EMBO/sd-character-level-ner huggingface dataset:
Examining potential essential translation-related targets and the functional importance of phosphorylation on rates of protein synthesis upon TOR kinase inhibitionPhosphorylation kinetics of common phosphosites identified for 3 essential translation-related proteins, and the effects of the tif471_18A phosphosite mutant on rates of protein synthesis upon Torin1 treatment.Figure 5A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold. C, D Relative phosphorylation levels of the 10 common phosphosites of Tif471 plotted against time after Torin1 treatment. Phosphorylation profiles are grouped based on phosphorylation kinetics, where 6 sites showed more than 1.5-fold increase by 10 minutes (C), and the remaining 4 exceeded 2-fold by 30 minutes (D). Grey dashed lines indicate 2-fold threshold. E, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.
Figure 1(A) Schematic of the translation elongation cycle as studied here. (B) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates. (C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates. (D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1.
Figure 9(A) Examples of simulated hemifusion-to-fusion transitions in the presence of modified vacuolarSNARE complexes. Upper panel: Short spacer. Lower panel: Long spacer. These stirred molecular dynamics simulations illustrate that progression of fusion is sterically achievable for both of the fluorescent protein-tagged SNARE complexes, even when the geometry of the fusion zone is extremely confined, i.e. the fusion between two highly curved membrane dimples (1/8 nm-1). This scenario suggests that the fusion-inhibiting effect of a fluorescent protein added via a short peptide spacer is unlikely to result from a direct "steric clash" with the membrane. Vam3 in red; Nyv1 in blue; Vti1, Vam7 in green. (B,C) A scenario simulating the presence of four SNARE complexes near the fusion site. The modeled indentation corresponds to a relative indentation of -1.6 nm (see Fig. 2B). This corresponds to an exaggerated indentation force of about 80 pN which we exploit to demonstrate the clustering between "indentations" within our limited simulation time. The C-terminal amino acid of each Vam3 and Nyv1 is represented by a red or blue bead, respectively. (B) Isolated SNARE C-termini with short peptide tag. The C-termini concentrate in a small zone (similar membrane perturbations attract each other). (C) Steric hindrance by the addition of bulky proteins prevents collective, localized force transduction by multiple SNARE complexes.
VHHmASC ameliorates antigen-induced arthritis.Figure 7(A) Schematic representation of the experimental setting used for the mBSA-induced arthritis model. Mice were injected sub-cutaneously (s.c.) with mBSA (500µg/animal) in an emulsion containing 1mg ml−1 Freund's adjuvant on day 0 and day 7. Control mice received injections lacking mBSA. Mice were then immunized with an intra-articular injection of mBSA (100 µg, right knee) on days 21 and 26. From day 21 until day 26, mice were treated daily with an intra-peritoneal (i.p.) injection of VHHmASC (5 mg kg-1), IL-1RA (50 µg kg-1), or vehicle (PBS). B, C Mechanical allodynia threshold (B) and edema (C) were evaluated on day 27. Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n=4; PBS + VHHmASC, n=3; mBSA + Vehicle, n=6; mBSA + VHHmASC, n=6; mBSA + IL-1RA, n=5. (D) Flow cytometric assessment of infiltrating total leukocytes (CD45+), granulocytes (CD45+ Ly6G+ Ly6C-), and inflammatory monocytes (CD45+ Ly6G- Ly6C+) recovered in the synoival fluid of the knee joints of mice treated as in (A). Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n=4; PBS + VHHmASC, n=3; mBSA + Vehicle, n=6; mBSA + VHHmASC, n=6; mBSA + IL-1RA, n=5. (E) ELISA of IL-1β, TNFα and IL-6 in tissue homogenates of knee joints of mice treated as in (A). Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n=4; PBS + VHHmASC, n=3; mBSA + Vehicle, n=6; mBSA + VHHmASC, n=6; mBSA + IL-1RA, n=5.
CD8+ memory T cells in the lung parenchyma from aged mice are tissue residentAged mice (CD45.Figure 3.CD8+ memory T cells in the lung parenchyma from aged mice are tissue residentAged mice (CD45.2) were infected with PR8 and parabiosed with young naive (CD45.1) mice at 5 weeks after infection. 4 weeks later, CD45+ white blood cells were intravenously labeled in each host before sacrifice. A. Schematics of experimental procedure. B. Each animal was bled at time of sacrifice and CD8+ T cells were examined by flow cytometery for origin (representative flow plot). C. Percent of host or donor CD8+ T cells in aged or naive hosts where each line represents all the CD8+ T the cells in blood from one mouse. D. Representative flow plot of total parenchymal CD8+ T cells (CD69+CD8+ T cells protected from i.v. CD45 labeling) derived from host or donor. E. Enumeration of lung parenchymal (left panel) or vasculature (right panel; circ) CD8+ T cells in each host by mouse of origin. F. Representative flow plot of parenchymal Db-NP tetramer+ CD8+ T cells (CD69+CD8+ T cells protected from i.v. CD45 labeling) derived from host or donor. G. Enumeration of Db-NP+ cells that are resident (left panel) or circulating (right panel). H. Representative flow plot of parenchymal Db-PA tetramer+ CD8 T cells (CD69+CD8+ T cells protected from i.v. CD45 labeling) derived from host or donor. I. Enumeration of Db-PA+ cells that are resident (left panel) or circulating (right panel). Experiment was repeated twice with 3 pairs each and data was pooled. * p<0.05 Student's two-tailed t-test with unequal variance.
