EMBO/sd-character-level-ner huggingface dataset:
Transcriptional effects of ZnSO4 in the ileum and the role antibiotics and germ free aspects.Figure 2RNA sequencing on ileum samples of C57BL/6J mice treated with water or 25 mM ZnSO4 water for 7 days and then injected i.p. with 200 µg Dex (Rapidexon) solved in 200 µL PBS, or with PBS only. 2h after injection the ileum was isolated (N=3 per group). Overlap between genes (A) upregulated or (B) downregulated by ZnSO4 (Zn) or Dex (LFC>0.8, p<0.05). RNA sequencing on ileum samples of C57BL/6J mice treated with water or 25 mM ZnSO4 water for 7 days and then injected i.p. with 200 µg Dex (Rapidexon) solved in 200 µL PBS, or with PBS only. 2h after injection the ileum was isolated (N=3 per group). (C) Scheme of numbers of genes upregulated in ZnSO4, Dex or ZnSO4+Dex treated mice (LFC>0.8, p<0.05). Significant expression of genes in the RNA sequencing was assessed with a Wald test with negative binomial distribution in DESEQ2. (D) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC>0.8, p<0.05 are considered, while these genes with p<0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism. (E) C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week 25 mM ZnSO4 in the drinking water. During this week AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 µg TNF solved in sterile PBS and survival recorded. Combined data of 2 experiments. (F) Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N=5-8). Mice housed in germ-free (GF) or specific pathogen free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 µg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of 2 experiments. (H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N=3/4 per group). (I) Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week and 750 µL of this IS was injected i.p. in C57BL/6J GF mice.
a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins.figf5(a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size. (c) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.
Figure 7The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm. The effects of individually mutating the first two residues of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Pmt3-Ape4 fusions were used to allow alanine substitutions to be introduced at M1 and Q2 positions. Scale bar, 3 μm. The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm. The effect of preserving the M1 residue of Ams1 on its Nbr1-binding ability. Scale bar, 3 μm.
a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice.figf4a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase. b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid) c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1. d, Immunoblots of liver Hom and LDs from HFD-fed mice. e, Liver homogenate immunoblots. PDI, protein disulphide isomerase. f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817). g, Hepatic TG concentration (*P  0.05, n = 3). h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4). i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.
Figure 8A. The database enables inference of protein abundance across primary liver cell types and pathological conditions of liver disease.
Figure 4(A) Gene expression profile array analysis in HCT116 cells. Representative genes with higher fold difference (up or down) compared IDH1 K224R cells with IDH1 WT cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). (B) ChIP assays for HIF1α and its binding motif. Antibodies anti-IgG and anti- HIF1α were used in the ChIP assays. qRT-PCR was performed to quantify the binding activity. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B , statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant. (C-D) The transcriptional activity of the SRC promoter was greatly increased after co-expression of HIF1α and SRC reporter in wild type. SRC promoter constructs containing a potential HIF1α binding motif (-1636~-1629 bp and -1466~-1459 bp). Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant. (E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay. (F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated. HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For G statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant. HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for lactate levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.
MYC controls mTORC1 signaling through regulation of the TSC1.Figure 1Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells. Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet). Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed). qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control. Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control. Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.
Tap-1 mediates interaction between TseL and VgrG-1.Figure 4A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1. C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1. D Diagrammatic interpretation of (A-C). Arrows indicate interaction.
Figure 4(A) Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test. (B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test. (C) 2D human airway organoids were incubated with the fluorogenic substrate in triplicate for 1 hour in the presence of wtSPINK6 or mutSPINK6, and applied to fluorescence assay. The fluorescence intensity of mutSPINK6-treated organoids is arbitrarily set as 1. Data represent the mean and SD of a representative experiment performed three times. (D) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with wtSPINK6 or PBS in triplicate for 24 hours (left). Cell-free culture media were harvested for viral titration (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral titer data present the mean and SD of triplicated samples. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test. (E) 2D airway organoids were applied to fluorescence assay after incubation with α-SPINK6 or isotypic IgG in triplicate for 4 hours (left). Data present mean and SD of triplicated samples (right). Data present the mean and SD of triplicated samples. (F) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α-SPINK6 or isotype IgG in triplicate for 24 hours (left). Cell-free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral load data present the mean and SD of triplicated samples. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.
