EMBO/sd-character-level-ner huggingface dataset:
Figure 2A, Superposition of the complete and partial ColE9 translocon structures (grey) aligned on OmpF. TolB in red and orange denote the full and partial translocons, respectively, ColE9 T-domain is presented in crimson and pale orange for the full and partial translocons, respectively. B, Sideview comparison showing the relative positions of the ColE9 T-domain (residues 85-316) in the two structures and highlighting the 15o rotation that occurs transitioning from the full (crimson) to the partial (pale orange) translocon. C, Extracellular view of the ColE9 T-domain position, with the OmpF trimer shown in the background. The loss of BtuB from the translocon complex elicits a 12 Å movement along the axis of rotation (black arrow) that results in repositioning of the T-domain from a central location (crimson) to above subunit two of OmpF (pale orange). D, TolB undergoes both rotation and translation when transitioning from the full (red) to the partial translocon (orange). The C-terminal β-propeller domain of TolB, which binds the ColE9 TBE, moves toward OmpF in the OM by ~8 Å along the rotation axis. E, View along the rotation axis from the periplasmic side of OmpF (grey surface) highlighting the 50o rotation that TolB undergoes upon loss of BtuB from the translocon complex.
Rolling between the two constitutive ABD dimers boosts receptor activity.Figure 2Left, localization of site 5 at the interface between the two constitutive ABD dimers. Right top, crystal structures of the inhibited (PDB 5IOV; Zhu et al, 2016) and presumably active (5FXG; Tajima et al, 2016) states illustrating the differences in distances between GluN2B-L795 and GluN1-E698 or GluN1-R673 at interface 5. Those three residues are colored green, while GluN1 and GluN2B subunits are colored red and blue respectively. Right bottom, schematic representation of the GluN1-R673C/GluN2B-L795C disulfide cross-link capturing an inhibited state of the receptor, and of the GluN1-E698C/GluN2B-L795C disulfide cross-link trapping the rolling motion Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801 Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors
Fig.Figure 7(A, B, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). Effect of miR-143-3p or miR-145-5p overexpression on YAP (A) and MRTFA (B) nuclear translocation by immunofluorescence. Data are represented as scatter plots with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or with a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). Effect of BRAFi or BRAFi plus MEKi on YAP nuclear translocation by immunofluorescence. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm. M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or with a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). Effect of BRAFi or BRAFi plus MEKi on MRTFA (D) nuclear translocation by immunofluorescence. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm. E) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ****P≤0.0001. M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm. M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.
Interaction and colocalization of Glycogen synthase with Atg8 is disrupted in R593A and W609A mutants.Figure 6(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined. (B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h. (C) Venus-GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus-GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus-GlyS (S651A) was localized to the autophagosomes. (F) Venus-GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.
The negative feedback-loop between ECM and proliferation is necessary and sufficient to restore tissue architecture.Figure 6Quantification of fibroblast proliferation (left; n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates) and collagen density (right; n= 4 PW0, PW2; n= 3 PW4, PW7, PW10, PW14; n= 2 PW21 biological replicates) inside (A) and outside (B) the wound bed over time. System of partial differential equations describing the process of dermal wound healing. Temporal evolution of proliferating fibroblasts (PF) and dermal ECM density over time. Simulation of dermal wound closure showing the distribution of PF (blue), QF (yellow) and ECM (green) at the time of wounding and at two subsequent time points. From left to right, we performed a wound by imposing that both proliferating cells and ECM are zero in a specific region and simulate the wound healing process during time with the model equations In the schemes above: ECM, grey; wound bed, brown; epidermis, green; adipocytes, round red circle; QF, yellow square; PF, blue square; activated fibroblasts, triangles.
Dysfunctional Quality Control in Gaucher Disease With loss of gba, lysosome function is severely impaired, and autophagy does not occur.Figure 7Dysfunctional Quality Control in Gaucher DiseaseWith loss of gba, lysosome function is severely impaired, and autophagy does not occur. Dysfunctional organelles accumulate, including mitochondria. Due to loss of gba and the self-propagation of α-synuclein, the autophagic pathway is disrupted, compromising UPS function. The damaged mitochondrial respiratory chain cannot support the potential, which decreases to a level at which the ATPase reverses. A decreased ΔΨm limits their ability to re-enter the fission-fusion cycle; this low ΔΨm, maintained by the ATPase, appears to be sufficient in maintaining a potential above the critical level required to recruit Parkin, and thus mitochondria are not flagged for turnover, amplifying the defect.
Calcineurin inhibition leads to impaired cytokine production, reduced early neutrophil recruitment and delayed fungal killing during pulmonary aspergillosis.Figure 2Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM. Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM. Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i. Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen. Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3. Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.
TP53INP1 deficiency is linked with impaired PINK/PARKIN mitophagy.Figure 5After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin. HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control. Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin.
The recruitment of ubiquitin and LC3 to GFP‐Gal3‐positive damaged lysosomes.Figure 1(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.
GCN5 acetylates TFEB at K116, K274 and K279.Figure 3A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test). B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf. C Quantification of LAMP1 puncta in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test). D Immunoblot showing LAMP1 levels in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells. E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys). F TFEB-Flag acetylation in the stable TFEB-Flag-expressing HEK293 cells transfected with Myc-tagged GCN5 or GCN5-E575Q after incubation with GCN5 siRNA for 48 h. G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively. H Co-precipitation of GCN5 with TFEB. TFEB was immunoprecipitated from MEF cells using anti-TFEB, and the precipitates were analyzed using anti-GCN5. I In vitro acetylation assays of TFEB. Purified recombinant TFEB was incubated with Myc-GCN5 or Myc-GCN5-E575Q immunoprecipitated from HEK293 cells. J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.
mtON inhibits hypoxia-adaptation.Figure 5Schematic of HR experiments. Top shows control hypoxia and reoxygenation. Below is the hypoxic preconditioning (PC) protocol, survival for these two timelines shown in panel B. Third from the top shows FCCP treatment protocol, calculated protection shown in panel C. Fourth and fifth from the top show light treatment protocol + and - PC, Calculated protection data from these protocols shown in panel D. Bottom shows light treatment during hypoxia, calculated protection data from these protocols shown in panel E. Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM. Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM. Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM. light during hypoxia. 2 sample 2-tailed unpaired t-test, *p = 0.0276, n = 7 independent experiments, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.
Non-overlapping old versus new histone H3 distribution patterns in mitotic Drosophila female germline stem cells (GSCs), but not in cystocytes (CCs).Figure 1(A) A cartoon and corresponding immunofluorescence image depicting the Drosophila germarium. Terminal filament cells (TFC) and cap cells (CapC) create a niche for female germline stem cells (GSC), which divide asymmetrically to self-renew and produce a cystoblast (CB). The CB undergoes four mitotic divisions to create differentiating cystocytes (CC). The germline (gray) is supported by somatic (green) escort cells (EC) and follicle stem cells (FSC) that produce follicle cells that surround developing egg chambers. The spectrosome (Spec, red) is a specialized organelle in the early-stage germline, such as GSCs and CBs. The spectrosome structure is round while fusome is branched, which branches over the mitotic divisions in further differentiated germ cells, connecting the CCs within a cyst. (B) A cartoon detailing how the heat shock controlled dual-color system driven by GreenEye-nanos-Gal4 labels preexisting (old) histones with eGFP (green fluorescent protein) and newly synthesized (new) histones with mCherry (red fluorescent protein), respectively. (C) A scheme of recovery time and histone incorporation after heat shock that induces an irreversible genetic switch in the histone transgene. (D-F) Old (green) versus new (red) histone patterns for H3 (D), H4 (E), and H2A (F) in prometaphase female GSCs marked by anti-H3S10p (gray). (G) Old (red) versus new (green) histone patterns for H3Rev in prometaphase female GSCs marked by anti-H3S10p (gray). (H-J) Old (green) versus new (red) histone patterns for H3 (H), H4 (I), and H2A (J) in prometaphase female 4-cell CC marked by anti-H3S10p (gray). (K) Old (red) versus new (green) histone patterns for H3Rev in prometaphase female 4-cell CC marked by anti-H3S10p (gray). (L) (L) Quantification of overlap between old and new histones in late prophase and prometaphase GSCs and CCs using Spearman correlation: The measurement is from a single Z-slice at the center of each mitotic nucleus, which shows results similar to analyzing every Z-slice throughout the entire Z-stacks followed by averaging them (Fig EV2A, see Materials and Methods). Data was collected over 20, 16, 11, and 12 separate experiments for H3, H4, H2A, and H3Rev, respectively. Values are mean + 95% CI. P-value: pairwise ANOVA test with bonferroni correction. ****: P< 0.0001, n.s.: not significant.
FRAP analysis of coat-associated proteins at the endocytic site.Figure 5Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of
Figure 5A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7). B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm. C. LUC assays in protoplasts of wild-type, bes1-D or the hsfA1qM plants. LUC reporter constructs, driven by 2 kb of the HSP70.3, HSP70.4 and HSP90.1 promoters, were tested either alone (0, with empty effector plasmid) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed. Data show the mean ±SD. n=5. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test. D. Basal heat stress resistance of hsfA1qM and bes1-DxhsfA1qM plants. Plants were directly exposed to 45°C for the indicated time. Data show the mean ±SE. n=8. E. Working model for the contribution of BES1 to heat stress signaling. BES1 is de-phosphorylated and activated by heat stress, utilizing ABI1 and redundant ABA-repressed PP2C phosphatases. It then interacts with HSFA1s, which are also activated by heat, for binding to HSEs in promoters of HSP70s and HSP90s genes, which induces them and increases heat stress resistance. BRs, while not needed for the activity, can promote it.
Effect of YAP on the anchorage-independent growth of normal and cancerous cervical cells.Figure 3A Representative images showing colonies formed by Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells after growth in soft agar for 9 days. Scale bar: 500 μm.B Quantitative data showing colony formation in Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells. Each bar represents mean ± SEM of five independent experiments. Bars with different letters are significantly different from each other (Ect1-MXIV vs. Ect1-YAP, P = 0.0003; Ect1-MXIV vs. Ect1-YAPS127A, P = 0.0002). C, E Representative images showing the anchorage-independent growth of YAP-expressing HT3 (C) and ME180 (E) cervical cancer cell lines. Anchorage-independent cell growth was determined by the soft agar colony formation assay. Scale bar: 500 μm.D, F Fluorescence-based quantitative analysis showing the differences of anchorage-independent growth in HT3-MXIV, HT3-YAP, and HT3-YAPS127A (D) and ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells (F). The anchorage-independent cell growth was determined using a CytoSelect 96-Well Cell Transformation Assay kit, and data are presented as relative fluorescent units (RFU). Each bar represents mean ± SEM of four independent experiments. Bars with different letters are significantly different from each other (HT3-MXIV vs. HT3-YAP, P = 0.0002; HT3-MXIV vs. HT3-YAPS127A, P < 0.0001; ME-MXIV vs. ME-YAP, P = 0.0011; ME-MXIV vs. ME-YAPS127A, P = 0.0013).
Hepatocellular carcinoma in P240 PR-SET7-deficient mice is composed of cells expressing hepatic progenitor cell markers.Figure 6A, B Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies. C-E Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.