EMBO/sd-character-level-ner huggingface dataset:
Two conformations of eIF3j in human and yeast 43S PICs.Figure 4A Overview and zoomed view on the model of human 43S PIC II (lacking eIF1A), focusing on the two protomers of the dimeric eIF3j 6-helix bundle in the ISS. eIF3j is in close vicinity to NDB1 of ABCE1 but only forms contacts to the 40S. The mRNA channel is indicated by a dashed gray line. B Two different views showing the interaction of the two Homo sapiens (H.s.) eIF3j protomers with the 40S. C Same views as in (A) on the model of the yeast 43S PIC. Here, eIF3j (Hcr1) is turned approximately 100 degrees around a pivot formed by the C-terminal helices contacting eS30 and uS12. Protomer 1 thereby contacts eIF1A and the FeSD of ABCE1 and protomer 2 contacts h33. D Two different views showing the interaction of the two S.c. eIF3j protomers with the 40S and ABCE1. E Overview and zoomed view highlighting the position of the eIF3j C-terminus in the yeast 43S-PIC-XL structure. F Zoomed views focusing on interactions of the eIF3j C-terminus with the 40S. The loop following the third helix of eIF3j protomer 2 in a pocket formed by the 40S h33, h34 and eS10. Lys230 of eIF3j C-terminus (protomer 2) and Phe59 of eS10 are sandwiching the flipped-out G1435 base of h34 (upper left); Lys231 and Lys234 interact with h33 (U1266 and G1267) and h34 (G1438) (upper right); salt bridges between Arg220 and Glu70-Glu71 of eS10 further stabilize the loop (lower left). Following the loop, the eIF3j C-terminus bridges the 40S body and head in the latch and contacts are formed with h18 (via Lys236) and via hydrophobic interactions with uS3 (lower right). See Appendix Text 1 for more molecular details. G Position of the ultimate eIF3j C-terminus in the mRNA entry tunnel (upper panel) and steric clash with mRNA as positioned in an 80S ribosome stalled during translation (PDB 5MC6); for clarity, in the lower panel only eIF3j and mRNA are shown, A/P/E respectively indicate the positions of aminoacyl, peptidyl, and exit site in the 80S ribosome.
Figure 3A) Representative isothermal titration calorimetry (ITC) binding curves of CEACAM-N domains and β-IgSF duplicates. Experiments were performed using β-IgSF and N domains of (r)CEACAM1 (CC1), CEACAM3 (CC3), CEACAM5 (CC5), CEACAM6 (CC6) and CEACAM8 (CC8). B) Representative flow cytometry plots analyzing the interaction of rCC1, CC3, CC5, CC6 or CC8 (10 μg/ml) with GBS A909. Fluorescence of bacteria was measured by flow cytometry. Representative data from n = 3 independent experiments.
Model of pre‐RC assembly and redistribution of Mcm2‐7 in G1Mcm2‐7 is loaded at ORC binding sites immediately after entry into G1.Figure 6Mcm2‐7 is loaded at ORC binding sites immediately after entry into G1. As G1 progresses, increasing cyclin E/Cdk2 activity promotes the loading of additional Mcm2‐7 complexes resulting in the full complement of Mcm2‐7 being loaded by the end of G1. All Mcm2‐7 loading is dependent on Cdc6 and Cdt1. Prior to or coinciding with the entry into S‐phase, the full complement of Mcm2‐7 redistributes along the chromosomes and is displaced from transcribed genes.
Novel polySer RAN protein encoded by the higher expressing ATXN8 transcript in SCA8 A Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand).Figure 1Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted. B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads. C Known and predicted homopolymeric protein expressed across SCA8 CAG expansion (ATXN8) transcripts. D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort). E Schematic of a sagittal section of mouse brain shows the distribution of polySer RAN protein in SCA8 BAC expansion mice. Red circles represent variable size and frequency of polySer aggregates throughout different brain regions. F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.
Figure 1A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments. B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments. C Experiment design for the UACC257 phosphoproteomic study, showing time points, cell line derivatives, replicate structure and GIST composition. The GIST was generated by pooling all heavy SILAC plates (blue) and spiked into all UACC257 (red) and UACC257 EDNRB-KO (green) samples. The A2058 cell line was also analysed in biological triplicates and a GIST but without EDNRB-KO cells. D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.
Figure 2(A) Boxplots showing repeats significantly upregulated (>2 fold where p=<0.05 using DESeq2) in knockout (N=3) compared to wildtype (N=3) HeLa cells based on mRNA-sequencing data. P values = 0.0174 (HERV-S), 0.0047 (HERV-K14C), 0.00013 (HERV-T LTR6B) and 1.90E-06 (HERV-T HERVS71). HERV-T and HERV-S but not HERVK14C also reached significance when only adjusted p-values were considered, where differences are compared to the whole of repBase. KAP1 binding (ENCODE data) is shown in Fig S1A. All numbers above bars depict fold changes compared to control cells (to one decimal place). (B) Evolutionary tree showing the age of KAP1-repressed HERVs identified in A). Here, HERV-K refers to HERVK14C. Estimated ages of stated lineages are given and marked with a star. Myr: million years (C) The 38 upregulated genes identified (>2 fold where padj=<0.05) were converted to DAVID IDs and used for gene ontology analysis. 3 gene clusters were significantly enriched (p-value = <0.05, drawn on the plot as a dotted line) in the data set: zinc finger proteins (p=2.2 x 10-7), negative regulators of metabolic processes (p=0.0036) and angiogenesis (p=0.011). Venn diagrams on the right show numbers of upregulated genes, ZNFs and KZNFs and the overlap. (D) The nature of conserved KAP1 binding sites between human ESCs [11] and 293Ts (ENCODE) (see Fig EV2D) is shown (left pie chart) compared to their abundance in the genome (right pie chart). (E) The 614 KAP1 common binding sites (see Fig EV2D) were interrogated for their nearest gene and this gene list was converted to DAVID IDs and used for gene ontology analysis. 4 gene clusters were significantly enriched (p-value <0.05, drawn on the plot as a dotted line): zinc finger proteins (p=1.1 x 10-19), fibronectin folding (p=0.016), protein phosphatase (p=0.033), and synapse (p=0.047). Venn diagrams on the right show numbers of KAP1-bound loci, ZNFs and KZNFs and the overlap. (F) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP-seq correlation analyses using ChIP-Cor software (see methods). Each plot shows duplicate ChIP-seq experiments from ENCODE. See Fig EV3B for complete data.
Reduced GATA4 levels entail diminished cardiomyocyte proliferation in vitro.Figure 3(A) Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 hours. (B) Number of cardiomyocytes (CM) in a defined low magnification (50x) microscopic field before the adenoviral infection and 48h later. The number in the bars indicates the number of cell culture plates analyzed. ****p<0.0001. (C) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU, troponin I (Trop I) and DAPI. Scale bar: 50µm; p=0.0169. (D) Representative pictures and quantification of cardiomyocytes positive for phospho-histone H3 (pH3) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3, troponin I and DAPI. Scale bar: 50µm; *p=0.0145. (E) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B (Auro B), troponin I and DAPI. Scale bar: 50µm. (F) Representative pictures, single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3, troponin I and DAPI. Scale bar: 100µm. (G) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later. Quantification of outgrowing cardiomyocytes is shown on the right. The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 50µm. **p=0.0037. (H) Representative pictures of cardiac explants like in (G), stained for pH3 in addition. The quantification is shown on the right. Scale bar: 50µm. *p=0.0215.
Figure 2A-H Immunostaining of representative xenografts derived from naïve (control) and Omomyc expressing BT168FO cells. A-D) Flag, Ki67, CD31, and DCX (doublecortin) immunostaining of adjacent sections in control and Omomycxenografts. E-G) Differentiation phenotype investigated by GFAP, ß-tubulin III, and OLIG2 staining. H) Immunostaining for anti-human nuclei. Representative images are shown. Scale bar = 100μm.
TRaMs reduce accumulated substrate following xenotransplantation.Figure 3A, B. Levels of globotriaosylceramide (Gb3) were measured by LC/MS in plasma and extracts of liver, spleen, heart, and kidneys of mice engrafted with healthy donor (HDo)-derived (A) or Fabry donor (FDo)-derived (B) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice. C, D. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.
Figure 7(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm. (B,C) The size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).
Alveolar macrophages do not produce an IFN response upon SARS-CoV-2 challenge.Figure 2Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1. Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2. (D) An overview of the timeline of the experiment. (A-D) Each colour corresponds to a donor; 6 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 6(A) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells. (B) Western blot analysis of the protein levels of HIF1A in siCtrl, siIgf2bp1, siIgf2bp2 and siIgf2bp3 3T3-L1 cells. (C) Western blot analysis of the protein levels of YTHDC2 and HIF1A in siCtrl and siYthdc2 3T3-L1 cells. (D) Western blot analysis of the protein levels of YTHDC2 and HIF1A in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (E) Western blot analysis of YTHDC2 in input and FLAG-RIP groups. (F) RIP-qPCR analysis of the association of Hif1a with FLAG in 3T3-L1 cells overexpressing FLAG-tagged Vec or YTHDC2. FLAG was measured by qPCR and normalized to input. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001. (G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells. (J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 1(A) Mice bearing established A375 were treated with vehicle (n=7), PLX4720 (n=5) bevacizumab (n=7) or COMBO (n=5). Tumor growth is expressed as % change of the initial tumor, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; P = 4.86E-10) ŦP < 0.05 compared to PLX4720 (P = 0.015).(B) Mice bearing established COLO205 were treated with vehicle (n=7), PLX4720 (n=8) bevacizumab (n=7) or COMBO (n=8). Tumor growth is expressed as % change of the initial tumor, ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07) Ŧp < 0.05 compared to PLX4720 (P = 0.039). (C) Melanoma MC-1 cells (5 x 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were: start point, n=6; vehicle, n=7; PLX4720, n=6; bevacizumab, n=6; COMBO, n=7, *P < 0.001 versus vehicle (P = 0.031). (D) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n=4 tumors), ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08).(E) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n=3 tumors). ***P < 0.001 versus vehicle (P = 6.2.7E-10).
