EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Experimental set-up for administrating control liposomes (Ctrl-Lipo) or macrophage-depleting clodronate liposomes (Clod-Lipo) as well as vehicle control (Veh) or Tamoxifen (Tam) for inducing whole body NLRP3A350V expression in Nlrp3iR26-A350V/WT mice. (B) Body weight of Nlrp3iR26-A350V/WT cohorts with indicated treatments (Ctrl-Lipo + Veh n=5; Ctrl-Lipo + Tam n=6; Clod-Lipo + Veh n=4; Clod-Lipo + Tam n=6). Data information: Statistics were analyzed by student t-test Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01; *** p<0.001. (C) Relative spleen weights of Nlrp3iR26-A350V/WT cohorts with indicated treatments at day 6. Data information: Statistics were analyzed by student t-test (C, Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01; *** p<0.001. (D-G) (D) Flow cytometric quantification of CD11b+F4/80+CD64+ macrophages, (E) IL-1β and (F) IL-18 protein levels, and (G) flow cytometric quantification of CD11b+Ly6C+Ly6G+ neutrophils in spleen, liver and kidney from Nlrp3iR26-A350V/WT cohorts with indicated treatments at day 6. Data information: Statistics were analyzed by student t-test , D, E: spleen, kidney, F: spleen, liver, G: spleen, liver) or Mann-Whitney test (E: liver, F: kidney, G: kidney). Dots represent individual biological replicates. Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01; *** p<0.001.
, Model calibration with IFNα-induced signal transduction in Huh7.Figure 3Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.   Model calibration with time-resolved IFNα-induced feedback transcripts upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=14) using a combined scaling and error model. Model trajectories are represented by lines. Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9 and USP18 determined 24 hours after prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD-camera-based device. Averaged values (N=4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model. Model calibration of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=4 to N=22) using a combined scaling and error model. Model trajectories are represented by lines.
Figure 5Col-0 and pifq seedlings were grown under short days at 22ºC and 28ºC, and samples were collected at the indicated time points. Expression of BR biosynthetic genes was analyzed by qRT-PCR, using the constitutive PP2A gene as internal control. Error bars represent SD of three technical replicates. The experiment was repeated twice with similar results Activation of the XTR7 PIF+BES-UP target was studied as a positive control of the experiment. Error bars represent SD of three technical replicates. Dark periods are shown in grey. The experiment was performed three times with similar results Increased levels of de-phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis. Col-0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC, on half strength MS media (mock) or media supplemented with 0.5 µM BRZ. BES1 was analyzed by western blot hybridization with an anti-BES1 antibody. RPT5 was used as a loading control.
Raptor phosphorylation by AMPK is required for AICAR-induced autophagy in TSC2−/− MEF cells.Figure 5(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm. (B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1. C) Schematic representation of AMPK-mediated suppression of the inhibitory effect of mTORC1 on the ULK1 autophagic complex.
Assay to screen for compounds that inhibit SARS-CoV-2 replication.Fig. 6. Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown).
Figure 5A (Upper) Schematic representation of ER-located fluorogenic probe Halo-tag K73T/L172Q mutant (AgHaloER). SP: signal peptide. The carboxyl-terminal KDEL motif is the ER retention sequence. (Lower) Diagram of AgHaloER that detects ER proteostasis in live cells. B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm. C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm. D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm. E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm. F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).
Impact of TFEB on muscle fibre ultrastructure in GAA−/− mice.Figure 8A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).
TOR-dependent phosphoproteomic changes identifies conserved proteins with potential roles in modulating rates of protein synthesisInvestigating the protein expression levels for the phosphoproteomics time course study and validation of experimental conditions based on known indirect TOR phosphorylation targets.Figure 2A Scatter plot showing the protein levels in wild type cells after 40 minutes of Torin1 (5 µM) addition plotted against the corresponding values for the same protein in Torin1-resistant cells after 40 minutes. Dark grey dashed lines at x = ±1 indicate the 2-fold threshold, of which no proteins exceeded in the study. The light grey dashed line represents y = x, plotted as guide to indicate positive correlation in protein levels between wild type and Torin1-resistant cells. B Frequency distribution of the proteins levels after 40 minutes of Torin1 treatment in wild type cells (n = 3380), with 98% of all proteins (3314/3380) within a 1.3-fold boundary, as indicated by the grey dashed lines at ±0.38. C - F Phosphorylation changes of the conserved phosphosites, S235 and S236, on Rps601(C) and Rps602 (D - F), over 85 minutes of Torin1 (5 µM) treatment wild type (green) and Torin1-resistant (brown) cells. M-values (in brackets) represent the different multiplicities of the same phosphosites. Lines were fitted with the curve fitting function on Prism9 using the the non-linear regressions exponential one-phase decay (C, D) or plateau followed by one-phase decay (E, F) functions respectively. Grey dashed lines indicate 2-fold threshold.
