EMBO/sd-character-level-ner huggingface dataset:
In vivo installation by base editing of oncogenic mutations in a model of triple negative breast cancer.Figure 1Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K. EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors. Overview of the intraductal injections performed in WapCre;Brca1F/F;Trp53F/F;Col1a1invCAG-BE3/+ (WB1P-BE3) females with high-titer lentiviruses encoding Myc cDNA and either a non-targeting (NT) sgRNA (Lenti-sgNT-Myc) or the sgRNA targeting Akt1 (Lenti-sgAkt1E17K-Myc). Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc (n=12) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (58 days after injection vs 72 days after injection, P < 0.01 by Mantel-Cox test). Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc.
a) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.f4(a) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance). (b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test). (c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments. (d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student's t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown). (e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student's t-test). (f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student's t-test).
Effects of XEN-D0101 and XEN-R0703 on APs of hESC-ventricular and hESC-atrial CMs.Figure 8A Representative APs of VM and AM CMs in the absence, presence and following washout of 3 μmol/l XEN-D0101. AP parameters are shown in Supplementary Table S7. B Representative APs (1 Hz) of VM and AM in the CCh, to activate IK,ACh and subsequent addition of XEN-R0703. AP parameters are shown in Supplementary Table S9. C, D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1, 3 and 10 mg/kg XEN-R0703 show (C) mean right AERP values (left), mean Van de Water's QTc (right) and (D) AF inducibility plotted as a function of dose.Data information: For RAP dog experiments, n = 5; statistical significance tested with paired t-test. Data are presented as mean ± SEM. AERP = atrial effective refractory period; AF = atrial fibrillation; RAP = rapid atrial pacing; N.S. = not significant. Other abbreviations as in Figs3, 6 and 7.
Figure 1A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM, 10 µM, or 20 µM CDDP for 3 days. The explants were labeled with Myosin 7A (red) to identify hair cells and NF200 (green) to highlight auditory nervefibers. Scale bar=20 µm. nf: nerve fibers, OHCs: outer hair cells, IHCs: inner hair cells. B Dose-response curves of CDDP-induced loss of OHCs (blue line) and IHCs (red line) in basal cochlear regions. Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point). C Effect over time on OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively). Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point). D Representative Western blot analysis using antibodies against β-actin and cleaved caspase 3 in whole cochlear extracts.E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group (n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P ≤ 0.028, **P=0.0007, cochleae at the different times after CDDP exposure vs. control 12h). F, G Confocal images showing the basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 (F) or 2 (G) days. Hair cells were identified using Myosin 7A (red), phosphatidylserine sites on the cell membrane surface were detected using fluorochrome-labeled Annexin V (green in F), and apoptotic DNA fragmentation was identified using a TUNEL apoptosis kit (green in G). The white arrow heads indicate cell surface Annexin V-positive labeling (lower left) and TUNEL-positive nuclei (lower right). Scale bar=15 µm.
Figure 3(A) Main T6SS cluster of A. baylyi. Genes marked in yellow are proteins with potential regulatory functions. (B) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in the absence of TslA. Data information: Scale bars and time intervals are indicated. (C) Time-lapse 3D-SIM imaging of sheath dynamics (TssB-sfGFP) in ΔtslA A. baylyi strain with ectopically expressed tslA (tssB-sfGFP clpV-mCherry2 ΔtslA ampC::PtslA-tslA-KanR). Yellow arrows indicate examples of contact-dependent sheath assembly. Data information: Scale bars and time intervals are indicated. (D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed. (E) Cellular localization of TslA in A. baylyi cells. Cells of the strain (tssB-sfGFP) were fixed by 2% paraformaldehyde and treated with GTE buffer, then probed with anti-TslA antibodies, followed by incubation with secondary antibodies conjugated with Alexa Fluor Plus 594. Signals were detected using a widefield microscope. Phase contrast, GFP, RFP and merged images are shown (GFP: TssB; RFP: TslA). Data information: Scale bars and time intervals are indicated.
Anaerobic growth of the different strains.Figure 1A) Time course of biomass concentration. B) Time course of glucose concentration. C Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD. C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). D) Yield of Eth, Ace, For, Lac, and Suc. Data information: The reaction rates in C were calculated for the exponential phase under assumption of quasi-steady state. As changes in glucose and fermentation products are rather small during cultivation of the HC ATPase strain, a higher initial biomass concentration of 0.1 gDW/L was used for this strain to get more robust data for the calculation of the metabolite exchange rates. The means ( and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD.
