EMBO/sd-character-level-ner huggingface dataset:
Figure 3The number of distal DNAse I hypersensitive sites (DHSs) of mtDNA maintenance genes and genes in the genomic POLG locus correlating >0.85 with target promoter DHS across 125 cell lines. Abbreviations: TWNK, Twinkle protein; POLG, DNA Polymerase gamma, catalytic subunit; POLG2, DNA Polymerase gamma, accessory subunit; SSBP1, Single stranded DNA binding protein 1; TFAM, mitochondrial transcription factor A; FANCI, Fanconi anemia group I protein; RHCG, Ammonium transporter Rh type C; TICRR, Treslin. Distribution histogram of DHSs for mtDNA maintenance genes. Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region. Genes surrounding LINC00925. Conservation of regulatory elements of POLG genomic locus in species: LINC00925, POLG EEs, MIR9-3. Abbreviations: PanTro, Pan troglodytes; MM, Mus musculus; MonDom, Monodelphis domestica; Xen, Xenopus levis. Adapted from https://rvista.dcode.org. Expression of LINC00925 and POLG in different human tissues and cell types. Quantitative PCR amplification of cDNA. Abbreviations: iPS, induced pluripotent stem cell; SH5Y, neuroblastoma line; HepG2, liver hepatocellular carcinoma line; U2OS bone osteosarcoma line. Error bars indicate standard deviation in 3 technical replicates. AU; arbitrary units. Polg and long-non-coding RNA Ai854517 (mouse homologue of human LINC00925) correlate tightly in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS). Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; cerebellar molecular layer (M), Purkinje cell layer (PC), granular cell layer (GC). Scale bars: hippocampus 760 µm, cerebellum 300 µm. Expression levels of MIR9-3 and Ai854517 correlate in mouse cerebellar development. Time points: E18, postnatal day 0, 3, 6, 9; three mice per time point.. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS). Predicted targets of MIR9. Six targets were predicted by all prediction programs, TargetScan, PicTar, miRDB and PITA: low density lipoprotein receptor adaptor protein-1, LDLRAP1; methylene-tetrahydrofolate-dehydrogenase-2, MTHFD2; follistatin-like-1, FSTL-1, cytokine; capping-Actin Protein of muscle Z-line alpha-subunit 1, CAPZA1, involved in actin polymerization; PR-domain zinc-finger-domain-1, PRDM1, a repressor of beta-interferon expression; PRRX, a homeobox protein regulator of bone development. One cut homeobox 1 and 2 (ONECUT1 and ONECUT2) and nuclear receptor subfamily 2 group E member 1 (NR2E1) are recently discovered MIR9 targets. RNA expressions of MIR9, NR2E2, LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre-MIR9 or scrambled RNA. Shown is mean with standard error of the mean. Statistical testing was performed using 1-way ANOVA with Dunnet's correction for multiple comparisons. Abbreviations: AU; arbitrary units.
Figure 2A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± s.d., N = 3. B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control. C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post control or Ad-Cre infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test. E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA. F C-circle assays shows increased C-circle formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection. G, H 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (H). Scale bar, 10 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.
a–c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min.figf3(a-c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody (a) and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified (b) or subjected to immunoblotting (c) using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (c). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 2A Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n=5) or healthy controls (n=5) by RNA-seq. Data information: All data are shown as mean ± SD. For (A), P values are determined by Student's t-test B Illustration depicts generation of the animal model with SARS-CoV-2 spike RBD-induced intestinal inflammation. Mice were fasted for 24 hours before acetic acid clysis. 16 hours after acid challenge, mice were given Control-Fc or Spike RBD-Fc intraperitoneally. Six hours later, TRITC-dextran or Evans Blue dye was injected intravenously for evaluation of permeability. C, D Representative H&E images (C) and the quantitative analysis of inflammation (D) of the intestinal tissues from animals treated by the protocol shown in (B). The inflammatory infiltrates were indicated by a yellow arrow. Scale bar, 100 µm. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). E qRT-PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test. F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (F) P values are determined by Student's t-test G ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test
Metabolic rewiring by iron depletion is hallmarked by impaired lipid homeostasis.Figure 2a. Hierarchical clustering heatmap analysis of the 60 most altered metabolites in cells treated with DFP for the length of time indicated. Each coloured cell on the map corresponds to a z-score value. The list was generated using MetaboAnalyst software and determined with an ANOVA test with a P<0.05 threshold followed by post-hoc analysis. The hierarchical clustering method of one minus Pearson correlation was applied (n=3, with 4 technical replicates per biological replicate). b-d. Volcano plots representing the difference in the mean of metabolites from DFP treated cells compared to control cells at acute (30 min), 8 h and overnight (24 h) time points, generated by unpaired T test with an FDR of 0.01. Coloured points represent significantly altered features. e-g. Pathway analysis generated in MetaboAnalyst using the significantly altered metabolites from the volcano plot comparisons. Statistical analysis was determined by hypergeometric test and the library used for pathway analysis was SMPDB.
