EMBO/sd-character-level-ner huggingface dataset:
a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP–LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right).figf6(a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm. (b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control. (d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control. (e) Quantification of autophagic flux from three experiments in different cell lines. Normal dividing cells (MBd poor) typically have high autophagic flux, whereas stem and cancer cells (MBd rich) have low autophagic flux. The data are presented as mean±s.d. (b-e). Uncropped images of blots are shown in Supplementary Fig. S6.
Figure 2Fluorescence Recovery After Photobleaching (FRAP) experiment on AcGFP-Siwi and AcGFP-Siwi-D670A. Red dotted lines represent recovery traces of Siwi-D670A foci (n=36) and black dotted lines represent recovery traces of Siwi foci (n=36). Solid lines represent locally estimated scatterplot smoothing (LOESS) curve and the grey areas represent 95% confidence level interval. Fluorescence intensity is quantified with the softWoRx software and normalized to minimum and maximum in a 0-1 scaling. (Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 minutes incubation at RT. Z-projections of the middle 6 μm stacks are then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n=30 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test. Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n=6 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test. Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n=32 cells) or BmSpnE (n=32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared to control (dsRLuc, n=32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.
Figure 2(A) The number of proteins changed in CSN2 or CSN7 RNAi revealed by quantitative proteomics. Red: the number of up-regulated proteins; green: the number of down-regulated proteins. Candidate proteins from the quantitative proteomics are listed along with protein fold changes in CSN2 or CSN7 RNAi. (B 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions. (C) Quantification of the lipid droplet size in (B). HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant. (D) In Drosophila S2 cells expressing RDH/CG2064-GFP, treatment with the proteasome inhibitor MG132 increases the RDH/CG2064-GFP protein level detected by anti-GFP antibody in a western blot. (E) The RDH/CG2064-GFP protein level is increased in ppl>DGAT1 larvae with CSN2 RNAi, as detected by anti-GFP antibody in a western blot. , F 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (F) RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. (G) Quantification of the lipid droplet size in (F). data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (H) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. (I) RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.
Figure 2A, B Total protein was extracted from 4-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings, respectively. WT and dja6 dja5 used in (A) were grown on MS medium with 2% sucrose, while WT, dja6, dja5 and RNAi mutants used in (B) were grown on soil. 10 μg protein was loaded, except for detection with anti-SUFE1 and anti-SUFA antiserum, for which 25 μg were loaded. Specific bands were identified by immunoblotting and by their molecular weight, and are indicated by asterisks. Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right, respectively. The Fe-S types of Fe-S proteins are shown in parentheses. Actin served as controls to normalize protein levels. For each protein, three independent biological replicates were performed and a representative one is shown.
Figure 4Schematic representation of a soluble receptor Neuropilin-1, consisting of five domains; two CUB motifs (A1, A2), two coagulation factor domains (B1, B2) and the MAM domain (C). Rate constant and binding affinities of Sema3A and VEGF to immobilized trap obtained using a one-site Langmuir binding model. C, D. Representative SPR sensorgrams for various concentrations of Sema3A (C) and VEGF (D) binding to immobilized Trap. E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap). I. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap). M. Proposed therapeutic paradigm. From a therapeutic perspective, intravitreal injection of NRP1-derived traps reduces pathological angiogenesis associated with CNV.
3D model and in vivo live imaging of dermal wound repair.Figure 7cross-section of the computational simulation of wound healing in adult dermis. Experimental design of in vivo imaging of wound healing. Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative wound bed Z-stack stitched maximum projections of the entire wound bed (upper panel) showing fibroblasts (green) and collagen with second harmonic generation (SHG) in purple at indicated time points after wounding and before live imaging. Dotted line indicates wound edge and boxed area indicates the position of the magnified area below. Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative time-lapse images of adult wound bed at PW4, where Z-directional movement is visualized by pseudo colouring the fibroblast nuclei (red, movement towards the basement membrane; blue, movement away from the basement membrane). In vivo lineage tracing of upper (Blimp1Cre) and lower (Dlk1CreER) dermal fibroblasts during wound healing. Experimental strategy for in vivo lineage tracing. In vivo lineage tracing of upper (Blimp1Cre) and lower (Dlk1CreER) dermal fibroblasts during wound healing. Immunofluorescence image of Blimp1Cre x Confetti (upper panels) and Dlk1CreER x Confetti (lower panel) back skin inside and outside the wound bed at 10 days post wounding. Colours show YFP (green), RFP (red) and CFP (blue) with bright field image overlaid. (G) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n= 400 clones of 4 biological replicates). (H) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n=300 clones of 2 biological replicates).
