EMBO/sd-character-level-ner huggingface dataset:
Figure 2Circos plots of ChIP-seq data showing genome-wide enrichment of dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer grey bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment for intron- or promoter-targeted dCas9 (normalized to non-targeted dCas9) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 1,200 RPM for the promoter-targeted dCas9 (rings represent increments of 200) and 400 RPM for the intron-targeted dCas9 (rings represent increments of 66.7). var genes are represented by red bars. An asterisk indicates an unintended binding event. ChIP-seq data show enrichment of dCas9 in strains at ring stage expressing non-targeted "Control" (grey), var intron-targeted (blue), or var promoter-targeted (red) sgRNAs. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted ChIP enrichment. q-values for promoter-targeted dCas9 peaks shown are 2.22x10-289 for PF3D7_0413100 and 7.07x10-257 for PF3D7_1240400. q-values for intron-targeted dCas9 peaks shown are 2.03x10-76 for PF3D7_412700, 6.81x10-46 for PF3D7_0412900, 1.53x10-62 for PF3D7_0413100, and 8.37x10-4 for PF3D7_1240900. ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, grey). One replicate was performed for ChIP-seq and RNA-seq and one replicate was performed for HA and IgG control RIP-seq.
CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL).f7CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann-Whitney U test.
Sequence features of SARS-CoV-2 nucleocapsid proteinFigure legend: A.Figure 1:Sequence features of SARS-CoV-2 nucleocapsid proteinFigure legend: A. The complete whole genomic features of SARS-CoV-2 isolate Wuhan-Hu-1 (Genebank: MN908947). UTR: untranslated region; orf/ORF: open reading frame; TRS: transcriptional regulatory sequences; S: spike glycoprotein encoding region; E: envelope protein encoding region; M: membrane protein encoding region; N: nucleocapsid protein encoding region. The figure is illustrated by SnapGene Viewer; B. Domain architectures of coronavirus nucleocapsid protein. NTD: N-terminal RNA-binding domain; CTD: C-terminal dimerization domain; C. Multiple sequence alignment of SARS-CoV-2 N-NTD with SARS-CoV N-NTD (UniProtKB: P59595), MERS-CoV N-NTD (UniProtKB: R9UM87), HCoV-OC43 N-NTD (UniProtKB: P33469). Red arrows indicate conserved residues for ribonucleotide binding site, dash boxes indicate variably residues in the structural comparisons.
Elevated levels of Snai1 result in reduction of secretory enteroendocrine and Paneth cells and an increase of CBC intestinal stem cellsThe effect of elevated levels of Snai1 was examined using a conditional transgenic mouse model (Rosa26Snai1) that permits expression of Snai1 when exposed to Cre recombinase.Figure 6A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS). B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003). C Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027). D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD. D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD. F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm. G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013. H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.
Figure 6(A) Western blot analysis after overexpression of Hel2 in strains expressing HA-tagged ribosomal proteins using an anti-HA antibody. Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3. (B) Structural overview of the ubiquitination sites on eS7. The general overview on the disome interface is depicted according to Fig 4A. Individual ubiquitination sites are highlighted as red spheres in the detailed cartoon model representation. (C) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination. (D) Western blot showing that Not4 was required for Hel2-mediated polyubiquitination of eS7 in Hel2-bound ribosomal complexes: Cells expressing HA-tagged eS7A or the eS7-4KR mutant, as well as PTH-tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7-ubiquitination. Either cell lysates or affinity-purified Hel2-ribosome complexes were used. The levels of the ubiquitinated eS7A in the lysates and in the affinity-purified samples with PTH-Hel2 were determined by Western blot analysis using an anti-HA antibody to detect eS7. (E) Western blotting of in vitro ubiquitination assays of eS7A. The reactions were performed with the indicated components including His-tagged ubiquitin. Ribosomes were purified from Hel2Δ cells expressing HA-tagged eS7A. Polyubiquitinated HA-tagged eS7A (eS7A-3HA) was detected by Western blot analysis using an anti-HA antibody. We observed Hel2-mediated polyubiquitination of the monoubiquitinated eS7A. (F) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2-mediated polyubiquitination of eS7 was mainly K63-linked and required Not4-dependent monoubiquitination: These assays were performed similarly as described in (E) except yeast strains were lacking Not4 (not4Δ). Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants and eS7A-ubiquitination was monitored using an anti-HA antibody.
