EMBO/sd-character-level-ner huggingface dataset:
Figure 2A. Schematic illustration of annexin A4 based co-translocation assay of potential mATG8 binding peptides and their target mATG8 to membranes upon calcium influx induced by ionomycin treatment. B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2. B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2. D. Biotinylated octameric peptides representing a single amino acid walk-through along the complete 16mer peptide AS_67 and AS_M67 are immobilized on streptavidin. Binding of GST-LC3C fusion-protein to the immobilized peptides is detected by a GST specific antibody (HRP-conjugate) and shown as absorbance at 450 nm. AU=arbitrary unit B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2. F. Translocation efficiency of the unmodified peptide AS_67, triplicate AS3_67 and charge optimized peptide AS_M67 is classified as % of cells that show no translocation '-' to very good '++++' translocation with EGFP-LC3C; n = total number of analyzed cells. Data are presented as mean of three biological replicates ±SD.
Figure 1Relative expression levels of the HIF1A-AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT-PCR analysis and represented as 39 minus Ct values. Sanger sequencing validation of RNA editing events on HIF1A-AS2. The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1. Asterisks mark the sites undergoing statistically significant increases in RNA editing. The distributions of the confirmed HIF1A-AS2 RNA editing sites (red ticks) are shown in UCSC Genome Browser representation, in relation to the HIF1A-AS2 gene locus. The transcript structure of the HIF-1A, HIF1A-AS2, and HIF1A-AS(m) (a splicing form of HIF1A-AS2) genes are also presented, with the identified exons indicated by blue boxes. Secondary structure of the full-length HIF1A-AS2 transcript was predicted by using the online database RNAfold. The region encompassing the double-stranded structure is shown with the RNA editing sites, as indicated. RNA-IP of ADAR1 in HUVECs, followed by real-time PCR analysis of the HIF-1A and HIF1A-AS2 RNAs. The relative enrichment of transcript association was normalized to the IgG. GAPDH was used as a non-binding control. Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR. Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in subcellular fractions (G), were determined by qRT-PCR. FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.
Figure 4Representative (n=3) WB analysis demonstrating that while GAPDH-HA control do not bind (A FEZ1-Flag control pelleted with in vitro assembled HIV-1 CA-NC complexes. Representative (n=3) WB analysis demonstrating that while EB1-Flag do not bind pelleted with in vitro assembled HIV-1 CA-NC complexes. Representative (n=3) WB analysis demonstrating that while Flag-CLIP170 pelleted with in vitro assembled HIV-1 CA-NC complexes. (D) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates (n=3) from C. Statistical significance was determined by t test (mean ±SEM) *** = p<0.001. (E) Representative WB analysis (n=3) showing the levels of HIV-1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation. Fractions 1-3 contain free p24 not associated with the intact cores, while fractions #8 and # 9 harbouring intact HIV-1 cores. (F) Flag-CLIP170, but not Flag control, also binds to fractions containing intact HIV-1 cores (Virus #8 and #9) but not with Mock fractions. (G) HIV-1-WT-luc infection is increased in CHME3 cells stably expressing Flag-CLIP170, but not control Flag or Flag-CLIP170ΔCAPgly. Molecular weight markers (in kDa) are shown to the right of WBs. (H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.
MCK1 is necessary for secondary wave of Msn2/4 activity Epistatic interactions of Δmck1 in the production rate parameter (See 3E).Figure 4Epistatic interactions of Δmck1 in the production rate parameter Epistatic interactions of Δmck1 in the total open time parameter Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).    Δmck1 effect on the response of all stress genes. The integral over mRNA values in WT strain (X axis) vs. the integral in the Δmck1 strain (Y values). The inset shows the integral for the HSP12 gene.   Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints (See 2C). Each gene is presented relative to its pre-stress level and normalized by the median over WT values to visualize the dynamic range in all genes. The genes are separated into MSN dependent and independent genes (Figure EV4B). The MSN-dependent genes are sorted based on the onset time to three groups (Early, Average and Late onset). The behaviour of early and late onset genes. Similar to (C) but showing the average over the genes in each group.
Figure 6The colors corresponding to each structure are indicated in the legend. Note that the cartwheel structure protrudes proximally from the microtubule wall; here, one unit has been depicted that corresponds to an external cartwheel of about 25nm. The cartwheel's structural unit consists of 3 ring-pairs, from which emanate 6 radial spokes that merge into a single D1 density before contacting the D2-rod adjacent to the pinhead structure. The pinhead and the A-C linker are connected through the triplet base. The A-C linker extends more distal than the cartwheel and co-exists with the inner scaffold structure.