a–e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.figf4a-e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.5): a, midbrain; b-d, liver; c, d, developing autophagosomes; e, heart. Arrows indicate the autophagic vacuoles. f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria. h-j, m-o, The numbers of autophagic vacuoles (h, m) and autophagic vacuoles containing mitochondria (i, n) and persisting mitochondria (j, o) in reticulocytes (h-j) and erythrocytes (m-o). Error bars indicate s.d. (*P  0.05; n = 55 cells). f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.
Overall structure of post-fusion 6-HB in SARS-CoV-2.Fig. 2.Sequence alignment of HR1 and HR2 domains in SARS-CoV and SARS-CoV-2. b. Structure of SARS-CoV-2 6-HB is shown in cartoon representation with HR1 colored in green and HR2 in cyan. The structural dimensions are indicated in angstroms. c. HR1 trimer of SARS-CoV-2 6-HB is shown in electrostatic surface, and HR2 domain is shown in cartoon representation, the important binding residues of which are shown in sticks and labeled. d. The superposition of 6-HB structure of SARS-CoV (PDB entry 1WYY), MERS-CoV (PDB entry 4NJL) and SARS-CoV-2 is shown in ribbon. The RMSD between structures is indicated. e. The sequence comparison of 6-HB structure of different HCoVs is shown in cartoon representation with different colors for HR1 and HR2. The helical fusion core regions are indicated.
Figure 4IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right). IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition. The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.
Schematic of how fibroblast state switching determines tissue architecture.Figure 8During development the tissue scale behaviour of dermal fibroblasts is achieved and maintained by a feedback loop between ECM deposition and fibroblast switching from proliferating to quiescent. Upon wounding, quiescent cells re-enter the cell cycle and tissue regeneration occurs via the sequential coordination of proliferation, active migration and cell displacement in the wound bed.
Figure 2Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (A) Typical confocal images from neurons stained for MAP2, Synaptobrevin/VAMP2 and Munc18-1. Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using confocal microscopy (B) Average synaptic Munc18-1 intensity (M18WT: 389 ± 85 a.u., n=21; M18Y473D: 261 ± 49 a.u., n=20, t-test with Welch correction, p = 0.2000), somatic Munc18-1 intensity (M18WT: 328 ± 49 a.u., n=21; M18Y473D: 188 ± 21 a.u., n=20, t-test with Welch correction, p = 0.0138), synapse number (M18WT: 129 ± 14, n=21; M18Y473D: 159 ± 14, n=20, t-test, p = 0.1203) and dendrite length (M18WT: 0.433 ± 0.028, n=21; M18Y473D: 0.454 ± 0.037, n=20, t-test, p = 0.6613). Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology(C) Left: typical examples of evoked release upon action potential stimulation. Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (D) Left: typical responses to hyperosmotic sucrose application (500 mM, 3.5 sec.) used to assess the RRP. Average RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473D: 0.08 ± 0.02 nC, n=10, t-test with Welch correction, p = 0.0274). Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (E) Average Pves (EPSC charge / RRP charge) (M18WT: 6.75 ± 0.77%, n=13; M18Y473D: 0.24 ± 0.20%, n=10; Mann-Whitney U test, p < 0.0001). Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (F) Left: example traces of spontaneous release of single SVs (mEPSCs). Average mEPSC frequency (M18WT: 20.18 ± 5.18 Hz, n=15; M18Y473D: 0.28 ± 0.13 Hz, n=17, Mann-Whitney U test, p < 0.0001), mEPSC amplitude (M18WT: 26.0 ± 1.7 pA, n=15; M18Y473D: 27.8 ± 2.2 pA, n=17, t-test, p = 0.5372) and mEPSC decay time (M18WT: 1.98 ± 0.10 ms, n=15; M18Y473D: 2.84 ± 0.41 ms, n=17, t-test with Welch correction, p = 0.0549).
