EMBO/sd-character-level-ner huggingface dataset:
Suppression of neurodegeneration associated with diverse misfolded proteins in invertebrate models by ufd-2 and spr-5 loss-of-function mutations.Fig 2(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A). (D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var) 3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4. (E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Figure 1A Experimental design. Laser coagulation was performed in C57BL6/J control and Ifnar1-/- mice. Animals were analyzed 3, 7 and 14 days after laser treatment. B, C Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in C57BL6/J controls (B) and Ifnar1-/- (C) mice. Scale bar: 20 µm.D Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 11-12 retinas; unpaired Student's t-test: ***p = 0.0004).E Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 42-62 cells; unpaired Student's t-test: ***p < 0.0001). F-K Representative fundus fluorescein angiography images of C57BL6/J (F-H) and Ifnar1-/- (I-K) mice 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in C57BL6/J controls versus Ifnar1-/- mice. Values show mean ± SD. (n = 14-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p < 0.0001). M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in C57BL6/J control mice (M, N) and Ifnar1-/- animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.Q Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.0281).
Figure 1Characteristic micrographs for both data sets, respectively (left and right). Local resolutions mapped on the respective 3D reconstructions. The center of the coat protein is resolved up to 1.8 Å with some features of approx. 5 Å resolution at the C-terminus. Both maps show well-resolved protein features including water molecules (left, right). Side-chain features of Y70, I121, R46 and F35 residues at Ca2+/acidic pH. Snapshot of RNA map features with Mg2+ ions in water conditions.
Figure 1 Brains dissected from Drosophila larvae (A were immunostained with anti-ALIX (red), Asl (white) and α-tubulin (green) and Hoechst (blue). Representative confocal micrographs of NBs in different mitotic phases (prometaphase to early telophase) (A The centrosome with more accumulated ALIX is marked (*) in (A). Scale bars, 5 μm (B) Brains of w1118 larvae were stained with anti-ALIX (white), anti-Cnn (white) or anti-Asl (white) and Hoechst (blue) and their centrosomal intensities were calculated in 41, 44 and 40 metaphase NBs, respectively. The average ratios of centrosomal intensities (strongest/weakest) (±S.E) of ALIX, Cnn and Asl calculated from 77, 76 and 56 NBs, respectively, are indicated below the micrographs (from at least three experiments). Scale bars, 5 μm ovaries dissected from adult flies (C), were immunostained with anti-ALIX (red), Asl (white) and α-tubulin (green) and Hoechst (blue). Representative confocal micrographs o FECs in metaphase are presented (C) (D) HeLa cells were immunostained with anti-ALIX (red), anti-glutamylated tubulin (green) and Hoechst (blue). A widefield micrograph of a representative metaphase cell is shown in the left panel (scale bar, 5 μm) and the insets show close ups of the centrosomes The white arrow indicates the direction of the line scan analysis performed in (E) (E) Line scan analysis of the fluorescent distribution of ALIX and glutamylated tubulin at the centrosomes. The average intensity (±S.E) of 26 centrosomes from three independent experiments is shown graphically
a, Electron microscopy analysis of wild-type (WT) and Smurf1−/− (KO) MEFs grown in normal media or EBSS (starvation) for 4 h.f3a, Electron microscopy analysis of wild-type (WT) and Smurf1-/- (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm. b, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype. c, Quantification of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P<0.001 against wild-type, Students t-test. d, Representative confocal micrographs of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &amp;amp;mgr;m. e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 µm. For a-e, similar results were obtained in 3-5 independent experiments.
Figure 1Identification of Rtt105-TAP associated proteins. Purified proteins were resolved on a 4-12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). *Non-specific band also in control. Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel). MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control. GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining. Rfa1-TAP purification was performed using Rfa1-TAP rtt105∆ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc).
Figure 3Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n=3 experimental replicates) Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA. Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line. Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat- inactivated in cell extracts, in the last lane no cells were added in lysis buffer. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC) (n=3 experimental replicates). AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates). HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).
restricts HIV-1 transcription to induce HIV latency.Figure 4A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids. Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. levels of p24 in the supernatants (C). D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h. E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR. F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.
Growth defect in DHR3 LBD mutant cells.Figure 6(A) Normalized eye size (mm2) of control (black bar; n = 20), DHR3W284X (grey bar; n = 18) and DHR3K243X (white bar; n = 18) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant eyes exhibit a highly significant size reduction (***P<0.001). (B) Ommatidia size of control (black bar), DHR3W284X (grey bar), and DHR3K243X (white bar) mutants. As compared to control recombined eyes (Co), DHR3W284X and DHR3K243X mutant ommatidia exhibit a significant size reduction (**P<0.01). (C-E) Comparison of y-marked homozygous clonal bristles (black arrows) to their neighboring heterozygous bristles (white arrows) at the wing margin. Note that control clonal bristles are of normal size and orientation (C), whereas those of DHR3W284X (D) and DHR3K243X (E) are reduced in size and are misoriented. (F) Size measurements (µm) of the length of homozygous clones (y−) and their neighboring heterozygous (y+) bristles (n = 51 for each bar). The size reduction of clonal versus neighbor bristles is highly significant for DHR3K243X (***P<0.001) and DHR3W284X (***P<0.001) mutants, but not for the control (Co) bristles (P = 0.028).