Figure 3A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days. B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student's t test: ***p<0.001. C: reduced versus oxidized cytochrome spectra. Peaks at 550, 560 and 602 nm correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline is an index of cytochrome content. D: respiratory chain complex activities. Biochemical activities of Succinate Quinone DCPIP Reductase, SQDR (CII), NADH-cytochrome c oxidoreductase activity NCCR (CIII), and cytochrome c oxidase (CIV) were measured on a mitochondrial-enriched fraction from cells grown at 37°C as in panel B. Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD. Statistical analysis was by unpaired, two-tail Student's t test: **p<0.01; ***p<0.001. E: Western-blot using an anti-HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C-terminus. Western-blot analysis on total protein extract. Total protein extracts were obtained by strains expressing HA-tagged CYM1wt and cym1R163Q. PGK was used as a loading control and signals were normalized to the wt. The quantification was performed on 5 independent blots.F: prolonged exposure of a Western-blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes (arrow). G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.
Figure 1(A) Measurement of maximum glycolytic capacity of MOTS-c in dystrophic muscle cells after oligomycin and 2-DG treatments (n=6; *p<0.05). (B) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS-c (n=6; *p<0.05). (C) ATP assay for dystrophic muscle cells in the presence of MOTS-c (n=6; *p<0.05). NC refers to untreated H2K or H2K mdx cells. (D) Tissue distribution and quantitative analysis of Rhodamine B-labeled MOTS-c in mdx mice after single intravenous injection (500 μg) (n=6; *p<0.05, *p<0.001). Tissues were harvested and examined 2 h after injection. This experiment was repeated twice. NC refers to untreated mdx mice. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle, D-diaphragm, H-heart, Li-liver, S-spleen, Lu-lung and K-kidney. (E) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS-c (500 μg) (n=6; *p<0.05).
Figure 6A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value. B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value. D Model for RCAN1 suppressive control over two prominent mechanisms of mammalian nonshivering thermogenesis (NST): one in white adipose tissue, where uncoupling of the mitochondrial membrane potential by UCP1 increases heat generation at the expense of ATP production; the other in skeletal muscle, where SLN increases ATP consumption by SERCA. In adipose, RCAN1 suppresses the conversion of white adipocytes to UCP1-positive, beige adipocytes by acting as a brake on calcineurin (CN)-dependent activation of Pgc1α-1b expression, thereby blunting downstream activation of Ucp1 expression. In skeletal muscle, RCAN1 suppresses CN-dependent expression of Sln directly. Loss of these repressive functions underlies the elevated metabolic rate and lean phenotype of the Rcan1-KO animals.
Using the enzyme-constrained model for metabolic engineering applications.Figure 6Figure 6: Using the enzyme-constrained model for metabolic engineering applications. (A) The physiological response of knocking out NDI1 can be reproduced using ecYeast7. Wild-type and knockout experimental data are shown, together with model simulations (continuous and segmented lines, respectively). (B) Succinate yield (grams of succinate per grams of substrate) versus biomass yield (grams of dry weight biomass per grams of substrate) simulations under batch conditions, compared with experimental data. Both glucose and ethanol phases are shown. (C) Farnesene yield (grams of farnesene per grams of glucose) versus biomass yield (grams of dry weight biomass per grams of glucose) simulations under fed-batch conditions, compared with experimental data.
Figure 1Schematic representation of the smFRET assay using crystal-structure snapshots of McjD. On the right side McjD is also shown as a cartoon. In the crystal structure nucleotides and ATP-Mg in orange sticks, the peptide MccJ25 in pink. Each half transporter is colored in dark and light grey. McjD is shown in a view along the plane of the membrane which is depicted in light grey. Colored balls show the position of the cysteines used for probing conformational changes. PBD codes for McjD in inward-occluded conformation are 5OFP (left) (Bountra et al, 2017) and in the outward-occluded conformation 4PL0 (right) (Choudhury et al, 2014) Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified protein Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency
Figure 3A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss). B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm C Principal Component Analysis (PCA) based on bulk RNA-sequencing of C57#1 DLP and C57#3 DLP mouse prostate organoids cultured in normal conditions (ENRAD) (n = 3 biological replicates, C57#1 DLP; n = 3 biological replicates, C57#3 DLP), upon acute A83-01 depletion (-A83-01, 24 hours) (n = 3 biological replicates, C57#1 DLP only) or adaptation (-A83-01, long-term) (n = 3 biological replicates, C57#1 DLP; n = 3 biological replicates, C57#3 DLP). D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21. F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).
Figure 6A Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin. Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B) a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C). D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin. Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains. Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains. Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors. Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors. K Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors. L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).
Atg5‐deficient goblet cells accumulate mucin.Figure 1(A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm. (B) Quantification of average mucin area/goblet cell in control and Atg5VC mice (n=5-7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A). (C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse). (D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm. (E, F) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test. (G, H) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.
Figure 5Representative images (A), cfu (B and qRT-PCR quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0.5 hpi Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria