EMBO/sd-character-level-ner huggingface dataset:
Knockout of Tefm in heart causes mitochondrial dysfunction.Figure 2Transmission electron micrographs of heart tissue in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 2 μm (upper panel) and 1 μm (lower panel). Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Upper 2 blots; n= 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Bottom 2 blots; ; n= 6 mice at each time-point for each group). VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA. Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The analyzed enzyme activities are NADH coenzyme Q reductase (complex I), NADH cytochrome c reductase (complex I/III), succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV). Barplot of the significantly changed proteins (Benjamini-Hochberg adjusted P <0.05) in Tefm knockout mice compared to control mice (n= 5 mice for each group), organized by individual OXPHOS complexes. Proteins are organized in alphabetic order based on gene name. Bars represent mean log2 [L/L, cre / L/L] ± 95% confidence interval. Boxplots of the protein intensity (LFQ) of TEFM (top) and POLRMT (bottom). Red dot: mean; blue dots: intensity value for each biological replicate (n= 5 mice for each group). Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks (n= 12 mice for each group). VDAC was used as loading control.
Developmental arsenic exposure leads to cognitive deficits and hippocampal genome-wide DNA hypomethylation in mice.Figure 1A Test procedure of NORT (left panel) and the bar graph showing the exploratory preference for novel object of offspring mice (right panel). F, familiar object; N, novel object. B, C 5meC enrichment analysis of the IAP (B), and Xist, H19 loci (C) in the hippocampus of offspring mice from each group, as measured by MeDIP-qPCR. Each of the biological replicate was run in three technical replicates and means were shown as cycles in the graph. D Relative CpG methylation level [log2(fold change)] at each chromosome in the hippocampal DNA samples (ATO vs. Ctr), as determined by MethylC-seq. E Number of hypo- (lower methylation in the ATO group) and hyper- (higher methylation level in the ATO group) differentially methylated tiles (1-kb), as determined by MethylC-seq. F GO enrichment analysis for the DMRs based on the biological process GO categories. Green dot indicates gene number in each term. G Volcano plot showing DEGs in the offspring's hippocampus upon 5000 ppb ATO exposure (n = 2 mice for both control and ATO groups). Dash lines indicate fold change ≥ 2 or ≤ 0.5 and adjusted p value < 0.05. Genes to be further validated for expression levels are labelled. H GO enrichment analysis for the 153 DEGs in the ATO group based on the biological process GO category. Green dot indicates DEG number in each term. I PPI network for the 153 DEGs based on STRING database. Node colors indicate fold changes; node sizes indicate link numbers. J Heatmap showing expression levels (Z-score) of the 12 cognition-related DEGs with node size ≥ 6 in the PPI network. K RT-qPCR validation of the 12 selected cognition-related DEGs.
Figure 3A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue). C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f D A diagram showing the co-culture experiments. shCtr and shM2 H1299 cells stably expressing Mito-GFP were used as donor cells to co-culture with H1299 cells expressing mCherry as recipient cells at 1 : 1 ratio either in direct contact or separated by a filter in transwells. E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter. F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f G Representative images of co-cultured shCtr and shM2 H1299 cells. Cells were fixed after 2 days of co-culture, and confocal images were acquired with 100× objective. The boxed areas are enlarged to show the donor-derived mitochondria in recipient mCherry cells with the corresponding orthogonal views along the thin dotted lines. Scale bar: 20 μm.
Figure 8 The VA-A (purple) appears as an isolated, etv2-expressing, clo/npas4l-dependent angioblast in the anterodorsal yolk sac region at 29 hpf, before migrating ventrally towards the VA and expressing fli1 and kdrl by 34 hpf, and lyve1b at 36 hpf. Around this time, facial lymphatic development in the head begins as the FLS (green) arises from the dorsolateral region of the CCV, while lymphatic progenitors become specified in the PHS (orange and yellow cells). At 42 hpf, the dorsal portion of the VA-A differentiates into the VA-L (pink) as it acquires a lymphatic cell fate by turning on Prox1 expression, while the ventral portion of the VA-A remains Prox1-negative (purple). Both the VA-L and the FLS begin migrating dorsally and anteriorly, respectively, towards the PHS, which at this time contains the PHS-LP (orange and yellow) domains. By 54 hpf, the VA-L has reached the FLS tip, which now consists of the PHS-Ls (orange and yellow) that have sprouted from the PHS and fused to the FLS. By 72 hpf, the VA-L has fused to the FLS to form the LFL, while the ventral Prox1-negative VA-A (purple) fuses to its contralateral counterpart and to the first aortic arch to form the HA. At 120 hpf, all components of the facial lymphatic network are present, with the OLV, MFL and LAAs sprouting from the existing LFL
Figure 7A qRT-PCR analysis of ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3). B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm. C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm. D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3). E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3). F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3). G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.
Determination of translational accuracy in cell-free translation assays.Figure 1A, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity. read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes n=4 read-through), mutant A1555G mitohybrid ribosomes n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity. C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test). D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography. E Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity. F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized. G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.
