EMBO/sd-character-level-ner huggingface dataset:
Figure 4A GST pulldown assay was performed to monitor the interaction between 6×His tag-fused VAPB_ΔTM and GST-fused PTPIP51_TPR (or GST-fused PTPIP51_ΔTM). VAPB_ΔTM pulled down by PTPIP51_ΔTM is marked with an asterisk and highlighted with a red arrow. Schematic representation of the FFAT-like motif of PTPIP51 and amino acid sequence alignment of the FFAT-like motifs of human, mouse, and chicken proteins. Results of fluorescence polarization (FP) experiments to monitor the interaction between the FFAT-like motif-containing peptide of PTPIP51 and VAPB_ΔTM. The Kd value of the interaction determined in the FP experiment was ~53 µM. Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested. The data are presented as the mean ± SD of technical quadruplicate experiments.
Figure 2PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker. Schematic diagram of the oligonucleotide used to direct insertion of the 3xHA tag into the 5' end of the human LRRK2 coding sequence and the corresponding coding sequence in the same colors (the LRRK2 start codon is underlined, as is the GGGGS linker). COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels. COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m. COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.
CRYST Na+/transmitter bound structureA, B.Figure 2A, B. Views of EAAT1CRYST trimer in outward-facing Na+/transmitter-bound state, including N-term helix TM1a'. C. EAAT1CRYST protomer viewed from the membrane with tranD orange, scaD teel (TM4a,b omitted), and 398NMDG motif green. Fo-Fc Na+-omit map contoured at 3.5σ (black mesh) around the tranD core. D. Coordination details of three Na+-bound (blue sphere) to EAAT1CRYST. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is in parenthesis.
Figure 1Representative microscopic views of Ndrg4+/+ and Ndrg4-/- murine intestinal sections (n=4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (Chromogranin A), Goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm. Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4-/- murine colonic sections labeled with either TuJ1 and S100 (B, n=3) or HuC/D (C, n=6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4-/- mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4-/- mice (n=6). Gastrointestinal motility assays reveal a similar whole-gut transit time (E; n=12 vs. 12), with a similar weight per stool and stool water content of the fecal pellets (F, G; n=11 vs. 10), a comparable small intestinal transit (H; n=10 vs. 12) and colonic propulsion time (I; n=7 vs. 6) in Ndrg4-/- compared to Ndrg4+/+ mice.
Hepatocyte-specific ASK1-knockout mice develop NASH and fibrosis (A) Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks.Figure 3Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm. Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice). Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)). Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)). Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice. Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm) quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice). Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm. Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice). Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #
Figure 5A) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the MoCA total score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included in the analysis. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red. B) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the UPDRS-III score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.
dTor and dLap1 differentially affect the fat body lipidome A) GL and GPL and enzymes.Figure 1GL and GPL and enzymes. Full abbreviations in Appendix Fig. S1A. dTor and dLap1 topology. Subcellular localization of dLap1mGFP and dTormGFP-E177Q in the wild-type (w-) or dLap1+/∆11 fat body of 3rd instar fly larvae, when expressed as UAS transgenes. Control, dTorKO and dLap1-/- larvae with pseudo-colored red fat body. The relative abundance of individual GL and GPL classes in the wild-type fat body, as detected by mass spectrometry (MS). n=10 individual analyses. TAG content of 3DO larvae measured by enzymatic assay. Bars show mean ± SEM measured in three sets of thirty larvae. One-Way ANOVA with Tukey's multiple comparison test. **** p < 0.0001. The abundance of individual lipid classes (mean ± SEM) within the set of six membrane GL and GPL extracted from 4DO fat body samples. w- n = 7, dTorKO n = 7, dLap1-/- n = 4. One-Way ANOVA with Bonferroni's post hoc test. * p < 0.05 and ** p < 0.01. Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI. % of adult females that hatch after crossing female dTor+/- ;; dLap1-/TM6C with male y/FM7i ;; dLap1-/TM6C flies. Two-tailed T-Test. **** p < 0.0001, compared with expected Mendelian ratio (n≥3).
