EMBO/sd-character-level-ner huggingface dataset:
TBK1 interacts with and phosphorylates mTOR within mTORC1.Figure 1A. The mTOR S2159 sequence fits TBK1 and IKKε consensus phosphorylation motifs. B. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro: In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST-mTOR substrate [200 ng] for 30 min at 30oC. Reactions were pre-incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX-795 [10 μM] or MRT-67307 [10 μM] and immunoblotted (IB) as indicated. C. TBK1/IKKε phosphorylate full-length mTOR on S2159: Myc-mTOR wild type (WT) and S2159A were immunoprecipitated (IP) from transfected HEK293 cells and incubated with re-TBK1 or re-IKKε. IVK assays were performed as above and immunoblotted (IB) as indicated. J. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed). E. TBK1 and IKKε overexpression increases mTOR P-S2159 within mTORC1: HEK293-TLR3 cells were co-transfected with Flag-TBK1 or Flag-IKKε, Myc-mTOR, and HA-raptor. HA-raptor immunoprecipitates and whole cell lysates (WCL) were immunoblotted (IB) as indicated. F. Flag-TBK1 and Flag-IKKε co-immunoprecipitate with endogenous mTORC1: HEK293T cells were transfected with Flag-TBK1 or Flag-IKKε wild type (+) or kinase dead (KD). Endogenous raptor immunoprecipitates and WCL were immunoblotted (IB) as indicated. G. mTOR is phosphorylated on S2159 in wild type but not TBK1 null MEFs: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.) and stimulated -/+ EGF [25 ng/mL]. WCL was immunoblotted (IB) as indicated. H. The TBK1- and IKKε-activating agonists poly(I:C) and LPS increase mTOR P-S2159 in a BX-795 sensitive manner in cultured macrophages: RAW264.7 macrophages were pre-treated with BX-795 [10 μM] (2 hr.) and stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). I. Poly(I:C) and LPS increase mTOR P-S2159 in primary bone marrow-derived macrophages (BMDMs): BMDMs were stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). J. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed).
SARS-CoV-2 convalescents make potently neutralizing N antibodies whose activity correlates with the number of active N-specific T cells.Figure 4(A) Capillary-based protein detection (Jess) of N-antibodies in convalescent sera, quantified by chemiluminescence. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. (B) Quantification of SARS-CoV-2 intracellular neutralization by electroporated convalescent sera using EDNA and RT-qPCR (as % of PBS condition). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. (C) Correlation between N-antibody titre, as calculated by Jess, and intracellular neutralization (fold decrease in SARS-CoV-2 replication), as measured by EDNA, in convalescent sera from multiple individuals. (D) Intracellular neutralization of SARS-CoV-2 by titrated serum from two strongly N-antibody positive individuals. ( Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. (E) ELISpot measuring IFNγ production in T cells from SARS-CoV-2 convalescents upon stimulation with a peptide library covering N-protein. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. (F) Correlation between N-specific T cell activity and intracellular neutralization activity. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical analysis was performed using a semilog non-linear fit (G) Correlation between N-specific T cell activity and N-antibody titre. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical analysis was performed using a semilog non-linear fit
Inhibition of Hsp90 delays the dissolution of SGs induced by different types of stressors.Figure 1(A, B) Kinetics of SG dissolution of living cells in absence and presence of Hsp90 or Hsp70 inhibitors. G3BP2-GFP HeLa-Kyoto cells were treated with sodium arsenite (50 µM) for 45 min. Then cells were allowed to recover in drug-free medium (Recovery Control) or in presence of GA (5 µM), 17AAG (5 µM or 0.5 µM) or VER (40 µM). Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 193 (Recovery Control, A); 340 (GA 5 µM); 187 (17AAG 5 µM); 209 (VER 40 µM); 363 (Recovery Control, B); 125 (17AAG 0.5 µM) and 353 (17AAG 1.5 µM). (C G3BP1-mCherry HeLa-Kyoto cells were lipofected for 72 hrs with a non-targeting siRNA control or siRNAs specific for Hsp90 α and β. (C) Efficacy of Hsp90 knockdown and expression levels of HSPA1A were verified in total protein extracts. TUBA4A was used as loading control. (D) Cells were treated with sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β). (E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted. Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA).
Figure 3A. Specific interactions between the upstream DNA (−25 and −26 nucleotides) and β′ ZBD. B, C. Effects of deleting β′ ZBD on transcriptional activities of RNAP on different promoters. The fliCp (B) was used as template for σ28-dependent transcription, and the lacUV5p (C) was used in σ70-dependent transcription. RNA products indicated by solid triangles were quantified and the signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test respectively. D. Transcriptional activities of ZBD mutated σ28-RNAP on fliCp. RNA products indicated by solid triangles were quantified. E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP-promoter open complex is indicated as "RPo", and the heparin-sensitive RNAP-promoter closed complex is shown as "RPc". Percentages of RNAP-promoter open complex (RPo) and closed complex (RPc) in EMSA performed using σ28-RNAP and fliCp (right). F. DNase I footprinting of the fliCp complex with σ28-RNAP. Promoter DNA was labeled on the non-template strand. The promoter region protected by RNAP is indicated by a red box at the bottom panel.