The relative level of TOR signaling pathway and autophagy genes RNA in the fly’s brain.Fig 2A-The Tor gene RNA cycles only in the brains of Canton S in LD 12:12, reaching the highest level 4 h before the end of the night (ZT20) and the lowest 4 h after the beginning of the day (ZT4) (mean RQ +/- SE) [Kruskal-Wallis Test: H (5, N = 25) = 15,10543 p =, 0099; post hoc multiple comparison test: * < 0.05]. B-The relative level of Atg5 RNA in the brain of Canton S male flies, held in LD 12:12 or in DD and in per01 mutants in LD 12:12 (mean RQ +/- SE). The Atg5 RNA cycles in the brains of Canton S in LD 12:12, reaching the highest level 4 h after the beginning of the day (ZT4) and 4 h before the end of the night (ZT20) [Kruskal-Wallis test: H (5, N = 35) = 23,34957 p =, 0003, post hoc multiple comparison test: * < 0.05]. C-The relative level of Atg7 RNA in the brain of Canton S male flies, held in LD 12:12 or in DD and in per01 mutants in LD 12:12 (mean RQ +/- SE). The Atg7 RNA cycles in the brain of Canton S in LD 12:12. The highest level was detected 4 h before the end of the night (ZT20) and at the beginning of the day (ZT1) [Kruskal-Wallis test: H (5, N = 31) = 23,07912 p =, 0003, post hoc multiple comparison test: * < 0.05; ** < 0.01].
PINK1 is required for parkin-dependent MDV formationRepresentative immunoblot of whole-cell lysates from HeLa cells transfected with non-targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 µM CCCP for 6 h in order to stabilize the PINK1 full-length band.Figure 5A Representative immunoblot of whole‐cell lysates from HeLa cells transfected with non‐targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full‐length band. B HeLa cells transfected with GFP−parkin (green) and siRNA targeting PINK1 (siPINK1) or non−targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin−positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm. C Quantification of PDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).
Figure 2A. Average miR expression levels in naive CD4 T cells (purity >97%) before and after 72h TCR-stimulation. Lines indicate changes of +/- 1 log2 fold between samples (n=2, paired pools of three and nine individual samples per library). Library normalization performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log2 fold change values in stimulated samples (log FC) and corresponding adjusted p value (p; Fisher test implemented in Deseq). The two most highly expressed miRs (mir-21-5p and miR-146b-5p) are also indicated. B. Relative miR-34c-5p and miR-155-5p expression levels (2deltaCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. mean ± SEM and p-value for differences in miR-155-5p expression (paired two tailed t-test) are shown. Comparison could not be performed for miR-34c-5p as it is undetermined in unstimulated samples. C. Time course quantification of miR-34c-5p expression (top panel, 2deltaCt values) in three different donors. C. Time course quantification of cell proliferation (middle panel) and cell activation (bottom panel) markers by flow cytometry, in three different donors.
Figure 8Figure 8. Scheme of FGF-dependent regulation of TGF signaling in smooth muscle cells and endothelial cells.In both smooth muscle cells and endothelial cells, suppression of FGF signaling leads to reduction of let-7 miRNAs expression that, in turn, results in increased TGFβR1 expression and activation of TGFβ-dependent transcriptional program. In SMC (left panel), activation of TGFβ signaling promotes SMC conversion from proliferative to contractile phenotype thereby reducing the number of SMCs in the plaque and reducing plaque growth. In contrast, in EC activation of TGFβ signaling promotes endothelial-to-mesenchymal transition thus increasing the number of plaque SMCs and promoting plaque growth.
Figure 2(A) The central channel in the apo-PfFNT protomer viewed from the parasite's membrane plane. The channel, calculated by HOLE (Smart et al, 1996), is indicated by blue dots. The two constriction sites are colored red. Residues forming the constriction sites are in green sticks. Residues that line the rest of the channel are in cyan sticks. (B) Representative figure of the calculated diameters of an apo-PfFNT protomer. (C) A cartoon of the central channel formed within a PfFNT protomer. The channel contains two constriction sites in this conformational state. Residues forming these constrictions and the K35-D103-N108 and K177-E229-N234 triads are illustrated as sticks. (D) Surface representation of the inside of the channel formed by a PfFNT protomer (red, negative -15 kT/e; blue, positive +15 kT/e). The five residues K35, F90, F94, K177 and H230, which are subjected for mutagenesis studies, are in green sticks.