Figure 6A. The stochasticity of deposit formation due to phase separation will define the foci size, which in turn will influence the amount of free protein in the cytosol. The abundance of the free protein (or the phase separated structure) will ultimately determine the cellular phenotype. The behavior and phenotypic outcome may also be influenced by the molecular structure and properties of the phase separated structure (e.g. complex coacervates, liquid droplets, etc.) B. Timeline for the different processes that are important for adaptation/response to an environmental change. Time estimates were obtained from published datasets
Figure 4(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion. (B) GO functional analysis of the 109 candidate gene-set from (A). (C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance. (E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).
Figure 7WT C57BL/6 mice were s.c. inoculated with PBS (mock, n=3) or 103 PFU of CHIKV (n = 3) in the left rear footpad. At 24 hpi, the dLN was collected and enzymatically digested into a single cell suspension. Cells were enriched for CD45- cells and analyzed by scRNA-seq as described in the materials and methods. (A) UMAP projection shows each replicate for mock- and CHIKV-infected mice; the number of cells obtained for each replicate is shown at the bottom. (B) UMAP projection shows annotated cell types (top) and the proportion of cells identified for each cell type (bottom). (C) UMAP projection shows endothelial subtypes (top) and the proportion of cells identified for each cell type (bottom). Non-endothelial cells are shown in white. (D) UMAP projection shows Marco expression. (E) UMAP projection shows the fraction of counts that align to the CHIKV genome. (F) UMAP projection shows cell types for cells classified as CHIKV-high. CHIKV-low cells are shown in white. The proportion of CHIKV-high cells belonging to each cell type is shown on the right. (G) The fraction of counts that align to the CHIKV genome is shown for CHIKV-high cells. Only cell types that include >20 cells are shown. (H, I) MARCO (H) and Mxra8 (I) expression is shown for MARCO+ LECs for mock-infected cells and CHIKV-infected cells classified as either CHIKV-low or CHIKV-high. P values were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction. In the boxplot, the central lines, the box limits, and the whiskers represent medians, the interquartile range (IQR), and min/max values that are not outliers, respectively. Outliers are shown as points and include any values that are more than 1.5x IQR away from the box.
| Conditional deletion of DND1 causes similar but not identical phenotypes to those of Nanos2-KO male gonocytes.Figure 4A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies. B-P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B-D), STRA8 (E-G), SYCP3 (H-J), activated-Caspase3 (K-M) or LAMININ (N-P) (green). Germ cells were immunostained with TRA98 (B-D) or DAZL (N-P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K-P, 50 m in E for E−J. See also Fig. EV3.
Figure 6(A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test. (C) Top: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of changes in ATAC-seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation, classified according to the underlying chromatin state in mouse ES cells; Bottom: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ChIP-seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions. Number of peaks in each group is indicated at the bottom. (D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks.
a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters.f3(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm. (b) Real-time imaging of an individual autophagosomal structure, showing one deconvolved image per 20 s. The ATG5 ring formation phase is defined as the steps up to establishment of the maximum diameter of the ring structure. The following steps, defined as the decay phase, being more dynamic, are depicted also at higher time resolution. Single-plane merged images of ATG5-GFP (green) and mCh-ATG8 (magenta) channels, deconvolved. Scale bar, 1 μm. (c) Representative FRAP experiment on mCh-ATG8 during the ring formation phase. Deconvolved images. Scale bar, 1 μm. The corresponding fluorescence recovery kinetic, measured on non-deconvolved images, is shown on the right. (d) Representative FRAP experiment of mCh-ATG8 during the ring decay phase, presented as in c. (e) Box and whiskers representations of fluorescence recoveries during ATG5 ring formation (n=11) and decay (n=17). Whiskers comprise the 10-90 percentile, the box the two central quartiles. The mean recovery values are indicated by an horizontal line. Recoveries during ATG5 ring formation and ring decay were significantly different according to the Mann-Whitney statistical test (***P0.001).
