EMBO/sd-character-level-ner huggingface dataset:
AosR is a novel redox responsive transcription factor.Figure 1Schematic outline of the strategy used for the identification of rv1332/aosR. Single-cell suspensions of Rv, Rv∆aosR, and Rv∆aosR::aosR strains were used to infect pΦ isolated from Balb/C at 1:10 MOI. Cells were lysed at indicated time points and the intracellular survival was enumerated. Data is presented as mean CFU log10/ml ± SEM and is representative of two biologically independent experiments, each performed in triplicates (n=3). Balb/C mice were aerosolically challenged with Rv, Rv∆aosR, and Rv∆aosR::aosR strains. Each data point represents log10 CFU obtained from the infected lung of mice and e­rror bar depicts the median with interquartile range for each group. Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group. Bacterial strains were treated with indicated oxidizing agents for 24 h and the survival was monitored. Control represents untreated cultures. Data is presented as mean CFU log10/ml ± SEM and is representative of two biologically independent experiments, each performed in triplicates (n=3).
Sphinganine accumulation accompanied by loss of genome integrity is an evolutionarily conserved hallmark of the aged vertebrate heart: (A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4).Figure 1(A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4). (B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine. (D) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates. (E) Heatmap of differentially expressed transcripts (Log2 fold change +/- 1, adjusted p-value cut off <0.05) between young and aged killifish hearts (n = 3). Significantly enriched relevant GO categories depicted as bar graph. (F) Transcriptional drift variance of genes involved in genome integrity, epigenetic regulation and sphingolipid metabolism. (G) γH2A.X staining of young, mid-aged and aged killifish hearts show signs of DNA damage, increasing with age (n = 4, # of sections per animal = 10 alternate sections, 10 µm thickness). (H) Quantification of γH2A.X+ cardiomyocyte nuclei. (I) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays. Representative images of single nuclei from young and old hearts along with comet length on the right (n = 4). (J) γH2A.X staining of young and aged human ventricles show signs of DNA damage accumulating with age (n = 2, # of sections per sample = 5 alternate sections, 10 µm thickness). (K) Quantification of γH2A.X+ cardiomyocyte nuclei is on the right. Scale bar = 50 µm.
Fig.Figure 6A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22). C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).
Fig.Figure 1 Fig. 1 Overview of integrative structural network biology of native cell extracts in a thermophilic eukaryote. We combined computational modeling approaches adapted from network biology (molecular profiling) with molecular biophysics, electron microscopy (EM) (structural profiling) and quantitative and cross-linking mass spectrometry (interface profiling) to systematically chart and characterize the organization of protein complexes into functional, local communities. Large-scale electron microscopy and cross-linking mass spectrometry are used as validation tools.
Selection and depletion of the edited T cells by exploiting an optimized hEGFRt gene.Figure 3A Schematics of donor DNA templates carrying corrective cDNA coupled to IRES sequence and hEGFRt selector genes. B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A). C Representative plots showing EGFR expression in bulk edited CD4+ T cells derived from male HD at 3 days after treatment with immunotoxin (left), antibody (middle) or toxin (right). D Bar plot showing percentage of EGFR+ T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin, antibody or toxin, measured by FACS Analysis. Friedman test with Dunn's multiple comparisons. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (**p=0.0052 and **p=0.0024 for immunotoxin vs antibody and **p=0.0010 and **p=0.0024 for immunotoxin vs toxin, referring to EGFRmod1 and EGFRmod2 respectively). Different dose-conditions were used as a unified group for statistical analysis (n=10 for each group). Median ± IQR.
a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.f10(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm. (c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm. (e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. 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 four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).
Figure 3HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay. HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay. C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media. D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot. E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4). F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells. G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells. H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot. I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot. J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.