Figure 7WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm. Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA). Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA). WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm. Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA). Schematic model of this study. Mammalian Atg8 proteins directly bind Stx16 through its LIR motif, which controls the proper localization of the Stx16 SNARE complex at the endolysosomal compartments. The Stx16 SNARE complex functions in the maintenance of lysosomal homeostasis upon autophagy induction, thus controlling mTOR activity.
Figure 7Cartoon cross section of nematode body showing neurons, intestine, and hypodermis. Our data suggest a model in which neurons release biogenic amines like 5-HT into the body cavity as a neurohormone. Molecules of 5-HT bind receptors like SER-1, resulting in augmented transcription of P450 enzymes like CYP-34A4. It is possible that 5-HT and other biogenic amines signal to another regulatory layer of neurons, which in turn release peptide-based signals (not shown). CYP-34A4 converts n-6 PUFAs into n-6 eicosanoids, which are released from the ER and allowed to diffuse extracellularly. Released eicosanoids could act through receptors in the hypodermis, triggering changes in UPS activity in this tissue. This triangle of signaling affords multiple points of regulation to coordinate external environment, internal physiology, and UPS proteostasis.
Figure 5Bar plots showing the X-Cell scores of specific cell types across control, mild and severe COVID-19 patients. Boxplot showing the selected cell type specific proteins among control (n=12), mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range. Systematic summary of the GO pathways enriched by tissue-enhanced proteins that exhibited altered expression among control, mild, and severe patient groups. The heatmap of each panel indicates expression patterns of tissues-enhanced proteins among control, mild, and severe patient groups. The top panel on the heatmap indicated the age and gender information of patients. The fold changes of tissue-enhanced proteins between mild/severe patient samples and control samples are shown on the right panel of heatmap.
a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49.f6(a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49. (b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).
Figure 2A-E. Behavioural tests of APP/PS1 mice. (A) Schedule of behavioural tests. Four-month-old male APP/PS1 mice were intravenously injected with Nec-1 (6.25 mg/kg, n = 7) or vehicle (2.5% DMSO in PBS, n = 6) for 12 weeks (2 times per week). As controls, age-matched male wild-type (Wt) mice were injected with Nec-1 (6.25 mg/kg, n = 7) or vehicle (2.5% DMSO in PBS, n = 7) for 12 weeks. A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test. A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg). F-I. Behavrioural tests of Aβ(1-42) infusion mice. (F) Schedule of behavioural tests. In first week, 8-week-old male ICR mice (n = 8 per each group) were intravenously injected with either Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) twice. Then 5 μl of Aβ(1-42) (100 μM) aggregates or vehicle (10% DMSO in PBS) were injected in intracerebroventricular region to generate Aβ(1-42) infusion mice model. In second week, same concentrations of Nec-1 or vehicle were intravenously injected twice again. F-I. Behavrioural tests of Aβ(1-42) infusion mice. (G) Intracerebroventricular (i.c.v.) injection site brain schematic diagram. F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.
S100A9 expression restricts HIV-1 infection of LC(A) MoLC were transfected with control (siCTR) or S100A9-specific (siS100A9) siRNA and checked for S100A9 expression by RT-qPCR (left) and western-blot (right).Figure 3MoLC were transfected with control (siCTR) or S100A9-specific (siS100A9) siRNA and checked for S100A9 expression by RT-qPCR (left) and western-blot (right). Shown is a representative experiment out of six. RT-qPCR data were represented as means of duplicates ± SD. MoLC were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry. The experiment shown is representative of data from six different donors (n=6). Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors. MoLC from six different donors were transfected with siCTR or siS100A9 and challenged with HIV-GFP vectors for 48h. S100A9 expression (left graph) and the percentage of GFP+ cells (right graph) were analysed by RT-qPCR and flow cytometry respectively. Flow cytometry data are presented as fold relative to siCTR-MoLC. Data represent means of fold values for each read-out from six donors (n=6).
Identification of Nix‐LIRs.Figure 2(A) Amino‐acid sequences of Nix and related BNIP3 protein from the indicated species were aligned using the ClustalW2 program (EMBL‐EBI, Cambridge, UK). The published LIR sequences were aligned manually alongside Nix/BNIP3 for comparison. The shaded regions indicate highly conserved residues in the two putative LIRs. Consensus symbols * (identical residues), : (conserved substitution) and . (semi‐conserved substitution) are valid for the Nix/BNIP3 alignment only. (B) A schematic of Nix showing its domain organization and the point mutations used in this study to identify LIRs. (C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.