To promote exclusion of non-cargo material, cargo receptors interact with Atg8 through multiple sites to wrap the membrane around the cargo material.figf7
Figure 1Experimental procedure: 114 compounds approved by the FDA or in clinical trials are assessed by the Opera High Screening system for their ability to reduce TRF1 protein levels in CHA9.3 lung cancer mouse cells. Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates. Schematic representation of the novel TRF1 regulatory pathways. Data are represented as mean ± SEM. n represents biological replicates. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3A and B. Molecular models show the position of mutated residues in the structures of two dimers on DNA: Sox2-Oct4 heterodimer (A) and Oct4-Oct4 homodimer (B). Ser in position 151 (asterisk) was mutated to Met in order to shift the preference of Oct4 for homodimerization. The individual helixes of the POU domains are numbered. C. On the left side, a schematic overview of Oct4 and its mutants used for iPSC generation. POUS and POUHD are shown as green bars connected by the linker. Sites mutated to the respective Oct6 residues and Oct6 linker are denoted in orange, and the mutation in the Sox-Oct interface is in black. The efficiency of these constructs for iPSC generation from MEFs is depicted as the absolute number of GFP-positive colonies on the right. Error bars represents standard deviations of biological replicates (n=3) and differences between selected samples were compared using ANOVA (p-values: Oct4WTxOct47D,22K p = 6.9e-3; Oct4WTxOct4LinkO6 p = 7.3e-4; Oct4WTxOct4151M p = 1.2e-2; Oct4151MxOct4LinkO6,151M p = 1.1e-2; Oct4LinkO6,151M xOct47D,22K,LinkO6,151M p = 7.1e-3) (*** p < 0.001, ** p < 0.01, * p < 0.05). D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.
MAKR5 loss-of-function confers CLE45 resistance.Figure 3(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments. (B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots. (C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm). (D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem. (F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings. (G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files. (H) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown). (I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.
Figure 1(A) Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day -1. All animals received in vitro-generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17. Mann-Whitney-U-test was performed on mean clinical scores (n=11PBS/13DTX); * p < 0.05, ** p < 0.01; see also Appendix Table S1. Pooled data from two independent experiments. (B) Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17. Flow cytometry was performed on surface stained cell samples. Cells were pre-gated on lymphocyte cells (FSC SSC gate) and PI-negative; pooled data of three animals; representative of two independent experiments. (C) Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFPTh17-transferred CD11c-DTR/GFP→C57BL/6mice 2-3 d after onset of the disease. Upper panels: cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-y, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3; pooled data from three animals, representative for two independent experiments.(D), (E) Quantification of cytokine expression data as shown in (C) for pooled data from 2 independent experiments; Mann-Whitney-U-Test * p < 0.05. (F) 2d2Th17 were transferred into lymphopenic Rag2-/-c-gamma-/-. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28.(G) GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2Th17 cells (see also (F)). Each dot/square represents a single animal; Kruskal-Wallis-test; * p < 0.05, ** p < 0.01, **** p < 0.0001.
Flow diagram of machine learning analysis.Figure 3The discover cohort consisted of only Illumina datasets and was used for feature selection using the GRRF algorithm. For the validation cohort Affymetrix datasets were also included. A cohort of unknown samples consisted of pre-challenge baseline samples of participants who stayed well following challenge, their respective nD7 samples (7 days after challenge), and febrile, culture-negative suspected enteric fever (sEF) cases from Nepal. Refer to Supplementary Table S2 for study identifiers. 03NP: Nepali cohort. T1: Oxford typhoid CHIM study 1. T2: Oxford typhoid CHIM study 2; P1: Oxford paratyphoid CHIM.
Figure 4(A) Generation of Reno1m/m and Bahcc1m/+ ES cells using CRISPR-Cas9. Primers used for colony screening marked by arrows. (B) Expression levels of Reno1 (both the spliced and unspliced isoforms) and Bahcc1 following four days of differentiation of the indicated cells. Mean ± SEM is shown for 3-4 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test). (C) Left: Oct4 staining of the indicated cells. Scale bar: 200 µm. Right: Cell count of the indicated ES cells grown in ES medium. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. (D) Left: Nestin staining of the indicated cells following four days of differentiation. Scale bar: 200 µm. Right: Cell count of the indicated cells following four days of differentiation. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. ** P<0.01 (unpaired two sample t-test). (E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity.
Figure 4Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end). (B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).
EBV-LMP1 fixes ANT1 at the m-state to inhibit ADP/ATP exchange.Figure 4(A-B) The ADP/ATP exchange rate in EBV-LMP1-positive nasopharyngeal carcinoma cells is decreased. The fluorescence value of Magnesium Green ™ (1 μM) was measured at 505 nm (Ex)/535 nm (Em) by enzymography (A), and the ADP/ATP exchange rate was calculated after conversion to ATP (B). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, ** p < 0.01). (C) LMP1-negative or -positive NPC cells were treated with BKA (1 μM) or CATR (5 μM) for 24 h, Mitochondrial OCR assessed with the XF24 extracellular flux analyzer (Seahorse) under sequential treatment with oligomycin, FCCP, and antimycin A. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (D) Oxidative phosphorylation-related metrics based on OCR datas. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05,** p < 0.01).