Figure 5A. Immunoprecipitation of OFD1 from HEK293 cell lysates expressing myc-OFD1, ubiquitin-HA and flag-praja2 (or flag-praja2rm). The precipitates were immunoblotted with anti-HA (ubiquitinated OFD1) and anti-myc antibodies. Flag-praja2 expression was evaluated in total lysates. B. Flag-OFD1 and ubiquitin-HA vectors were co-transfected with control siRNA or siRNA targeting praja2 (sipraja2). Serum deprived cells were left untreated or stimulated with FSK for 1 hour. Ubiquitinated OFD1 and the levels of endogenous praja2 were detected as in A. C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E E. Ubiquitination of flag-OFD1 or flag-S735A in HEK293 cells treated with FSK for 1 hour. F. Immunoblot analysis of flag-OFD1 or flag-S735A in HEK293 cells stimulated with FSK as in C. The levels of α-tubulin were used as loading control. G. Quantitative analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Student's t test *p<0,05 versus S735A mutant (3 hours and 4 hours, +FSK) and basal values (-FSK). H H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).
Elongator is a two-lobed asymmetric complex.Figure 1A. Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1. B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm. C. Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å. D. Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure. E. Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison. F. Analysis of Elp2 localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex. G. Elp3 localization analysis using a C-terminal MBP tag, denoted by the white arrowhead. H. Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead. I. Three dimensional reconstruction of the intact Elongator complex at 25Å. J. Schematic of the approximate subunit locations based on 2D EM analysis. K. Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.
a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation.f7(a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of the indicated cell types per total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. ***P0.001(χ2-test assessing the genotype effect in a generalized linear model with logit link). (c) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. **P0.01(χ2-test assessing the genotype effect in a generalized linear model with logit link). (d) FACS blots to detect CD25+FoxP3+ Treg cells in lungs 2 weeks after AdCre inhalation. Cells were gated on CD4+ populations. Numbers indicate percentages among total CD4+ cells in the lungs. (e) Mediastinal lymph node and lung cells were isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods. The left panel shows the percentage of CD4+ T cells among all lung-infiltrating CD45+ haematopoietic cells (±s.e.m.). The right panel shows the percentage of FoxP3+ cells among CD4+ T cells in mediastinal lymph nodes and lungs (±s.e.m.). n=4 per genotype; *P0.05 (unpaired, two-sided t-test).
vp1388-90 homologous operons are widespread in T6SS-encoding marine bacteria.Figure 1A Selected examples of the genetic structure of vp1388-90 homologous operons. SP, signal peptide; MIX, Marker for type sIX effector. B Distribution of vp1388-90 homologous operons in bacteria. A phylogenetic tree of bacteria encoding homologous operons, based on the DNA sequences of rpoB. The presence or absence of T6SS in each genome is denoted in the inner rings (black and dark green). Intermediate bars indicate the number of complete (triad) and partial (dyad or truncated) homologous operons identified in each genome. An external ring denotes the group to which the bacterial strains belong. V. parahaemolyticus (Vpara) were annotated separately (red), as were Aliivibrios (pink).
Figure 3Representative image ), cf and qRT-PCR quantification of intracellular Shigella in HeLa cells pre-treated with arsenite in the presence or not of amitriptyline, and corresponding controls Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells pre-treated wit anisomycin in the presence or not of amitriptyline, and corresponding controls Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection 3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance
Figure 1A Flow diagram of sample allocation for molecular profiling, primary culture, HTS and PDX drug studies. B Outcome of in vitro expansion in 46 fresh samples. Outcome of PDX establishment in 46 fresh samples. D Outcome of in vitro and in vivo drug testing attempts in 46 fresh samples.
