clear vacuolar GFP fluorescence. Data represent means ± SEM (n = 4). ∗∗∗p < 0.001.(C) Representative immunoblot analysis of cells shown in (A) and further aged to day 5 using anti-GFP and anti-GAPDH (loading control) antibodies to detect "free-GFP" indicative of autophagic flux (see also Figure S2). (D-F) Representative micrographs (D), respective quantification (E), and immunoblot analysis (F) of wild-type (WT) and MPC1 deleted (Δmpc1) yeast expressing GFP-Atg8p chimera as in (A)-(C) and aged to indicated time points. Data represent means ± SEM (n = 4). ∗∗∗p < 0.001.
PolII ChIL-seq detect active genes in tissue.Figure 3(A) Dynamic ranges of bulk-tissue RNA-seq and PolII S5P ChIL-seq. The cumulative proportion in total mapped reads at genes (red: ChIL-seq, blue: RNA-seq) were compared. Genes are ordered by the read counts on the exons for RNA-seq and on +/-750 bp from TSS for ChIL-seq, respectively. (B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes. (C) Coverage of expressed genes by PolII S5P ChIL-seq peaks. The stacked bar chart shows the proportions of detected genes in the RNA-seq only (RNA-seq: blue), PolII S5P ChIL-seq only (ChIL: red) and both (Common: green). (D) Higher expression levels at PolII S5P ChIL-seq peaks. The expression levels of all expressed genes (TPM > 0) are shown. (E) The tissue-specific genes identified by PolII S5P ChIL-seq. The IGV tracks of all replicates of PolII S5P ChIL-seq are shown at each specific gene (Trf, Myh6, and Meig for the liver, heart, and testis, respectively). Actb is also shown as the ubiquitously expressed gene in the three tissues.
Figure 6A) Density Power radial kymograph of a representative time-lapse. Each horizontal line displays for each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Values scale is on the right. Graph representing the mean (line) and standard deviation (shadow) of the power density radial kymographs (left scale) at different distances (bottom scale) by phases (in coded densities of purple) for all samples. Surface Stress radial kymograph of a representative time-lapse Phases are represented in different densities of brown. Graph representing the mean and standard deviation of the surface stress radial kymographs Values color coded in different densities of brown.
Figure 4(A) Cartoon of the strategy for generation of pseudotyped lentiviruses. HEK-293T cells were transfected with a lentiviral construct containing the EGFP marker gene and two more plasmids encoding for the gag and pol genes of HIV-1 and for the S protein of SARS-CoV-2. Alternatively, the latter plasmid was replaced by another one encoding the G protein of VSV. The cell supernatants collected after 48 h of transfection were used to transduce HEK-293T cells stably transfected with human ACE2. (B) Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum, refers to a control containing no human sera. Data represent the mean±SD of triplicates. (C) The presence of neutralizing antibodies in sera from donors #46 and #48, found negative by ELISA and positive by flow cytometry (Fig. 3C) was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample). (D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown. (F) Plot of the S/EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig. EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive.  (G) The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56 and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Unpaired two-tailed t-test comparing each serum to the No serum sample)
Figure 3(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown. (B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01. (C) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown. (D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (F) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown. (G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05. (H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test.
Figure 1A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left. B Cyclin C and Bax directly interact. Recombinant Bax and SUMO-His6-cyclin C were either incubated together or separately at room temperature then passed over Co++ resin. The elutions were subjected to Western blot analysis probing for Bax and cyclin C (lanes 4-6). Input controls for each sample (1/10) are included in lanes 1-3. Molecular weight markers (kDa) are indicated on the left. C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left. D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left. E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left. F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.
Figure 5A Representative flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h). B Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiacneutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001. C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13). D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13). E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13). F Mean fluorescence intensity (MFI) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype (WT) and CXCR2 KO mice. Student`s t-test. N = 6 mice in both groups; *P = 0.0001. G Percentage of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0001 (ZT13). H Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n= n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0461 (ZT13), ns = not significant.
Comparison of immunostained primary hippocampal neurons in STED resolution.Figure 7A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells. B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons. C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells. D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.
PHD1 hydroxylase favors p38α-mediated p53 phosphorylation.Figure 5Western blot for p53 pS15, p53, and vinculin (vinc) in HCT116 treated with 300 μM 5-FU upon silencing of PHD1 and p38α, alone or in combination. Vinculin (vinc) is used as a loading control. Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α, p38β, or p38γ of p53immunoprecipitated from cells silenced for a control (SIMA) or PHD1. Detection by Western blot of p53 and p38 from whole cell extracts (WCE) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5-FU.