Migrating interneurons express neurotransmitter receptors A.Figure 3A. UMAP depicting three pseudotime trajectories - two MGE trajectories and a CGE trajectory. Additionally, visualization of each trajectory on isolated UMAPs. After removal of the clusters relating to LGE/striatum and the TH cells, a total of 2968 cells were used for pseudotime analysis. B. Visualization of the relative gene expression for characteristic genes along the pseudotime of the three trajectories. The genes visualized are color-coded to represent their respective pseudotemporal expression. C. UMAPs depicting the relative expression of migration genes CXCR4 and EPHA5. Visualization of the gene expression of migration genes CXCR4 and EPHA5 along the pseudotime axis. D. UMAPs depicting their relative expression of neurotransmitter genes GRIA4 and GRIA2 along with visualization of their gene expression along the pseudotime axis. E. Heatmap depicting the expression of various neurotransmitter genes for all cells across the 7 clusters analyzed. Clusters are color-coded and labeled according to depiction in 2B. Clusters are labeled to reflect clusters with progenitors, migrating interneurons and mature interneurons.
Transcriptome profiling of pax2amKO2-High and pax2amKO2-Low thyrocytes (A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS.Figure 9(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression. (D , E) RNA-Seq. analysis of transcriptome isolated from pax2amKO2-High and pax2amKO2-Low subpopulations. (D) Volcano plot representing the differential expression of genes between the two thyrocyte subpopulations. Dots for pax2a, tg, mKO2 and EGFP are marked. Dots for genes that display significant differential expression (DESeq2, adjusted p-value < 0.05) are coloured. (E) Heatmap displaying the expression of selected genes in the two subpopulations. Biological replicates are represented by individual columns.
Let‐7 and miR‐125 miRNAs rescue astrocyte differentiationSchematic of screen procedure.Figure 4Schematic of screen procedure. Results of miRNA screen in Dgcr8Δ/Δ GPCs. Each dot represents one well in the screen. Mock‐treated wells were used as control and are shown in blue. Confirmation of let‐7 and miR‐125 screen hits with independently synthesized mimics. Quantification is shown on the right (n = 4). Synergistic rescue of GFAP expression by let‐7 and miR‐125. Total concentration of mimics in wells with miRNA was 50 nM (n = 3). Quantification of GFAP‐positive cells in each well normalized to the mean of the experiment is shown on the right. RT-qPCR for panel of astrocyte markers in mock and let‐7 + 125 treated Dgcr8Δ/Δ GPCs following 48 h of differentiation (n = 3). Representative blot of pSTAT3 levels 14 h post‐differentiation following let‐7 and miR‐125 addition in Dgcr8Δ/Δ GPCs (n = 3).
Lamplight expression in degenerate retinas can restore diverse and switchable responses to lightA) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV.Figure 3A) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV. Anti-mCherry staining (white) was widespread (Left, scale bar = 500µm). Fluorescent cell bodies found in different focal planes consistent with expression in ganglion (Top Right, scale bar = 50µm) and bipolar cells (Bottom Right, scale bar = 50µm). B) Representative single unit showing characteristic ipRGC response. C) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm). D) Representative single unit showing light responses over 20 stimulus repeats. E) Distribution of response latencies for 3 Lamplight-driven responses to 405nm, as well as reversal caused by 525nm in sustained inhibition units. Two outliers with slow latency (>2.5s) in transient excitation group are likely responding to stimulus offset (N = 35 for Transient Excitation, N = 6 for Transient Inhibition, N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation, all biological replicates). F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates).