Figure 7Diagram summarising the changes occurring at decommissioned or maintained enhancers during commitment from naïve pluripotency. Decreased NANOG levels, although reversible, facilitate subsequent decrease in the levels of other naïve pluripotency TFs, including Esrrb and Klf4. Downregulation of these TFs triggers irreversible changes in the pool of active enhancers and marks commitment to differentiation. Class I enhancers are regions where OCT4 occupancy depends on the robust binding of naïve TF. These elements are decommissioned during commitment, losing histone acetylation and accumulating DNA methylation. Other enhancers, on average showing less pronounced binding of naïve TFs, retain OCT4 and active histone marks in primed pluripotent cells. These elements constitute a core of 'primed' regulatory elements bound by OCT4 that may serve to nucleate the regulatory architecture through which lineage specific TFs collaborate with Oct4 to specify the early post-implantation epiblast identity.
Figure 5In the presence of cyst(e) ine, cysteine blocked the interaction of CARS with AMPKγ2, the AMPK complex is in an inactive state. In the deficiency of cystine, CARS is able to bind to AMPKγ2, thus recruiting CaMKK2 to the AMPK complex and leading to phosphorylation and activation of AMPK, which ultimately promotes cell survival.
a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4).figf3(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3). (b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker. (c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389. (g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM. (h) TAT-GluN2BCTM (25 μM; n = 5; P = 0.001), but not control TAT-GluN2B (25 μM; n = 4; P = 0.223), decreased native DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; P = 0.302). One-way ANOVA, P 0.001, F(5,24) = 13.591. Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control (white bar; *P 0.05, **P 0.01 and ***P 0.001) or NMDA-treated group (gray bar; ΔP 0.05, ΔΔP 0.01 and ΔΔΔP 0.001). Membranes reprobed for β-actin were used as a loading control. Bars represent relative means ± s.e.m. Sample sizes represent number of individual experiments. Full-length blots are presented in Supplementary Figure 9.
CGA-CCG and CGA-CGA induce stalling through decoding-incompatible mRNA conformations in the A site (A-C) Cryo-EM structural characterization of the pre-state 80S RNC with A site tRNA in the decoding center.Figure 4(A-C) Cryo-EM structural characterization of the pre-state 80S RNC with A site tRNA in the decoding center. (A) Schematic representation of the decoding situation (top) and molecular model for the pre-state RNC with A site tRNA in the decoding center. (B) General overview of the A, P and E sites with A/A and P/P tRNAs and mRNA. (C) Detailed view of the mRNA in the A site using sticks model with cartoon phosphate backbone representation. The 18S rRNA bases A1755 and A1756 recognize the minor groove of A site tRNA - mRNA interaction during tRNA decoding. (D-F) Cryo-EM structural characterization of the CGA-CCG stalled 80S RNC. (D) Schematic representation of the stalling situation (top) and molecular model of the CGA-CCG stalled RNC (bottom). (E) General overview of the A, P, and E sites with P/P and E/E tRNAs and mRNA. (F) Detailed view of the mRNA in the A site using sticks model with cartoon phosphate backbone representation. The mRNA positions +2 to +5 and their interactions are shown. The C +4 is flipped by approximately 95° degrees towards the wobble A:I base pair in the P site and stabilized by interaction with the C1637 of the 18S rRNA helix 44. The C +5 is stabilized by stacking interaction with the A1756 of the 18S rRNA which normally recognizes the minor groove of A site tRNA - mRNA interaction during decoding (C). (G-I) Cryo-EM structural characterization of the CGA-CGA stalled 80S RNC. (G) Schematic representation of the stalling situation (top) and molecular model of the CGA-CGA stalled RNC (bottom). (H) General overview of the A, P and E sites with P/P and E/E tRNAs and mRNA. (I) Detailed view of the mRNA in the A site as in (F). Downstream mRNA is indicated by the dotted line. Note the rotation of the C+4 base compared to the CGA-CCG mRNA.