Our animal model of PF-PH exhibits similar vascular wall cell proliferation and Slug expression to PF-PH patients.Figure 5(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model. Relative expression of Slug mRNA (B) normalized to GAPDH. Relative expression of Slug protein (C) normalized to GAPDH. (D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05) (E) CD68 mRNA quantification in Bleo and Bleo-MCT rats (F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.
Induction of OCT4+/BLIMP1+ PGC precursors from human iPSCsEffects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction.Figure 1Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale. Gene expression dynamics of selected pluripotency, PGC, and mesodermal genes during prolonged PGC‐precursor induction of up to day 7. Y‐axes are in log2 scale. Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm. Quantification of respective cell populations shown in (C).
Figure 3A Heatmaps representing normalized ChIP-seq intensities at MycER-associated promoters or distal sites, as indicated. Each row represents a genomic site called in at least one of the experimental samples, with each column spanning a 4 kb-wide genomic interval centered on the union of MycER peaks. All sites are ranked according to the intensity of the MycERWT signal, and divided based on the presence of the indicated DNA motifs (#1: canonical CACGTG; variants #2-5: CACGCG, CATGCG, CACGAG, CACATG; no E-boxes: none of the above) in an interval of ±100 bp around their peak summit. The data for RNAPII, histone marks (H3K4me3, H3K4me1, H3K27ac) and DNAseI hypersensitivity are from 3T9-MycERWT fibroblasts without OHT (Sabò et al., 2014). B Frequency of peaks (as %) that contain the indicated motif within ±100 bp from the peak summit in each ChIP-seq sample (EV, WT, HEA and RA). I, As in (B) respectively, for the CACGTC motif. C ChIP-seq intensities for peaks (promoters + distal) containing the indicated motifs in an interval of ±100 bp around their summit. For most accurate quantitation, intensities were computed within ± 50 bp from the summit. p-values were calculated using Wilcoxon's test. The central band in the boxplot represents the median of the data, boxes the lower and upper quartiles (25% and 75%) and whiskers the minimum and maximum values. D Density plots showing the distribution of the indicated motifs in a ±100 bp interval from the peak summit. As in (D), respectively, for the CACGTC motif. De novo motif discovery analysis performed underneath the summit of the top 200 peaks called with (E) MycERWT, (F) MycERHEA, infected cells. The position weight matrixes of predicted DNA binding motifs are shown together with their E-values. De novo motif discovery analysis performed underneath the summit of the top 200 peaks called with (G) MycERRA and empty vector (EV) infected cells. The position weight matrixes of predicted DNA binding motifs are shown together with their E-values. H As in (F) for the top 200 MycERHEA -specific peaks (i. e. not bound by MycERWT).
Apg16p interacts preferentially with Apg12p‐modified Apg5p.Figure 4Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3. Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δcells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.
Figure 8(A) Quantification of indicated mRNA levels 2 days after TAC measured by RT-qPCR. Analyzed by One-way ANOVA. n=5-7 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001. (B) LV weight to body weight ratio (mg/g) of WT and Cand2 KO mice subjected to sham and TAC surgeries after 2 days. Analyzed by One-way ANOVA. n=5-7 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001. (C) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT-qPCR. Analyzed by One-way ANOVA. n=3-5 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001. (D) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice. Analyzed by t-test. n=5-8 mice per group. Cul1 neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001. (E) Model of Cand2-dependent stabilization of Grk5 by Cul1 neddylation inhibition in mTOR-dependent manner.
Figure 1. Experimental scheme for B-H. Whole-cell patch-clamp recordings were made from nearby pairs of uninfected and infected neurons in CA1 of organotypic hippocampal slices infected with lentivirus carrying shRNAs against GSK3α or GSK3β. EPSCs were elicited with CA3 Schaffer collateral stimulation. B. Representative traces for C-H, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and shRNA-expressing neurons (green traces). C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. F, G. As C, D, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. H. As E, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.
Figure 4(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM). (B) Heatmap displaying transcript wise scaled expression of NOTCH1, HES1, MYC and miRNAs from ex vivo isolated subsets correlating (spearman rho >= 0.5) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR-17~92 cluster (n=2). (C) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions (OP9-control, OP9-JAG1, OP9-JAG2, OP9-DLL1 and OP9-DLL4) and time points (day 0, 5 and 10). (D) NOTCH1 correlating miRNAs (spearman rho >= 0.3) of in vitro cultured thymocytes across multiple culturing conditions and timepoints. The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR-17~92 cluster (n=2). (E) CHIPseq data for Notch1 and RBPj binding at the miR-17~92 locus in CUTTL1 cells. Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample.
Figure 4(a) Proteomics workflow. (b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29). (c) The heatmap shows the correlation coefficients between proteins (rows) and clinical parameters (columns). (d) The heatmap illustrates the computationally derived meta lung function variable combining multiple lung function parameters. (e) The volcano plot shows the multivariate regression coefficients (x-axis) and the -log10 p-value (y-axis) for BALF protein abundance with the meta lung function.
Substrate stiffness affects motility patterns of ookinetes.Figure 1(A) Ookinetes sandwiched between two hydrogel surfaces. (B) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B. (D) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint. (F) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels. (G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.