Fig.Figure 3Neural differentiation of hESCs was induced on a feeder layer of MS5 stromal cells. Representative colonies differentiated from the WT (+/+, c42) and homozygous mutant (m/m, c38) hESCs are shown (left). Neural induction efficiencies were estimated by % neuroepithelial colonies out of total colonies (n=5~7 dishes for each group). Neuroepithelial cell colonies were identified by neuroepithelial cell structure (neural rosettes) and by staining of markers specific for primitive neuroepithelial cells (PLZF, N-CADHERIN, and SOX2) and rostral embryonic brain (OTX2). Scale bars, 100 µm. Ventral midbrain (VM) patterning was assessed during 24 days of +/+ and +/m hESC differentiation (chemically defined protocol). Each dot represents mRNA expression detected by qPCR; boxes and bars represent means and SEs of +/+ and +/m lines. The gene expression levels of +/+ and +/m at day 24 are compared in the last graph (n=4, *p<0.05 or **p<0.001, t-test). Neural stem cell (NSC) expansion estimated by cell growth curve and PDL during NSC passages (n=12, *p<0.001, t-test). NSCs were derived from neuroepithelial cell colonies (shown in (A)).
Figure 1Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.
Figure 1A TUNEL assay comparing apoptotic cells in Miwi+/- and Miwi-/- testis during diakinesis. Blue, DAPI; Green, TUNEL labeling. Asterisk indicates TUNEL-positive stained cells in the meiotic metaphase stage. Scale bars, 10 μm. B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm.E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm.H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm.K, L Mitotic metaphase spread preparations of Miwi heterozygous (K) and knockout (L) testes. Scale bars, 5 μm. M Frequency of mitotic metaphase cells with chromosomal abnormalities. Chromosome counts were defined as the number of chromosomes per mitotic metaphase cell. Three mice were used for each genotype, total numbers of counted mitotic metaphase cells in Miwi heterozygous and knockout are 52 and 71, respectively.
Oligo#5 reduces food intake after delivery to the blood.Figure 6A. 50 µg of cy3-labelled-oligo#5 was delivered through a carotid artery catheter and one hour later the cy3 signal was detected. ChP: choroid plexus, RSGc: retrosplenial granular cx,c, 3V: 3rd ventricle; D3V: dorsal 3rd ventricle. B. Change of RNA2/RNA1 ratios after carotid artery injection.The area around the arcuate nucleus was microdissected at the times indicated post injection. The ration of RNA1/RNA2 was determined using real-time PCR. Changes became apparent statistically significant after 4 and 8 hrs ( 2 hrs: p=0.3; 4 hrs: p=0.003, 8 hrs: p=0.0006, n=5) C. Effect of oligo#5 delivered through a carotid artery catheter on food uptake in wild-type mice. C57BL/6 wild-type mice with a pre-implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated. D. Long term effect of oligo#5 action. Food intake was measured for 70 hrs after one injection of 100 µg oligo#5 through a carotid catheter at time zero. E. Model of oligo#5 action. The 5HT2C receptor generates two major RNA isoforms generating a truncated receptor and a full-length receptor. The truncated isoform is retained in intracellular membranes. Due to heterodimerization, it sequesters the full-length receptor inside the cell. The loss of the truncated receptor isoform caused by oligo#5 releases this sequestration and causes an accumulation of the receptor on the cell surface, which results in an anorexic response.
Figure 2Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top. Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line). Left: HADDOCK model of interaction of DFL (CPK representation) with CXCL12 (grey surface and cartoon). CXCL12 residues with CSP > avg + sd, located around the sY21 binding site and on the β1 strand are in red and orange, respectively. Right: Zoom in the binding site, CXCL12 residues (sticks) involved in hydrophobic and electrostatic interactions with DFL (sticks) are explicitly labelled (right). DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFL DFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).
Figure 4A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm. D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.
NO triggers mitochondrial fission.Figure 1(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1. (B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers. (C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m. (D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).