EMBO/sd-character-level-ner huggingface dataset:
– Tau protein deficiency affects basal synaptic activity in granule neurons.Figure 3A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice. B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice. C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test). D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test). F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).
Inhibition of Cbl-b, c-Cbl and SHP-1 enhance NK cell function.Figure 1(A) YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or Cbl-b siRNA, c-Cbl siRNA and SHP-1 siRNA, using Amaxa electroporation. After 48 hours, cells were lysed, and subjected to western blot with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of three independent experiments.(n=3) P values were calculated by One-sample t-test, and are indicated by asterisks *P < 0.05. (B) YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments. (C) YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments.(n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P<0.0001 . (D) Granzyme release. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified using standard recombinant granzyme B concentrations Data are means ± SEM of eight independent experiments;(n=8); P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks *P < 0.05.. (E) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .
Figure 3(A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue). Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. (B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. (C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.
Figure 3(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm (B) Quantification of caspase-positive particles (anti Dcp-1, red) within macrophages (anti-SIMU, green). Values from at least five independent experiments are represented as mean ± SD total volume of caspase-positive particles (** P<0.01, by Mann-Whitney test). (C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm (D) Quantification of caspase-positive particles in the central brain area of larval brain. Values from at least five independent experiments are represented as mean ± SD total volume of caspase-positive particles (** P<0.01, by Mann-Whitney test).
Figure 1Week 10 cerebral organoids (COs) stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV n=4 from 1 batch). Scale = 100µm (left), 75µm (right). Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches). Quantification of markers that represent the different populations that compose the ventricular-like zone (VZ), sub-ventricular zone (SVZ) and the cortical plate (CP), as depicted in Appendix Figure S2B-C. NeuN marks mature neurons, TBR2 marks intermediate progenitors (IPs) and SOX2 the ventricular radial glia (vRGs). The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV n=4 from 1 batch). qPCR analysis for the assessment of expression levels of different neural markers in 15 weeks COs: SOX2 and PAX6 (progenitor cells), TUBB3 (pan-neuronal) SLC17A6 and SLC17A7 (VGLUT2 and VGLUT1; glutamatergic neurons) and GAD1 and GAD2 (GAD67 and GAD65; GABAergic neurons). Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.
a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.figf6(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection. (b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400. (d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap). (f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice). (g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)). (g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).
Effects of EmGFP-Tm1-I overexpression on oskar mRNA localization.Figure 7(A-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3'UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.
Figure 2Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCR Two days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4). The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3). The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).
Figure 7Graphical summary, depicting the molecules involved in sensing, signaling and raising an adaptive response in MSCs. MSCs sense bacterial intruders as modelled by the key molecule LPS, a widely distributed PAMP and wall component of Gram-negative bacteria. LPS is sensed via the TLR4 receptor and the signal is relayed via MyD88 to downstream effectors which, in consequence, shape the adaptive MSCs response. This response is enforced by fundamental transcriptomic reprogramming which is responsible for the release of factors critical for neutrophil and macrophage recruitment to the wound site. The adaptive response of LPS primed MSCs depicts a bifurcation with the activation of neutrophils with enhanced microbicidal NET formation and ROS release to directly counteract invading bacteria. In addition, in a second line of tissue protection and repair, apoptotic neutrophils, are phagocytosed by macrophages. Neutrophil engulfment constitutes a strong signal for macrophages to release TGFβ-1 which subsequently enhances differentiation of myofibroblasts and accelerates wound contraction and thus wound closure. Acceleration of wound closure together with enhanced NET formation and ROS release effectively counteracts microbial invasion. These data may stimulate new avenues to refine MSC-based therapies for difficult-to-heal wounds and/or infected wounds.
Figure 1Yeast two-hybrid interactions between SR-Gal4 activation domain (AD) and Gid5-DNA binding domain (DBD). Growth on -His-Ade+Aureobasidin A (AbA) is indicative of an interaction between the two test proteins. Spots represent 1:5 serial dilutions. Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt. Tetracycline reference-based chase performed during transition from ethanol to glucose media with wildtype, ∆Gid4 and ∆Gid10 strains, and in wildtype and ∆Gid4 strains overexpressing (OE) Gid10. Points represent mean, error bars represent standard deviation (n>3 biological replicates). Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK. Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK. Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.