Urm1 reaction scheme and its comparison to canonical UBLs.Figure 5Working model of the Uba4-catalyzed reaction cycle of Urm1 thiocarboxylation. After adenylation by the Uba4 AD (ochre), Urm1 (red) forms a thioester intermediate with the catalytic cysteine (C202) located within the crossover loop. The RHD (blue) connected to the AD by a flexible linker reaches Urm1 and replaces the thioester linkage by an acyl-disulfide bond by a nucleophilic attack of the persulfide on the active cysteine (C397). Thiocarboxylated Urm1 is released and serves as sulfur donor for tRNA thiolation. Structural comparison of various E1-UBL system focusing on the position of their catalytic cysteine residues, crossover loops and nucleotides. Distances between C-terminal glycine (red) and catalytic cysteine residues are indicated unless they are covalently linked. PDB identifiers, nucleotide types and residue numbers are labeled.
Actin clearance and dynamic re-localization of Aurora B in early anaphase upon centralspindlin depletion.Figure 1(A) Stills from time-lapse sequence of representative Hela cells expressing Lifeact-GFP and H2B-mCherry, exiting mitosis under different conditions i.e. Control siRNA and knock-down of centralspindlin protein RACGAP1. Scale bar- 10µm (B) High magnification view of boxed regions in (A) pseudo-colored to show actin depletion in anaphase. Scale bar -1 µm. (C) Schematic representation for quantifying actin intensity along the cortex as in D. Segmented line was drawn along the cortex from one equatorial region to the other. Actin intensity across the line is plotted in D. (D) Quantification of actin intensity profile of representative siControl and siRACGAP1 treated cells shown in (A). While actin levels were uniform across the cortex at metaphase (black line), they reduced from the polar regions (Blue line - segments 2-8) in both siControl and siRACGAP1 treated cells at early anaphase. (E) Consolidated quantification of actin intensity at the poles of cells at 6 mins post anaphase onset under different conditions as in (A). Despite reduced elongation, actin is cleared from the poles in both Control and RACGAP1 depletion. Data represented as mean±SD. siControl-n=44, 0.98±0.051, siRACGAP1-n=48, 0.99±0.041. Unpaired Welch's t-test comparing siControl and siRACGAP1 shows no significant difference between them, p=0.5199. One sample t test comparing siControl and siRACGAP1 to theoretical mean 1 (implying no change in actin compared to metaphase levels), shows significance for both treatments, with *p=0.0195 for siControl and *p=0.0392 for siRACGAP1. With means lower than 1 (red dashed line) for both treatments, it suggests significant clearance of actin from poles. (F) Stills from time-lapse sequence of representative Hela cells expressing mNeon-Green Aurora B and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm. (G) Maximum projection of 2-z slices of Hela-Cdk1as cells stained with anti-RACGAP1 antibody when treated with DMSO and 2µM ZM447439 (ZM) at metaphase and early anaphase, showing the localization of RACGAP1 at spindle midzone even upon Aurora B inhibition at early anaphase. Scale bar - 10 µm
Figure 1A Images show collagen matrix gel contraction by HDF (human dermal fibroblasts), skin-MAF (melanoma associated fibroblasts isolated from skin lesions), LN-FRC (lymph node fibroblast reticular cells), and LN-MAF (melanoma associated fibroblasts isolated from metastatic lymph node). Dashed circles represent the diameter of the gel. Data are representative of n = 3 independent experiments. B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition. C Atomic force microscopy (AFM) measurement of the elastic properties (apparent Young's Modulus, Εapp) of fibroblast-derived ECMs. Each dot represents a specific Young's Modulus obtained by fitting the corresponding individual force-curve acquired on a determined point of the sample. A representative experiment from 2 independent experiments is shown. Scatter plots show mean ± SEM. The black bars represent the median and the interquartile range. ****P<0.0001, Two-tailed Mann-Whitney test.
homodimer in both states.Figure 2Interaction interface of the MICU2 homodimer in the Ca2+-bound state. The left box (gray dotted line) shows residues involved in electrostatic interactions, the right box (gray line) residues involved in hydrophobic interactions. Molecule 1 and molecule 2 in the dimer are colored in purple and yellow, respectively. Side chains of residues that make crucial intreractions are shown in stick representation. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.
The microRNA-generating complex localizes at the neuronal endoplasmic reticulumComponents of the RLC co-precipitate with ER markers in density gradient fractionation.Figure 1Components of the RLCco-precipitate with ER markers in density gradient fractionation. The post-nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers Calnexin, Ribophorin I and Climp63, co-sedimented with ribosomes (RPS6, ribosomal protein subunit 6), mitochondria (Tim23) and RLC proteins at fractions 3-5. The bottom graph shows the density of each fraction. B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm). C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm). D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively. E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively. F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.