a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice.figf1(a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice. Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum (Fed). Bars represent mean ± s.d. for n = 5 mice; *P≤0.05; **P≤0.01; ***P≤0.001. (b) Representative β-gal staining of liver and kidney frozen sections isolated from fed and 24-h-fasted heterozygous Tcfeb- β-gal mice. c) Time-course expression analysis of TFEB in wild-type HeLa cells, MEFs and hepatocytes after the addition of starvation media (time 0). Data represent mean ± s.d. for n = 3 independent experiments. (d) Expression levels of transfected hTFEB-FLAG, Tcfeb- β-gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control (+/+) and heterozygous Tcfeb- β-gal/+ mice. Data represent mean ± s.d. for n = 3 mice; *P≤0.05. (e) Time-course expression analysis of TFEB mRNA in control (Ctrl) or TFEB-overexpressing HeLa cells during fasting and re-feeding. Primers specific for the endogenous TFEB were used. Data represent mean ± s.d. for n = 3. (f) Time-course expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control MEFs during fasting and re-feeding. Specific primers for the endogenous Tcfeb were used. Data represent mean ± s.d. for n = 3 mice. (g) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control hepatocytes in fed and after 24 h fasting. Data represent mean ± s.d. for n = 3 mice. (h) ChIP analysis from liver of mice fed ad libitum or 24 h fasted. The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1. Red rectangles represent exons, and the ATG indicates the first codon (from the mouse Tcfeb isoform b). The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Data represent mean ± s.d of 3 independent experiments.
a) Chemical structures of SMERs 10, 18 and 28.figf1(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28). (c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death). (d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a). e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Figure 1(A) The XccbphP gene (XC_4241) is located downstream the heme oxygenase gene (XccbphO) as a bicistronic operon. Specific primers (small numbered arrows) were designed to evaluate the physical linkage between XccbphO and XccbphP mRNAs. (B) The confirmative RT-PCRs were resolved in a 2% agarose gel. Untreated reverse transcriptase RNAs (-RT) and genomic DNA served as negative and positive controls, respectively. (C) The XccBphP domain architecture. The photosensory module, composed by the PAS2-GAF-PHY domain triad, binds to the chromophore biliverdin IXα (BV) from a conserved cysteine residue indicated by a red arrow (Cys13). The output module comprises a single PAS9 domain. (D) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.
TAG .Figure 3(A) Rescue of cholesterol depletion on the plasma membrane (PM) reverses SARS-CoV-2 entry block in Calu-3 cells. 25HC induces the depletion of accessible cholesterol on the plasma membrane and supplement of additional cholesterol can reverse the depletion and viral entry. (B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy. (D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting. Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection. Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.
a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.figf5(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins. (b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL. (c,d) Molecular model of interaction sites for CL binding to LC3 by docking analysis. CL is coloured magenta in the top-ranked binding site conformation (c), and cyan in the alternative conformation (d), with the phosphates as spheres (oxygen, red; phosphorus, orange). Amino acids interacting with CL are represented as spheres along the LC3 ribbon structure, coloured blue>orange from N>C terminus. (e) Thirty-nine LC3 family sequences including isoforms A-C were aligned with ClustalW and displayed using WebLogo, with symbol heights corresponding to relative amino acid frequency. Asterisks denote residues predicted to contact CL in the favoured (magenta) and alternative conformations (cyan). (f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle. (e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).
Figure 5A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5). B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition. C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration. D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.
a,b) Subcellular fractionation of FAO (a) or HepG2 (b) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments.figf2(a,b) Subcellular fractionation of FAO (a) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments. Catalase, PMP70 and lamin A/C were used as subcellular markers for the peroxisome (P) and nuclear (N) fractions, respectively. EEA1, LAMP1 and VDAC were used as markers for endosomes, lysosomes and mitochondria, respectively. WCE, whole-cell extracts; M, membrane; C, cytosol. (a,b) Subcellular fractionation of HepG2 (b) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments. Catalase, PMP70 and lamin A/C were used as subcellular markers for the peroxisome (P) and nuclear (N) fractions, respectively. EEA1, LAMP1 and VDAC were used as markers for endosomes, lysosomes and mitochondria, respectively. WCE, whole-cell extracts; M, membrane; C, cytosol. (c) Subcellular fractionation of TSC2+/+ and TSC2−/− mouse embryonic fibroblasts (MEFs) demonstrating the localization of TSC2, TSC1, Rheb and AKT. PMP70, catalase (peroxisome fraction, P), LDH (cytosolic fraction, C), β-integrin (membrane fraction, M) and lamin A/C (nuclear fraction, N) were used as subcellular markers. Uncropped images of western blots are shown in Supplementary Fig. S6.
Figure 1(A) Licochalcone B (LicoB) structure. (B) Cell Counting Kit 8 (CCK-8) was used to assess the viability of BMDMs treated with different doses of LicoB for 24 h. (C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.