EMBO/sd-character-level-ner huggingface dataset:
CircRNA abundance is reduced in the PD SN and negatively correlates to RNA editing levels (A) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.Figure 3(A) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.02 p*=0.026 and p*=0.022 for comparison between PD and CT in SN, MTG and AMG. T-test for total SN vs MTG p*=2.27E-8 and SN vs AMG p*=5.86E-10. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. P value was calculated by T-test. (B) Global editing levels in the 3 brain regions is based on the Alu editing index, representing the weighted average editing level across all expressed Alu sequences, Wilcoxon, corrected p*=0.038, 0.055 and 0.42 for Amygdala, MTG and SN. Wilcoxon test between the Alu editing index of control and PD samples. (C) Global RNA editing levels based on the Alu editing index, studied only in Alu elements within circRNA exons and their flanking introns, in the 3 brain regions, for circRNAs corrected p*= 0.025 and 0.040 for the AMG and MTG and p=0.3 for SN. For flanking introns p*= 0.02 and 0.040 for AMG and MTG and p=0.34 for SN, Wilcoxon test. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. (D) Negative correlation between total RNA editing and circRNA abundance, correlation = -0.37, Correlation test p value = 0.0015. (E) Adar1 expression levels in all tissues. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. (F,G) Correlations between circRNAs detected in each sample and the age of the control (green, correlation = 0.68, p-value = 0.032) and PD donors (blue, correlation = -0.14 p value = 0.62). (H) Volcano plot of DE circRNAs in PD vs control tissues, red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test (DEseq2 analysis). CircSLC8A1 is marked is red.
Rate of change in Florbetaben PET vs baseline GE180 PET: A, B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.Figure 2Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15). B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15). C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.
h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h.Figure 4MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test). MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm. Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).
controls airway allergy inflammation in reovirus infection murine model (A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse).Figure 3(A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates. Cell number of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates. Cell number and category of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates. (F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm. (G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.
Figure 4A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test). B-D, Averaged poststimulus time histograms ±SEM from OtofI515T/I515T mice (n=25-32) and Otof+/+ littermate SGNs (n=13-27) to stimulation with 50ms tone bursts at the characteristic frequency (CF) of each fiber, 30dB above threshold at indicated stimulus rates. E, Quantification of onset (largest 0.5ms bin, Mann-Whitney U test) and adapted responses (averaged 35-45ms from response onset, t-test) from data in B-D. F, The jitter of the first sound-evoked spike was significantly increased (p<0.001, Mann-Whitney U test) but retained its inverse correlation with spike rates. G, Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T/I515T SGNs, both for repetitive stimulation with 50ms tone bursts (left, p=0.014, t-test) and for continuous stimulation with amplitude modulated tones (right, p<0.001, Mann-Whitney U test). H, Phase locking to amplitude modulated tones (assessed as the maximal synchronization index) was typically less precise in OtofI515T/I515T SGNs than in Otof+/+SGNs (p=0.09, Mann-Whitney U test). I, 100ms masker tone and 15ms probe tones (both at CF, 30dB above threshold) were separated by silent intervals of variable duration. Inter-masker intervals were 500ms for Otof+/+, and 1000ms for OtofI515T/I515T. The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T/I515T (pink; mean ±SEM red) compared to Otof+/+ (grey; mean ±SEM black; for 4ms interval: p=0.001, t-test) and a slowed time course of recovery (x: half time of recovery, taken from normalized recovery functions; p<0.001, Mann-Whitney U test). K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.
Figure 1A Domain structure of FAK. Important phosphorylation sites are indicated and the FAK region used in the cryo-EM study is boxed with a dashed line. B Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5) P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image. C Local resolution map of the refined Apo and AMP-PNP particle, as calculated with Blocres (Heymann & Belnap, 2007). D 14 FAK molecules, each containing a FERM and kinase domain are fitted into the EM maps obtained from the AMP-PNP dataset. Each molecule is in a different color. Middle: Membrane distal view; right: Membrane proximal view; left: zoomed view of central symmetric dimer from the membrane distal view with locations of FERM (F) and kinase (K) domains indicated. The equivalent figure for the Apo structure is shown in Fig. EV1d. E Linear oligomeric assembly of FAK formed by three symmetric FAK dimers in the AMP-PNP particle shown from the membrane distal view. FERM (F) and Kinase (K) domains are labelled. FERM and kinase of the same molecule are in the same color with the kinases shaded in a lighter tone. F Model of the FAK oligomeric assembly placed on a lipid bilayer. The membrane is placed by considering the KAKTLRK basic sequence in the FERM F2 lobe as main membrane binding site (Cai et al., 2008; Goni et al., 2014).
Figure 1A. Whole mount in situ hybridization (WISH) of evi1at 20 (left) and 32 (middle) hpf. evi1expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1co-localizes with the endothelial marker flk1 (right). B, C. WISH of runx1/c-myb ;in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.D-G. WISH of globinin erythrocytes of 6 dpf old embryos (D), of rag-1in the thymus of 5 dpf embryos (E; red asterisk), of cd41at 52 hpf (F), and gata2bat 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***). I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.K. Graph displays quantitation of results from I and J using the same method as above.
Loss of FBXO7 in TH+ cells results in late-onset motor deficits.Figure 7A. Genotyping PCR of TH-Cre, TH-Cre;fl/+ or TH-Cre;fl/fl mice using primes for floxed allel, FBXO7 wild type and cre. B. Average body weight of 6 month-old TH-Cre and TH-Cre;fl/fl mice. n=16 and 15 respectively (unpaired t-test, ** p<0.01, mean + s.e.m.). C. Average body weight of the weight-corrected cohort of 6 month-old TH-Cre and TH-Cre;fl/fl mice. n=11 and 10, respectively (unpaired t-test, mean +s.e.m.). D. E. and F. Open field (D) of 6 month-old TH-Cre and TH-Cre;fl/fl mice. n=11 and 10, respectively (unpaired t-test or Mann-Whitney test (coordination score), mean +s.e.m.). D. E. and F. Balance beam (E) or pole test (F) of 6 month-old TH-Cre and TH-Cre;fl/fl mice. n=11 and 10, respectively (unpaired t-test or Mann-Whitney test (coordination score), mean +s.e.m.). G. Rotarod test of 6 month-old TH-Cre and TH-Cre;fl/fl mice. n=11 and 10, respectively (unpaired t-test, *p<0.05, ** p<0.01, mean +s.e.m.). H. DigiGait analysis of 8 month-old TH-Cre and TH-Cre;fl/fl mice. n=7 for both groups (unpaired t-test, *p<0.05, ** p<0.01, mean +s.e.m.). Data was visualized using a Partial Least Squares (PLS) regression with Orthogonal Signal Correction model. I. Average body weight of 12 month-old TH-Cre and TH-Cre;fl/fl mice. n=16 and 9, respectively (unpaired t-test, mean + s.e.m.). J. K. L. and M. Open field (J), balance beam (K), pole test (L), rotarod (M) of 12 month-old TH-Cre and TH-Cre;fl/fl mice. n=16 and 9, respectively (unpaired t-test or Mann-Whitney test (coordination score), * p<0.05, ** p<0.01, *** p<0.001, mean +s.e.m.).
Inhibition of endocytosis results in goblet cell mucin accumulation.Figure 4(A) Schematic for the generation of differentiated colonic epithelial cells grown in a monolayer. (B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm. (C, D) Quantification of average number of LC3β dots per cell (C) and the percentage of LC3β dots that co‐localized with EEA1 per cell (D) (n=3 experiments/group; 25-30 cells/experiment). (E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments. (F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm. (G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.
Superresolution analysis shows distinct features of type A and C cellular neoaggregates A-C) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue.Figure 4A-C) dSTORM images of cells seeded for 6 days with non-neurodegenerative control (non-ND control), FTLD-TDP-A and FTLD-TDP-C seeds extracted from post-mortem brain tissue. Staining shows localization of TDP-43 (HA tag, cyan) along with LaminB1 (yellow, A), nucleopore complexes (nucleopores, yellow, B) and nucleolus (yellow, C). Cells seeded with FTLD-TDP seeds show coaggregation of nuclear membrane proteins upon neoaggregate formation. All images are presented at the same scale, 2 μm as indicated in the non-neurodegenerative control top panels. D) dSTORM images of cells containing aggregates after 6 days of seeding with SarkoSpin fractions of postmortem brain from patients with FTLD-TDP-A or FTLD-TDP-C. Red arrows point to cytoplasmic aggregates. Cells were labelled with TDP-43 (HA-tag, cyan) and phosphorylated TDP-43 pS403/404 (pTDP-43, magenta). All images are presented at the same scale, 2 μm as indicated in the FTLD-TDP-A top panel. E) Violin plots showing the size of analyzed neoaggregates using TDP-43-HA area. Non-parametric unpaired t- test FTLD-TDP-A vs FTLD-TDP-C, p<0.0001. (Continuous line represent median, dashed lines represent quartiles , n = 3 independent experiments, 6 biological replicates for each condition) F) Bar graph showing the number of neoaggregates per cell. Non-parametric unpaired t test FTLD-TDP-A vs FTLD-TDP-C, p=0.0003. Error bars represent mean ± SEM, n=3 independent experiments, 6 biological replicates for each condition. G) Heatmap representation of the density of TDP-43-HA and pTDP-43 particles for four representative neoaggregates of FTLD-TDP-A and FTLD-TDP-C seeded cells. All images are presented at the same scale, 1 μm as indicated in the FTLD-TDP-C top right panel. H) Violin plots showing the density of particles TDP-43 per µm2. Non-parametric unpaired t test FTLD-TDP-A vs FTLD-TDP-C, p=0.0083. (Continuous line represent median, dashed lines represent quartiles , n=3 independent experiments, 6 biological replicates for each condition)
Figure 1A. Origin of the flight muscles. Left panel: Trachea, air sac primordium, IFM myoblasts and DFM myoblasts in the adepithelial layer overlaying the wing disc epithelium. Right panel: Lateral view of the adult flight muscles in the thorax. B. ﻿ Workflow for droplet-based scRNAseq. Wandering third instar larval wing discs were dissected, pouch was removed and dissociate into single-cell suspension. Droplets containing unique barcoded-beads and single cells were collected. Following library preparation, sequencing data were aligned, and a gene-cell expression matrix was generated and analyzed using Seurat for identification of variable genes and unsupervised cell clustering based on gene expression similarity. C. Annotated cell-type, including 6,711 epithelial, 272 tracheal and 4,544 myoblast cells, in UMAP plot of the reference single cell atlas. D. ﻿RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset. Cells (column) are clustered by the expression of the main marker genes (row). E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset. F. Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti-Zfh1 (red) and anti-Fas3 (green). G. Dot plot showing the expression levels of the marker genes identified for the myoblast, epithelial and tracheal cells across the 26 clusters of the reference cell atlas. Color intensity represents the average normalized expression level. Dot diameter represents the fraction of cells expressing each gene in each cluster. Gene expression for each cell was normalized by the total expression, and then the expression of each gene was scaled. H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.
NeoR accesses the endosomal/lysosomal compartment through autophagy.Figure 5(A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence. (B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity. (C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control. (D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels. (E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.
ZDHHC5, SFT2D2, or GRINA Depletion Affect Levels of Endosome-to-Golgi Cargo Proteins.Figure 6(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown. (B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.
Cytoplasmic USP42 functionally interacts with the Dishevelled interacting region of ZNRF3 A, Scheme showing the GFP-USP42 and ZNRF3-HA constructs generated for this study.Figure 2A, Scheme showing the GFP-USP42 and ZNRF3-HA constructs generated for this study. Note that ZNRF3∆C did not express properly, possibly due to misfolding at the ER. B, Immunofluorescence microscopy showing HEK293T cells transfected with USP42 constructs from (A). Note that depletion of the putative NLS or the whole C-terminal domain containing the NLS leads to increased cytoplasmic expression of USP42. Representative images are shown. Scale bar = 10 μm. C, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). Representative blots of n ≥ 3 independent experiments are shown. D, TOPflash reporter assay in HEK293T cells upon overexpression of the USP42 constructs shown in (A) or an empty vector. Cells were stimulated with control or Wnt3a and RSPO3 conditioned medium. Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as **P < 0.01, ***P < 0.001, or n.s.: not significant. E,F, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F) cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown. G, Scheme showing the proposed interacting regions of USP42 and ZNRF3.
Figure 3A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001. B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.