Figure 7One protomer extracted from the post-fusion trimer of TBEV sE (PDB 1URZ [15] + stem residues 402-404 modeled from the sE-linker* structure) displayed next to a protomer extracted from the complete trimer of hairpins of the RVFV Gc (PDB 6EGU [36]) class II fusion protein. Both proteins are color-coded by domains as in Figure 1 but with the two segments that make up domain II distinguished in yellow and brown. An arrow at the top marks a disulfide bond conserved across all class II viral fusion proteins (shown in black). In RVFV Gc, the stem reaches the FL and its C-terminal end is stabilized by interactions with a GPL head group (shown as ball-and-stick) bound in a cavity underneath the FL, with its glycerol moiety interacting with the conserved disulfide bond. The most C-terminal residue visualized in the TBEV sE-linker* structure, Gln404, is about 15 amino acids short of reaching the FL, which would correspond roughly to position 419 at the beginning of the CS. The location of CS within the stem is indicated in the panel below.
The Nem1-Spo7 complex and ESCRTs are key components for micro-ER-phagy.Figure 3Fluorescence images of ∆atg7 and ∆atg8 cells (A) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm. Fluorescence images of ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm. C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3. D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type. F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control. G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4.
Pharmacological inhibition of BCL-XL in combination with DNA damage-inducing chemotherapeutics or γ-radiation at clinically relevant doses does not impair kidney function and architecture (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose).Figure 7(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed. (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed. (C) Histological analysis of H&E-stained sections of the kidneys of drug-treated GFP-Chimeras or control mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group. (D) Histological analysis of H&E-stained sections of the kidneys of drug-treated mice or control untreated mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.
Figure 2A. Diagram illustrating the maternal-to-zygotic transition in zebrafish, depicting the analysis of mRNA fold changes between 2 and 6hpf. Embryos were bathed in cycloheximide (CHX) at 2 hpf or injected with the RNA PolII inhibitor α-amanitin at 0hpf. B. Cumulative distributions of mRNA fold change (6-2 h) in wild-type embryos, embryos injected with α-amanitin, and embryos treated with the translation elongation inhibitor cycloheximide. The different curves represent transcripts: enriched in non-optimal codons (top quartile, blue), containing at least one miR-430 complementary site (6- , 7- or 8-mer) (green), enriched in optimal codons (top quartile, red) and a control set of maternal genes that do not follow any of the described criteria (gray). P-values are indicated for the comparisons shown with the colored dots (Wilcoxon rank-sum test). C. qRT-PCR analysis for predicted optimal (red) and non-optimal (blue) transcripts relative to the controls (cdk2ap2 and taf15). IK Cytokine, constitutive Heat Shock protein 90 (hsp90ab), tRNA methyltransferase 12 homolog (trmt12) and Hermansky-Pudlak syndrome 4 (hps4). Results are shown as the average expression ± S.D.M. from three biological replicates (top panel) and six biological replicates (bottom panel). (Mo, embryos were injected with a morpholino complementary to the translation start site of the endogenous mRNA). * = p< 0.0001 compared to 2h. * = p< 0.005 compared to uninjected embryos. D. Schematic representing the maternal mRNA decay dynamics and the transcript groups analyzed.E. Barplot representing relative codon distributions between the most unstable (quartile) and the most stable (quartile, n = 1458) maternal mRNAs in wild type, α-amanitin, and cycloheximide (CHX) treated embryos. Codons were sorted by their CSC value (Fig 1D). P-values are indicated for the comparisons between enrichment/depletion in each condition and CSC (Spearman rank test). Optimal codons were highlighted in red and non-optimal in blue (light, weak; dark, strong, as Fig 1F). Note the depletion of optimal (red) and enrichment of non-optimal (blue) codons in unstable mRNAs in wild-type and α-amanitin treated embryos but not in CHX treated embryos, consistent with a translation-mediated mechanism of maternal mRNA decay through codon optimality.
a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L.figf6(a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L. plantarum (DAP; 100 μg/ml) or lysine-type PGN from S. epidermidis (Lys). (b) Dot- or ring-shaped GFP-LC3 signals in hemocytes after infection with wild-type or Δhly L. monocytogenes or treatment with rapamycin (5 μM) or with TCT, DAP-type PGN or lysine-type PGN. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).