Figure 6(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm. (I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.
Figure 5Schematic diagram of the experiment. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice after a 20 kHz/90 dB auditory stimulus (n = 4 for each group). Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice (n = 4 for each group, **P = 0.0022, ****P < 0.0001, n.s., not significant). Schematic diagram of the experiment. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice after a 20 kHz/90 dB auditory stimulus (n = 4 for each group). Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice (n = 4 for each group, P**** < 0.0001, P*** = 0.0002, P** = 0.0021).
L374P mutation abolishes STIM1 puncta formation and colocalization with ORAI1.Figure 2(A) Domain architecture of STIM1. Abbreviations: CAD: CRAC activation domain; CC1-3: coiled-coil domains 1-3; CCb9, coiled-coil fragment b9; cEF, canonical EF-hand; nEF, non-canonical EF-hand; PB, polybasic domain; P/S, proline/serine-rich region; SAM: sterile alpha motif; non-canonical EF-hand; S, signal peptide; SOAR: STIM1-ORAI1 activation region; TM: transmembrane domain. (B,C) Homology models of the STIM1 p.L374P mutation based on published crystal (Yang et al., 2012) and NMR (Stathopulos et al., 2013) structures of STIM1. (B) Location of the M374P mutation (red sticks) in the context of the triple mutant (L374M/V419A/C437A) human CAD/SOAR/CCb9 dimer structure relative to CC2 (light blue ribbon) and CC3 (light green ribbon). The template structure without the M374P mutation (grey ribbon) and the movement of the apex positions of both subunits are indicated. (C) Location of the L374P mutation in the context of the NMR structure of a bimolecular complex of two STIM1 CC1TM-distal-CC2 peptides (CC1TM-distal region shown as light pink ribbon, and CC2 shown as light blue ribbon) and two ORAI1 C-terminal (aa 272-292) peptides (yellow ribbons) (Stathopulos et al., 2013). The loss of the backbone amide hydrogen (NH) due to the L374P mutation results in an altered hydrogen bond network near the mutation (small red dashed box). The zoomed view of this area (large red dashed box) shows the backbone hydrogen bond network for the L374P protein (red dashed lines) compared to the WT protein (black dashed lines). The maintained backbone atom hydrogen bonds in this region for the L374P mutant compared to the WT protein are listed in red text while the lost hydrogen bonds are listed in black text. GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm. GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry.
The co-feeding of arginine and succinate promotes nitrogen fixation and ATP production in isolated bacteroids.Figure 1(A) Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represents the mean and standard error of the mean of at least eight independent replciates. (B) Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the 4 individual measurements (points) together with the regression trajectory of each experiment is shown.
Figure 7(A) A model for AS-NMD regulation of the sams genes in C. elegans. See text for details. (B) Modelled structure of C. elegans UAF-2 binding to 5'-UAGGU-3'. The structure was modelled based on sequence homology to U2AF23 from S. pombe (Appendix Fig S9) and crystal structure of S. pombe U2AF23/U2AF59 complex bound to the RNA (Yoshida et al, 2020). N-terminal Zn finger 1 (ZnF1), U2AF homology motif (UHM) and C-terminal Zn finger 2 (ZnF2) domains are colored in blue, yellow and green, respectively. Red spheres indicate zinc ions. The position of the amino group methylated upon m6A modification (N6) is indicated with an arrowhead.
Figure 5A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h. B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h. C The colocalization analysis of K63-Ub (Red), Flag-NLRP3 (Purple) and GFP- CCDC50 in HeLa cells; scale bars, 5 μM. D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM. F The colocalization analysis of NLRP3 and LC3B in CCDC50-WT and CCDC50- KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments. G The oligomerization analysis of NLRP3 in CCDC50 wild-type and knockout THP-1 cells stimulated with LPS as well as ATP. LPS-primed (1 μg/ml, 3 h) THP-1 cells were stimulated with ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE. H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50 or empty vector for 24 h, and then treated with DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE. I Co-IP (with anti-HA) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h. J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments. K Confocal microscopy analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.
Figure 2Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=54.22, LoD (RBD)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD.