EMBO/sd-character-level-ner huggingface dataset:
Figure 5A Schematic of FdoH and FdnH fusions. B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively. C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. D Alignments of HybA, FdoH, and FdnH in S. sonnei with P. stuartii TatA and with their homologues from phylogenetically diverse organisms (S. sonnei, Salmonella enterica, Yersinia enterocolitica, Proteus mirabilis, Pseudomonas aeruginosa, and Burkholderia pseudomallei) highlighting conserved glycine (green) and proline (orange) residues. E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively. F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI. rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red arrows, respectively. G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.
Figure 3A-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).
a) Autophagic machinery functions properly in WT condition through autophagosome and lysosome fusion, maintaining cellular homeostasis.f5
Figure 2Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles. Three-dimensional reconstruction of RNC FtsQ:SecYEG-ND complex. Primary structural elements of the ribosome and SecYEG-ND are indicated. Local resolution map of SecYEG-ND sub-particle. The cytoplasmic side of the translocon demonstrates higher resolution due to stabilization by the bound ribosome, while high-resolution at the periplasmic side is hindered by the SecYEG-ND dynamics within the complex. The associated ribosome is not shown for clarity. A planar slice through the SecYEG-ND core at different signal levels (blue/green/red) with indicated positions of SecYEG TMHs (SecY indicated in orange, SecE in purple, and SecG in green). A single helical turn could be fitted in a density in the area where SecG TMH 1 was expected (green asterisk).
Figure 1A) M. florum doubling time at different incubation temperatures measured by colorimetric assays. The bars represent the mean and standard deviation values obtained from three technical replicates. The asterisk indicates the absence of significant growth, preventing the calculation of a doubling time. B) M. florum growth kinetics at 34°C. Growth was monitored for 24 hrs by measuring the optical density at 560nm (black circles) as well as the cell concentrations using two different methods, colony forming units (CFUs, red squares) and flow cytometer (FCM, blue triangles). The four typical bacterial growth phases (lag, exponential, stationary, and death) are represented by gray shading. The dots and error bars indicate the mean and standard deviation values obtained from three independent biological replicates. CFU data points superimposed to the x-axis represent values below the limit of detection (2x10-2). C) Exponential growth fit on CFU (red squares) and FCM (blue triangles) counts shown in B. Calculated doubling times (Dt) and correlation coefficients (R2) are shown. The dots and error bars indicate the mean and standard deviation values obtained from three independent biological replicates. D) Representative images of SYTO 9 and propidium iodide (PI) double stained M. florum cells, harvested from an exponential or death-phase culture, observed by widefield fluorescence microscopy. The brightness of each channel was adjusted equally between conditions. Scale bar: 5 µm.
Fig.Figure 5In-situ hybridization for AAV-derived pDyn mRNA at 10 weeks post AAV-pDyn delivery in the left dorsal hippocampus is depicted for a single brain at five levels between 1.1 (left) and 2.3 (right) mm from bregma. The probe specifically detects the codon-optimized sequence, therefore the contralateral side represents a control devoid of the respective mRNA. At 10 weeks post AAV-pDyn delivery, markers of inflammation were analyzed as follows: Relative optical densities (ROD) were evaluated from film autoradiographs after in-situ hybridization for mRNAs of inflammatory markers (Interleukin-1β (B), Interleukin-6 (C), Interleukin1a receptor agonist (D), Tumor Necrosis Factor-α (E) and Nitric Oxide Synthase-1 (F)) in 3 principal sub-regions of the hippocampus in AAV-pDyn (n=7; red) or AAV-eGFP (n=5; blue) injected animals. GC = granule cell layer; CA = cornu ammonis.
Figure 3A. Control (Ctrl) or STING-KO MC38 cells were treated in vitro with STING agonist cGAMP (left) for 6 hours or 5-FU (right) or relevant vehicle controls for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3. B. Ctrl or STING-KO CT26 cells were treated with 5-FU or vehicle control for 24 hours. The RNA expression levels of indicated genes were determined by qRT-PCR. N=3. C. Ctrl or STING-KO YUMM1.7 cells were treated with (left panel) vehicle control (DMSO) or DTIC, or (right panel) PBS or 5-FU for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3. D. Western blot analysis was performed with Ifnα and Gapdh antibodies, for control (Ctrl) and Ifna1-Ifnb1-double-KO (Ifn-DKO) MC38 cells. Two independent KO clones are shown. E. MC38 Ctrl or Ifn-DKO cells were treated with (left panel) control or the STING-agonist DMXAA for 4 hours, or (right panel) control or 5-FU for 16 hours. Ifnβ levels were determined in harvested culture media by ELISA. N=2. Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (F) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (G) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (H) Tumor and spleen weights at the endpoint for (G), with each dot representing a mouse.
KRIT1-ablated cells display autophagy suppression.Figure 1Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments. Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear). Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments. Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14. Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments. Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments. Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments. Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.
Figure 7(A, B) eEF3 binds to (A) non-rotated 80S in a pre-translocation (PRE) state bearing classical A/A- and P/P-tRNA (PRE-1) as well as to (B) non-rotated state occupied by A/A- and a hybrid P/E-tRNA (PRE-2). (C, D) (C) Rotation of the ribosomal subunits leads to unstable binding of eEF3 to a ribosome with A/A- and P/E-tRNA (PRE-3) as well as to (D) a fully rotated ribosomal species bearing hybrid A/P- and P/E-tRNAs (PRE-4). (E) Binding of eEF2-GTP facilitates 40S-head swiveling and translocation of the hybrid A/P- and P/E-tRNA into the chimeric ap/P and pe/E tRNA positions (POST-1). (F) After dissociation of eEF2-GDP and the resulting back-swivel and back-rotation of the ribosome, eEF3 binds stably to the non-rotated ribosome with a classical P/P- and E/E-tRNA (POST-2) with L1-stalk in the ´int´ position. (G) The eEF3-CD directly interacts with L1 stabilizing an ´out´ conformation that facilitates release of the E-site tRNA. eEF3 remains bound to the non-rotated ribosome bearing a P/P-tRNA (POST‑3). Transition from states E to F and F to G are accelerated by eEF3. Binding of eEF1a-ATP-tRNA ternary complex and subsequent peptide bond formation results in formation of PRE-1, as seen in (A). (H) In the absence of eEF3, we suggest that POST-2 ribosomes formed after eEF2 dissociation can bind an A-tRNA, but cannot translocate further because of the presence of three tRNAs on the ribosome. Data information: Volumes (A-G) are representing the cryo-EM reconstructions from the eEF3-TAP pull-out, whereas volume (H) shows a potential scenario based of the results of the eEF3-depletion studies. All maps are filtered to 6 Å. The outline shown in (C-E) assigns the disordered eEF3 ligand present in these volumes.
a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag–ΔN85-ATG16L1(green) and infected for 10–20 min with S.figf6(a) Immunofluorescence microscopy of HeLa cells transfected with HA-Nod2 (red) and Flag-ΔN85-ATG16L1(green) and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments. (b) Immunofluorescence microscopy of endogenous Nod2 and ATG16L1 in Mode-K cells and infected for 10-20 min with S. flexneri M90T. Blue, DAPI. Arrows indicate colocalization of Nod2 and ATG16L1 around invading bacteria. Scale bars, 5 μm. Data are representative of one of three independent experiments.
Figure 6 (A) Steady state levels of LETM1 protein in fibroblasts from controls (C1-C3), WHS LETM1+/- (S1, S3, S4) and S5 the sole case of WHS in this study whose deletion does not encompass LETM1 (boxed in blue). GAPDH is shown as a loading control  (B) Immunofluoresence analysis of control (C1), and WHS LETM1+/- S1-S4, fibroblasts with anti-TOM20 antibody. Scale bars are 8 µm in the main images and 3 µm in offset merged magnifications  (C) Electron micrographs of controls (C1 and C2) and WHS LETM1+/+ (S5) cells; side panels show zooms. Scale bar: 0.2 µm. Black arrows indicate the areas shown offset at higher magnification (D) Electron micrographs of WHS LETM1+/- S1, S3 and S4 cells. Black arrows indicate elongated and branched mitochondria; side panels show magnifications. Scale bar: 0.2 µm
Overview of the results obtained from all immunostainings, in all of the laboratories testing glyoxal.Figure 16 Figure 16 Overview of the results obtained from all immunostainings, in all of the laboratories testing glyoxal. Various cellular targets, ranging from the nucleus to synapses of hippocampal neurons, were tested after fixation with either PFA or glyoxal by us and 11 additional laboratories. Overall, 51 targets were better stained after glyoxal fixation than after PFA fixation, 12 targets were stained worse, and 19 targets were equally well stained.
Figure 5HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). To confirm successful depletion of endogenous β-catenin and the expression of exogenous genes, the resulting cells were immunoblotted with the indicated antibodies (A). HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). Band intensities were quantified using the ImageJ program and normalized with α-Tubulin, and the relative values of band intensities are marked. Cytokinesis-defective cellular phenotypes of each stable cell line were observed under a microscope using immunostained samples (B) HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm. (D) HeLa CCL2 cells infected with either control luciferase (shGL) or β-catenin (shβ-catenin) knockdown virus were subjected to FACS analysis. ***, p<0.001; *, p<0.05; NS, not statistically significant (unpaired two-tailed t test). Error bars, S.D. from three independent experiments. HeLa CCL2 cells transiently transfected with either GFP-β-catenin WT or GFP-β-catenin S60A were monitored during cell cycle progression by live cell imaging (E). The subcellular localizations of GFP-tagged β-catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti-GFP antibody. The cells were additionally stained for Plk1 (see Appendix Figure S5A). The specific localization of GFP-β-catenin WT at the midbody was observed under a confocal laser microscope with high magnification (E, left panel). Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were monitored (E, right panel) Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were counted using video clips (F). mCherry-Histone H2B was co-transfected with each GFP-β-catenin construct to visualize chromosomes. All live cell images and video clips show representative data of more than 50 telophase cells in each experimental group. Arrowhead indicates GFP−β-catenin WT located at the midbody in telophase HeLa CCL2 cells. Scale bars, 10 μm.
Cacna1a mutant neurons display lysosomal fusion defects.Fig 6A. LysoTracker Red staining of the primary cerebella neurons from Cacna1atg-la mice and WT controls. The control cells show big bright punctae, while the mutant cells have much dimmer and smaller punctae. B, C and D. Quantification of LysoTracker relative intensity ("n" represents the counted cell number), LysoTracker positive punctae number and size per cell ("n" represents the counted image number) . E. Lysosomal marker LAMP1 co-localizes with autophagosome marker LC3 in CTL but not Cacna1atg-la mutant cells. Scale bar, 20 μm. F. Quantification of LC3 and LAMP1 co-localization. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001).
Fig.Figure 4A Schematic outline of the experimental design. B. The distance traveled (upper panel), the speed (middle panel) and the time spent in the center region (lower panel) during a 5 min open field test were similar amongst groups (n=10/group). One-way ANOVA. C. Latency to enter the escape hole during Barnes maze training (Two-way ANOVA). n = 10 per group. D. Plots showing the different search strategies across training trials. Each strategy is labeled with a unique color. E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.
Figure 1CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC). CSF PGRN is not associated with age in either NC or MC. CSF PGRN is increased in males compare to females. CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.
Figure 2A. Crossover frequency normalised by the number of F2 individuals, and SNP density in 100 kilobase (kb) adjacent windows were calculated for each population and ranked into percentiles according to SNP density. Centromeric regions were excluded from analysis (Underwood et al, 2018). The Spearman's rank correlation coefficient (r) between SNP density and crossover frequency is printed inset. Trend lines were fitted in ggplot using a generalized additive model (GAM) with the formula y~poly(x,2). B. Generalized Linear Model (GLM) plots showing observed Col/Ler crossovers per megabase (red) for SNP intervals grouped into hexiles or quintiles, by increasing values (left to right along the x-axis) of a given explanatory variable. Data were modeled with the glm2 function in R, using the binomial family with the logit link function. The formula used for the model was Crossovers ~ (SPO11-1 + nucleosomes + H3K4me3 + DNA methylation + SNPs/kb + width)^2. Predicted crossovers per megabase for each group of intervals were derived by sampling from the binomial distribution based on the probabilities of intervals within each group overlapping a crossover. The black box plots represent 100 samples, where the central band represents the median, the box represents the interquartile range (IQR), and the whiskers represent the minimum and maximum values that are no more than 1.5 times the IQR from the box. The final stepAIC-selected model was:
Figure 2 A. DNA unwinding by DnaB was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 10). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dSaCas9 binding positions. B. DNA unwinding by DnaB was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 11). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. C. DNA unwinding by BLM was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 12). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. D. DNA unwinding by BLM was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 14). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. E. Phi29 DNAP strand-displacement synthesis was initiated from the upstream side of the PAM (n = 15). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. F. Phi29 DNAP strand-displacement synthesis was initiated from the downstream side of the PAM (n = 19). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.
Normal phenotypes of the nbr1 mutant plants in age- and darkness-induced senescence.Figure 8(A) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.
The Salmonella SPI-2 system triggers FAK activation and suppresses the autophagic capture of intracellular Salmonella.Figure 7
BiFC analysis of NBR1 interaction with ATG8a in planta.Figure 1Fluorescence was observed in the transformed N. benthamiana leaf epidermal cells, which results from complementation of the N-terminal part of the YFP fused with ATG8a (ATG8a-N-YFP) by the C-terminal part of the YFP fused with NBR1 (NBR1-C-YFP). No fluorescence was observed when ATG8a-N-YFP was coexpressed with unfused C-YFP or with mNBR1-C-YFP or when unfused N-YFP was coexpressed with NBR1-C-YFP. YFP epifluorescence images, bright-field images and overlay images of the same cells are shown.
Alternative form of BRAFV600E is correlated with p-ERK and p-MEK reactivation in M032R3.Figure 3Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases. Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.