Figure 3(A) Schematic illustration of proteins involved in TGFβ storage in the extracellular matrix. The Small Latent Complex (SLC) is a non-covalently linked tetramer of 2 LAP (Latency Associated Peptide) (40 kDa) and 2 TGFβ1 (12 kDa). LTBP1 + SLC together form the Large Latent Complex (LLC) through covalent bonds between LAP and LTBP1. LLC interacts with the ECM through non-covalent interactions of LTBP with fibrillins and fibronectin. (B) Immunoblot analysis of LAP in mammary whole tissue lysates from Wt and Hpn-/- mouse. The 90 kDa band represents unprocessed pro-TGFβ and the 40 kDa band corresponds to mature TGFβ LAP peptide. (C) Schematic representation of the mouse experiment, showing orthotopic transplantation of Wt Wap-Myc tumor cells into syngeneic Wt and Hpn-/- recipients. (D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each). (E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated. (F) Immunoblot analysis of conditioned medium (LAP peptide) and cell extracts (hepsin and vinculin) from control (DOX) or hepsin overexpressing (DOX+) MCF10A pIND20-HPN supplemented with 100ng/ml latent-TGFβ (Small Latent Complex). Ponceau was used as a loading control for the cell culture medium. (G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns). (H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).
Fig.Figure 3A. and B. Binding of ASC filaments (A) or α-synuclein PFFs (B) to multiple proteins. Serial dilutions of ASC filaments and α-synuclein PFFs were performed and bound fractions were detected using anti-ASC or anti-α-synuclein (MJFR1) antibodies. ASC filaments did not interact with any of the proteins (A). Conversely, α-synuclein PFFs strongly bound LAG3, CD4, and MTBD tau and showed moderate binding properties to PrPC, APOE3, and TDP-43. No binding between α-synuclein PFFs and nAra h 2 or BSA could be detected. Values represent means ± SD of technical duplicates. C. and D. Diffusional sizing of α-synuclein PFFs with fluorescently conjugated LAG3, CD4, BSA, and MJFR1 α-synuclein antibody (C) and of fluorescently labelled LAG3 with monomeric α-synuclein, α-synuclein PFFs, FGL1, and Relatlimab anti-LAG3 antibody (D). An increase in the hydrodynamic radius (Rh) of α-synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of the control proteins, also not at a lower scale (see insert) (C). Similarly, the hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab and FGL1 but does not change with α-synuclein PFFs or monomeric α-synuclein (D). Values are given as means ± SD of technical triplicates. E. and F. Co-immunoprecipitation of LAG3 and subsequent immunoblotting with anti α-synuclein antibody (MJFR1) (E) and co-immunoprecipitation of α-synuclein (monomeric or PFFs) and subsequent immunoblotting with anti-LAG3 antibody (D2G40) (F). Using the anti-α-synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar α-synuclein. The usage of anti-LAG3 for pulldown led to the specific detection of α-synuclein PFFs, although the PFF fraction predominates also in the input and supernatant. Bands for α-synuclein (E) and LAG3 (F) are indicated by arrows.
Figure 7. Northern blot analysis to detect the endogenous huORFchop-tagged gfp (uORF-gfp) mRNA and its cleaved form in zebrafish embryos of transgenic line huORFZ treated with non-stress condition (NS) or heat-shocked (HS) stress. Zebrafish 18S rRNA served as an internal control. The RNA fragments cleaved from huORFchop transcript were detected. . Northern blot analysis to detect endogenous CHOP mRNA in HEK 293T cells treated with either DMSO (control group) or thapsigargin (TH). Human GAPDH RNA served as internal control. The RNA fragments cleaved from CHOP mRNA were detected. . The predicted RNA sequences of Endouc recognition site on huORFchop transcript from 72 to 91 nts are illustrated. The predicted cleavage sites at uridylates (U) are indicated by arrowheads. D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe. F. Electrophoretic mobility shifted assay of RNA. Biotin-labelled huORFchop transcript was reacted with the increased amount of Endouc. The positions of free RNA (free probe) and RNA-protein complex (shifted band) were indicated on the right. The luc 105-nt RNA and single-stranded huORFchop 105-nt DNA served as negative controls.