Figure 6Primary Siwi-piRISC is produced through the primary pathway (Nishida et al, 2018). The piRISC binds transposon mRNAs and cleaves them into pieces. Of those, one fragment is loaded onto unloaded Ago3, which is anchored to mitochondrial Papi with help from Vasa (Nishida et al, 2015). Zuc processes the RNA to give rise to mature Ago3-piRISC. Vret associates with Ago3-piRISC and assembles Ago3 bodies. During this process, the Vret−Ago3-piRISC complex binds transposon transcripts in the antisense orientation. Unloaded Siwi enters into the bodies and joins the Vret−Ago3 complex. In this context, Ago3-piRISC and unloaded Siwi are held in close proximity with assistance from two (or more) Vret molecules, facilitating secondary Siwi-piRISC production in the ping-pong cycle.
PF-PH patients exhibit increased proliferation of vascular wall cells and increased Slug expression.Figure 2(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001). Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients. Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients. (D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification. (E) Relative CD68 mRNA expression normalized to GAPDH (F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.
LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections.Figure 5.LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections. Cotyledon cells, SGs, and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods. (A) Differential interface-contrast image of a broken cell. (B) Fluorescent image of A. (C) Differential interface-contrast image of a broken cell. (D) Fluorescent image of C. (E) Differential interface-contrast image of cells of a section from attached cotyledons. (F) Fluorescent image of E. (G) Magnified image of E. (H) Magnified image of F. (I) Differential interface-contrast image of an SG. (J) Fluorescent image of I. (K) Differential interface-contrast image of an SG. (L) Fluorescent image of K. (M) Differential interface-contrast image of cells of a section from detached cotyledons. (N) Fluorescent image of M. (O) Magnified image of M. (P) Magnified image of O. Bars: (A-D, G-L, O, and P) 20 μm; (E, F, M, and N) 50 μm.
Figure 1(A)-(C) Secondary structure of the complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C). The stem-loop structures (SLI and SLII), SL3 of WNV, pseudo-dumbbell (ψ-DB1) structure of ZIKV, duplicated dumbbell structures (DB1, DB2), as well as the essential 3' SL are indicated. Sequences involved in pseudoknots (PK1, PK2, PK3, PK4) formation are indicated with red lines. SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B) SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).
Figure 4A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). G. Quantification of α-SMA immunofluorescence shown in (A-F) H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0.
Conditional knockout of MCL‐1 in the forebrain activates an exacerbated autophagic response.Figure 5(A) Control and MCL‐1Δ/Δ mice. (B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes. (C) Brain morphology of MCL‐1Δ/Δ. Brain sections were stained with cresyl violet and imaged using a × 1 objective. (D-G) Cortical neurons around the lesion in MCL‐1Δ/Δ animals are positive for the autophagic marker LC3. Brain sections from animals with the indicated genotypes (P14 for E, G; P7 for H) were stained for LC3 (Green) and the neuronal marker NeuN (in H, red), along with the nuclear stain Hoechst (Blue). Confocal images were taken using × 10 (E) or × 63 (G, H) objectives. Scale bars=200 μm (E); 50 μm (G, H). Quantification of the LC3 staining is shown in D. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of LC3‐positive cells in at least three animals per genotype±s.d. *P0.01. (F) Quantification of the total surface area occupied by autophagosomes. Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype±s.d. P0.005 (I) Representative EM images from MCL‐1+/Δ and MCL‐1Δ/ΔP14mice. N, nucleus; *, autophagosomal structures; arrowheads, double membrane; arrow, mitochondrion inside a vesicle.
Nucleosome turnover at Su(H)-bound enhancers is increased in Notch-ON cells and is dependent on the BRM complex.Figure 5Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2. brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi. Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars). brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.