BMDMs derived from Atg5−/− mice are defective in clearing the WT L.Figure 4(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test. (B) Western blot showing the absence of Atg 5 in the knockout. Data are representative of three independent experiments.
a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.figf6(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC. (b) Immunoassay of differentiated THP-1 cells treated as in a with protein-dissociation buffer, followed by immunoprecipitation of ASC (24 kilodaltons (kDa)) and immunoblot analysis. (c) Confocal microscopy of differentiated THP-1 cells transfected for 2 h with poly(dA:dT), immunostained for ASC (red) and polyubiquitin (green). PolyUb, polyubiquitination. Scale bars, 10 μm. Brackets (right margins in a,b) indicate smear of ubiquitinated proteins. Data are representative of three (a,c) or two (b) experiments.
Figure 5A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3). B, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors). B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).
Figure 5A-C Live confocal images of ddaC dendrites expressing tdEOS::α-tubulin driven by ppk-Gal4. The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 (A, right panels) and WP stages (B, right panels), and increased in Efa6-overexpressing neurons at wL3 stage (C, right panels), compared to the control neurons (A-C, left panels). D-F Quantification of microtubule turnover rate as indicated by the remaining fluorescence intensity of converted EOS after 30 min.
Transcription factors, PGC-1α, and citrate synthase are differentially regulated in SOD1G86R mice.Figure 5A-D Relative mRNA levels of (A) Pparß/δ, (B) Foxo1, (C) citrate synthase, and (D) Pgc-1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Pparβ/δ **P = 0.0016 at 65 days; Foxo1 ***P = 0.0001 at 105 days, citrate synthase ***P < 0.0001 at 105 days, Pgc-1α *P = 0.0104 at 105 days (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test). E Representative Western blot of PGC-1α and respective actin that was used for normalization. PGC-1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age-matched WT. **P-values versus WT: 0.02 (n = 6/genotype at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test). F Relative mRNA levels of ERRα, Mfn2, and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: ERRα ***P < 0.0001, Mfn2 **P = 0.002 and Nrf1 **P = 0.009 (n = 6-7 and 7-8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
Figure 2(A) Sequence alignment of the WW domains derived from the WW domain-containing proteins that are known to bind the Hippo PY motif-containing proteins. The two conserved tryptophan restudies were highlighted in purple. Additional conserved amino acid residues were highlighted in yellow. (B) Summary of the residue difference in the identified 9-amino acid sequence for the control WW domains. The conserved two tryptophan residues are labelled in grey; the changed residues are labelled in orange; and the unchanged residues are labelled in white. (C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.
Figure 2Representation of the magnetic tweezers Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifen Quantification of PSCs stiffness Histogram shows percentage of gel contraction, n>10 gels per condition Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions Representative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions Histogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonist Histogram shows percentage of gel contraction after treatment with indicated agonist, n>10 gels per conditio
Figure 4Quantitative mRNA expression of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9). western blotting of ANG and PLXNB2 in isolated IECs or colonic laminar propria (cLP) cells from WT mice (n = 9). Representative images showing immunofluorescence staining of ANG, PLXNB2 and macrophage surface marker F4/80 in frozen colonic section from WT mice. Representative images showing immunofluorescence staining of GFP and PLXNB2 in frozen colonic section from WT mice infected with adeno-associated virus (AAV9). PLXNB2 expression in IECs from the AAV9-infected WT mice (n = 3). Body weight loss of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment. DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment. Colon length of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. serum FITC-Dextran level of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. representative HE staining image, and (K) histopathological score of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.
Development of antinuclear antibodies in lymphotoxin‐deficient mice is influenced by gut microbiotaLtbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA.Figure 3Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months. 16S rRNA gene analysis of luminal samples of wild‐type, ANA‐positive, and ANA‐negative Ltbr−/− mice. Left panel: Beta diversity plot using multidimensional scaling on Bray-Curtis dissimilarities distances. Right panel: Hierarchical cluster analysis using Pearson's correlation. Bacteria community compositions differ in the different groups. Please note that Candidatus arthromitus, according to Thompson et al (), should be renamed Candidatus savagella. Real‐time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA‐positive and ANA‐negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m. C3H/HeN germfree pregnant mice were treated with the LTβR‐Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day. LTβR‐Fc‐treated wild‐type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months