Figure.Figure 2A. Schematics of the live-cell imaging experiments. The embryo imaged from pronuclei until blastocyst stage and transferred to pseudo-pregnant females B. Representative live-cell images of dynamics MERVL::tdTomato expression during SCNT embryos preimplantation development. The selected images from a series acquired every 15 min. The time after starting observation is shown on the upper right corner of each image. Scale bar, 50 μm C. Representative DAPI staining of blastocysts of tdTomato+ and tdTomato- SCNT embryos after 115 hr. of culture in vitro. Scale bar, 50 μm. D. The tdTomato+ and tdTomato- SCNT blastocyst cell numbers were determined by counting the DAPI-stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value. E E. Immunofluorescence images of tdTomato+ and tdTomato- blastocysts derived from SCNT. Nanog (ICM) and Cdx2 (TE) were used as lineage markers. Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown. Scale bar, 50 μm F. Representative image of tdTomato+ and tdTomato- SCNT embryos retrieved at E6.5. The tdTomato+ SCNT embryo displays normal egg-cylinder morphology. By contrast, the tdTomato- SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm G. The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts. The efficiency was calculated based on the total number of blastocysts used for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student"s  t H. The bar chart shows the efficiency of ntES derivation. The efficiency was calculated based on the total number of attached blastocysts for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student"s  t I. RT-qPCR analysis for somatic cell genes in SCNT 1-cell embryos, 2-cell tdTomato+, and 2-cell tdTomato- embryos. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. ***P < 0.001 by two-tailed Student"s  t
Figure 5Panels (A and C): the pre-host attachment state. Panel (B): the post attachment state. The atomic structure of the conserved tube-baseplate core complex (derived from the T4 tube-baseplate complex) in the pre- and post-attachment states is fitted into the cryoEM maps. The color scheme is the same as in Figure 2. BW3 gp104 and its ortholog T4 gp7 are colored magenta. The putative chain of gp104 crossing three gp105 dimers is shown as a thick coil. (C) A thin slice through the pre-attachment state cryoEM map is shown. The plane of the baseplate is about 8° out of the plane of the paper. The distal part of the A511 tail fiber is formed by the gp108 trimer, whose structure is modeled with homologous domains that are fitted into the pre-attachment baseplate cryoEM map. The structures are shown as ribbon diagrams and colored in a rainbow pattern with N-terminus in blue and C-terminus in red. The numbers indicate the N- and C-terminal residues of the gp108 sequence that match the start and end of the homologous structures. The smaller insets show cross-sections whose positions are indicted with thicker black lines.
Rab25 increases glucose uptake.Figure 4A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment. B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT. C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25. D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescence microscopy staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM. E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells. E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT. E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.
The cleavage of VN by tc-uPA requires the binding of both uPA and VN to uPAR.Figure 4A Plasminogen activation results in the generation of N-terminal VN-fragments containing the SMB-domain. 293/uPAR cells were seeded on VN and treated with 10 nM sc-uPA and tc-uPA, 30 nM Plg and Pli, and 100 nM a2AP for 2 h at 37˚C. The supernatants were harvested and the level of SMB-containing VN-fragments was measured by immunoassay (see Material and Methods section). Values represent the mean ± SD of at least 3 independent experiments. The statistical significance was probed using Student's t-test (two-tailed). B Cartoon illustrating the mechanism of action of the different compounds/mutation used in panel C. The monoclonal antibody 8B12 is a competitive inhibitor of the interaction between uPAR and VN, having a binding site overlapping with that of VN [25]. The Arg91 residue in uPAR is part of the VN-binding epitope in uPAR. The GFD domain of uPA is a competitive antagonist of uPA-binding to uPAR. C The generation of soluble VN-fragments containing the SMB-domain requires binding of both uPA and VN to uPAR. Cells were plated on VN and treated with 10 nM tc-uPA. Where indicated, a 30 minutes pre-incubation with 300 nM GFD or 30μg/mL 8B12 was performed. Conditioned media were analysed as described in panel A. The statistical significance was probed using Student's t-test (two-tailed). D uPAR accelerates the generation of SMB-containing VN-fragments by uPA. VN immobilized in 96-well plates was incubated with 10 nM sc-uPA or tc-uPA together with increasing concentrations of soluble Fc-tagged uPAR (uPARWT) or a mutant form specifically deficient in VN-binding (uPARW32A/R91A). After a 1 h incubation, the supernatants were recovered and analysed for the presence of N-terminal VN-fragments by immunoassay. The release is reported as percentage of the maximal signal observed in the experiment (10 nM uPARWT + tc-uPA). Data points represent mean ± SD of three independent experiments. E Dose dependent binding of soluble uPAR to immobilized VN. The same experiment as in panel D, but here quantifying the levels of VN-bound uPAR. After the recovery of supernatants for VN-fragment quantification, the 96-well plates were washed and probed for bound uPAR-Fc using an Eu3+-labelled anti-Fc antibody. The specific binding is reported in % of the maximal binding observed in the experiment (10 nM uPARWT + sc-uPA). Data points represent the mean ± SD of three independent experiments. F Levels of uPAR binding to VN and the release of VN-fragments are strongly correlated. All the individual data points recorded in the three independent experiments described in panel D and E were analysed by plotting uPAR-binding versus SMB-release. Pearson correlations are shown.
Rapamycin affects intracellular Ca2+ signaling.Figure 2A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated. B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA. C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA. D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3. E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.