Figure 8A. Protein sequences were extracted from UniProt accession number BP08_BPT4 for Enterobacteria phage T4 (gp8; Bacteriophage T4) and the Pseudomonas Genome Database (TssK1; reference strain PAO1). Conserved positions are shown in black and grey background. The secondary structural elements corresponding to the 3D structure of gp8 (PDB code 1PDM) are shown above the alignment. Regions with significant sequence conservation are indicated with a corresponding block number. B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.
'-phosphopantetheine causes brain biomarkers to return to pre-treatment levels.Figure 7Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),
Heterologous AID expression does not promote carcinoma development.Figure 2Schematic of the constructs used for conditional expression of AID in epithelial cells. An AID-IRES-GFP cassette preceded by a transcriptional STOP flanked by LoxP sites was introduced by homologous recombination within the endogenous Rosa26 locus (R26AID+/KI mice, top). R26AID+/KI mice were bred with Villin-CRE and p48-CRE mice to achieve specific AID expression in colon and pancreas, respectively (bottom). GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm. qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells. Kaplan-Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).
HIV-mediated autophagy and lysosomal gene expression is dependent upon TFEB.Fig 6(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4. (B) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4. (C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.
Figure 6Left side: V-ATPase activity is regulated by reversible disassembly in a nutrient dependent manner. Upon glucose withdrawal, active V1Vo dissociates into subunit C and autoinhibited V1-ATPase and Vo proton channel subcomplexes in an ATP dependent manner. Reassembly of active V1Vo from autoinhibited V1 and Vo and C subunit requires the "Regulator of the H+-ATPase of the Vacuolar and Endosomal membranes" (RAVE) complex. Right side: The here presented data show that disassembly of V1Vo can also be induced by presence of Oxr1. The physiological role of Oxr1 induced disassembly, and whether there is a mechanism in vivo to release Oxr1 and reactivate V1 for reassembly, is currently not known.
Non-apical division occurs in the posterior epiblast and can give rise to one or two mesoderm cells.Figure 3(a) Z-projection (a) and 3D cell reconstruction (a') of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells showing a division giving rise to an epiblast and a mesoderm cell that will exit from the epiblast layer. White dotted lines outline basal (top) and apical (bottom) borders of the epiblast at 0min, 25 min, 50min, 1h15min and 1h40min. (b) (c) Z projection of stills from two-photon live imaging of an E7.25 embryo mosaically labelled for epiblast cells. Non-apical division can be observed in the posterior epiblast only, without (b) or with (c) attachment to the apical side. Stills were taken at 0min, 25 min, 50min, 1h15min and 1h40min for (b) and 50min, 1h15min, 1h40min, 2h05min and 2h30min for (c). White dotted lines outline basal (top) and apical (bottom) borders of the epiblast. (d) Mitotic Index (GFP+ dividing cells over total number of GFP+ cells) in the anterior and posterior region, as well as among cells exiting the epiblast at the PS. The posterior region includes the PS region, as it could not be precisely discriminated. (e) Ratio of non-apical rounding (non-apical GFP+ rounding divided by the total GFP+ rounding) and expressed in percentage for the anterior and posterior regions. (f) Ratio of non-apical division (non-apical GFP+ division over total GFP+ division) and expressed in percentage for the anterior and posterior region of the embryo. (g) Quantification of posterior cell division outcome for apical and non-apical divisions. Outcomes are 2 epiblast daughter cells (epi/epi), one epiblast and one mesoderm daughter cells (epi/meso) or 2 mesoderm daughter cells (meso/meso).
Analysis of the effects of Rubicon and UVRAG on HCV yield.Fig 5Cells transfected with the control siRNA (siNC), the Rubicon siRNA (siRb), the Rubicon expression plasmid (pRubicon) or the UVRAG expression plasmid (pUVRAG) were infected with HCV (m.o.i. = 1). The incubation media were harvested at 24 and 48 hours post-infection and used to infect naïve Huh7.5 cells. Cells were either fixed and stained for the HCVcore protein for the determination of viral titers (A) Cells transfected with the control siRNA (siNC), the Rubicon siRNA (siRb), the Rubicon expression plasmid (pRubicon) or the UVRAG expression plasmid (pUVRAG) were infected with HCV (m.o.i. = 1). The incubation media were harvested at 24 and 48 hours post-infection and used to infect naïve Huh7.5 cells. Cells were lysed for Western-blot analysis of the HCVcore protein (B) two days after infection. Actin served as the loading control in (B).