Three-dimensional analysis of interaction between the peroxisome and the ciliary pocket.Figure 4Maximum projection of z-stack sections of hTERT-RPE1 cells expressing MCHR1-GFP (blue), DsRed2-PACT (red) and ECFP-SKL (green) 48 h after serum starvation. The ECFP-SKL-positive cell with primary cilia, indicated by the red box, was analyzed by FIB-SEM. Top, differential interference contrast (DIC); bottom, merged image. Scale bar, 10 µm. The fluorescent image of primary cilia, enlarged the area boxed in red in panel (A). Arrow indicates the ECFP-SKL positive-peroxisome associated with primary cilium. Scale bar, 5 µm. Single FIB-SEM image (left) showing a cross-section the ciliary pocket membrane, and 3D surface model reconstructed from segmented FIB-SEM images (right). Region of interest was coated by a protection layer of platinum before FIB milling. Plasma membrane (PM), yellow (surface view 75% transmission); cilia, green (surface view); ciliary pocket membrane (CPM), red (surface view 70% transmission); mitochondria (Mito), orange (surface view); peroxisome (PO), purple (surface view); basal body (BB), white (surface view). Scale bar, 1 µm. Enlarged 3D view of peroxisome interacting with ciliary pocket and mitochondria. Left, x-y view; right, oblique view. Scale bar, 1 µm.
Enlarged salivary glands secretory granules, association of lysosomes and secretory granules, and secretion of the lysosomal acid phosphatase in the Trpml1-/- mice: (a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.Figure 5(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice. (c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin. (d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line. (e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.
Figure 2SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.   Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.   Western blots of the proteins in mascRNA S and AS pulldown samples (top 4 panels), and mascRNA and menRNA pulldown samples (Bottom 2 panels). β-tubulin was used as a loading control. RT-PCR analysis of RNAs extracted from ARS immunoprecipitation (RIP) samples (erro bars: mean ± SD; biological replicates n = 3). Eight complex-forming ARSs (DARS, EPRS, IARS, KARS, LARS, MARS, QARS and RARS) and GARS tagged with FLAG were overexpressed in HEK cells. ARS-RNA complexes were imminoprecipitated with anti-FLAG Magnetic Beads. The cell line constructed with PQCXIP vector (PQ) was used as a negative control. GAPDH was used as an internal control for qRT-PCR. Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control. Northern blot (top 2 panels) and western blot (bottom 3 panels) analysis of ARS immunoprecipitation samples (RIP). RIP was performed using EPRS, QARS and GARS antibodies. Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.
Figure 3A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated. B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration. C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05 (C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05 E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.
The flap domain of PPM1H is a determinant of Rab specificity.Figure 4(A) Domain organization of chimeric variants for in vitro and cellular assays. (B) 3 μg of recombinant proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. (C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates. (E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.
Figure 8The schematic illustrates the effect of in vivo edaravone administration in Skm from LowOXPHOS mice. Restraining OXPHOS generates a ROS-mediated rewiring of lipid metabolism through enhanced BCAA catabolism and lipid synthesis. Skm and WAT perturbations in lipid species are observed, defining a LowOXPHOS phenotype of adiposity and lipotoxicity. Edaravone treatment restores normal ROS and lipid metabolism, reducing v-WAT deposits and preventing the setting of IR mediated by mitochondrial dysfunction.
Metabolite reporting & vote counting analysis in tissue and bloodA, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites.Figure 2A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes). B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text. C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).
Figure 3Arginine 1285 of FANCI, which is mutated in some FA patients, is located in the Arm ID2 interface in our cryoEM IUbD2Ub-dsDNA structure. Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA. Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown. FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE. FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison. FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.
SV2 is reduced in i3 neurons from a C9orf72-ALS patient A, B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) or synaptophysin (green) (B).Figure 7i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) Shown are representative z-stack confocal images of neurites. B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for synaptophysin (green) (B). Shown are representative z-stack confocal images of neurites. Quantification of SV2 (C) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated. Quantification of Synaptophysin (D) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated. E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01. F) Whole cell lysates were generated from 3 independent wells of C9-ALS and isogenic i3 cortical neurons differentiated for 21 days. Lysates were immunoblotted for SV2, and normalized to total protein loading. Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for SV2 (G) Loading controls were β-actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used. Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for synaptophysin (H). Loading controls were total protein loading. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.