Figure 1(a) Schematic representation of U2-pre-mRNA interaction. Partial sequences/secondary structures of vertebrate and S. cerevisiae U2 are schematically shown. Also shown are the base-pairing interactions between the U2 branch site recognition region (BSRR) and the pre-mRNA branch site (BS). Pseudouridines (Ψ) are indicated. Enzymes responsible for yeast U2 pseudouridylation at the three conserved sites are also indicated. (b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom). (c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit "processive"). Lanes 1-8 and lanes 9-16 are from two separate gels. (d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].
Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase.Figure 5Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.
Bafilomycin A1 inhibits the 1,25D3 mediated inhibition of HIV and M.Figure 6MDM were pretreated with bafilomycin A1 (Baf A1) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7. (B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7. (C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
Hsp90 regulates DYRK3 activity by acting outside of condensates.Figure 4(A) G3BP1-mCherry HeLa-Kyoto cells were left untreated or treated with sodium arsenite (50 µM) for 45 min to induce SGs. Cells were then fixed and immunostained for DYRK3 and total Hsp90 antibodies. Quantitation of the percentage of SGs that show DYRK3 and Hsp90 enrichment (> 1.5). Automated imaging and SG segmentation is based on mCherry-G3BP1 signal. Number of SGs analyzed: 3171 (arsenite). Scale bar is 10 µm. (B) Confocal microscopy of HeLa-Kyoto cells lipofected for 24 hrs with cDNAs encoding for GFP-DYRK3 and either an empty vector or mCherry-Hsp90 α and β. The upper panel shows endogenous Hsp90, stained with a specific antibody for total Hsp90; the lower panel shows exogenous mCherry-Hsp90 α and β. Scale bar is 10 µm. (C) Quantitation of the fluorescence intensity recovery after bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of GA (5 µM) or of GSK (5 µM). FRAP was performed during the stress recovery period. A representative image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP curves (recovery GSK) is shown in red; the sem is shown in gray.
Figure 2(A) Neutralization assay of FD20 using SARS-CoV-2 pp harboring spike derived from four Variants of Concern as indicated. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate IC50 values in nM. (B) Microneutralization assay of FD20 against authentic SARS-CoV-2 Wuhan isolate (hCoV-19/China/CAS-B001/2020). The protective effect of antibody was assessed using cells infected with a high viral dose (2,000 TCID50 mL-1). Data are from a single experiment performed in octuplicates. Numbers in brackets indicate IC50 values in nM. (C) The plaque-reduction neutralization assay using two authentic SARS-CoV-2 strains: the Germany isolate (BetaCoV/Munich/BavPat1/2020) and UK/B1.1.7. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate IC50 values in nM. (D) IC50 (up, in nM) and normalized IC50 folds (bottom, normalized against the wild-type) of FD20 and control antibodies measured using different SARS-CoV-2 pp harboring S mutants or variants. E406W is an escape mutant from REG10933 and REG10987 cocktail generated in vitro (Starr et al, 2021). K417N is an escape mutant for CB6 (LyCoV.016) and is present in the B.1.351 lineage first identified in South Africa (Li et al, 2020; Starr et al, 2021). Y453F increases the ACE2 binding affinity, and with A475V and F490L, is found in independent mink-related SARS-CoV-2 variants (Larsen et al, 2021; Oude Munnink et al, 2021) identified in Denmark and the Netherlands (B1.1 and B.1.1.298) that may escape human antisera and REG10933 (Starr et al, 2021). D614G appeared early during the pandemic and is now the dominant form worldwide, and is associated with other mutations in new variants of concern (Korber et al, 2020). A222V and S477N are described in 20A.EU1 (B.1.177) and 20A.EU2 (B.1.160) that emerged in early summer 2020 and subsequently spread to multiple locations in Europe at the end of 2020 (Lemey et al, 2021). N439K is in B.1.141, B.1.258, and in mink strains; it increases affinity to ACE2 and reduces neutralization of sera from convalescent patients (Thomson et al, 2021). Finally, ∆69/70-N501Y-D614G are characteristic mutations in RBD for the UK/B.1.1.7 lineage (501Y.V1) (Leung et al, 2021), one of the three fast-spreading new variants of SARS-CoV-2 that have emerged in recent months. The B1.351 (501Y.V2) identified in South Africa has raised concerns that the efficacy of current vaccines and therapeutic monoclonal antibodies could be threatened (Tegally et al, 2021). The P.1 (Gamma or Brazilian variant) has contributed to a surge in cases in the northern city of Manaus and it has been reported in January 2021 in Japan. The highly contagious B.1.617.2, commonly known as the Delta, is currently responsible for over 90% of the cases in the UK and over 80% of the cases in the USA and has caused concerns about vaccine efficacy (Lopez Bernal et al, 2021). The background is color-colored based on the neutralizing activity (up) or change in neutralizing activity (bottom). IC50 values are the mean from at least two independent experiments.