a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast.figf1(a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast. Data represent means ± s.e.m. (n = 3; *P 0.05 and *P 0.001). (b) Survival determined by clonogenicity during chronological ageing of wild-type yeast (BY4741) cells with (o) and without (▪) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 5). (c) Intracellular levels of spermidine in chronologically ageing wild-type yeast cells cultured with (open bar) or without (closed bar) spermidine (4 mM) for 5 days. Data represent means ± s.e.m. (n = 3; **P 0.001). (d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown. (e) Survival determined by the age-specific number of dead individuals of female Drosophila with and without (control) supplementation of food with various concentrations of spermidine (as indicated). A representative ageing experiment of at least 50 flies per sample is shown. (f) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine, compared with normal food (control). Data represent means ± s.e.m. (n = 3; *P 0.01). (g) Survival determined by annexin V/7-AAD co-staining (unstained cells were considered as viable) of human immune cells (PBMC) cultured for 6 and 12 days in the absence (black bar) or presence (white bars) of various spermidine concentrations (as indicated). Data represent means ± s.e.m. of 3 independent experiments (each performed on PBMC from different donors). *P 0.05 and **P 0.01. (h, i) Free thiol group (RSH) concentration in serum (h) and intracellular spermidine concentration in hepatocytes (i) of ageing mice with (open bars) or without (closed bar) supplementation of drinking water with spermidine (0.3 and 3 mM) for 200 days. Data represent mean ± s.e.m. (n = 3; *P 0.05 and **P 0.01).
Figure 8Immunofluorescent analysis of TDP-43 in control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). Scale bar: 25µm. B Profile intensity plot of control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor. C Quantification of nucleo-cytoplasmic ratio (N/C) fluorescent intensity of TDP-43 in motor neurons, each dot represents one analyzed cell, unpaired Mann-Whitney-test. Western blot analysis of cytosolic fraction, quantification in E, ratio paired-t-test. Western blot analysis of nuclear fraction, quantification in G, ratio paired-t-test. Western blot analysis showing pTDP-43 levels and I quantification, ratio paired-t-test.
Characterization of AT+GCPs and ND+GCPsA.Figure 1A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs. B. Schematic illustration of AT+GCPs and ND+GCPs. KI67-positive GCPs in the EGL were distinguished by the expression of two bHLH transcriptional factors, ATOH1 (AT+GCPs) and NEUROD1 (ND+GCPs). C. Pax6-positive and Pcna-positive cells were extracted from published scRNA-seq data. UMAP-based dimensional reduction and cluster analysis grouped extracted cells into 5 clusters. D. Normalized expression of selected genes (Atoh1, Neurod1) were mapped onto the UMAP dimension. E. Featured molecules in each cluster were determined and visualized by heatmap. The values of each molecule were normalized by Z-score. Molecular features of each cluster suggested that clusters 0 and 1 were GCP lineage cells, cluster 3 was GCs and clusters 2 and 4 were likely to be interneuron precursors as Pax3 and Id3 expression were strong. In addition, the strong and featured expression of Atoh1 suggested that cluster 0 and cluster 1 might be AT+GCPs and ND+GCPs. F. Cumulative labeling of AT+GCPs and ND+GCPs in P6 mice. Total cell cycle length (Tc) and S phase length (Ts) were calculated as previously described. Calculated time of Tc, Ts, TG1, TG2 and TM were indicated in the table (F'). N=3 for each time point. Error bars are representing as Mean ± SEM. Two-way ANOVA analysis was performed against Cumulating rate from 0hrs to 9hrs in each cell, followed by Bonferroni test. *p < 0.05, **p < 0.01. Number of cells: 113,101 and 159 AT+GCPs from N=3 mice(0hrs), 116,115 and 95 AT+GCPs from N=3 mice(3hrs), 106,105 and 100 AT+GCPs from N=3 mice(6hrs), 122,168 and 170 AT+GCPs from N=3 mice(9hrs). 196,167 and 158 ND+GCPs from N=3 mice(0hrs), 139,110 and 101 ND+GCPs from N=3 mice(3hrs), 98,163 and 81 ND+GCPs from N=3 mice(6hrs), 136,124 and 115 ND+GCPs from N=3 mice(9hrs). Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67. (I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).
Figure 1A Schematic outline of the intraductal injection approach used to generate ILC xenograft models. ILC cells are transduced with RFP-luciferase expressing lentiviruses and injected into 3rd thoracic and the 4th inguinal milk ducts, highlighted in red, of 10-week-old NSG females. Tumor growth is monitored by bioluminescence imaging. B Bar graph showing percentage of engrafted mammary glands of non-ILC, ER+ HER2-; total number of glands injected with MCF7 (n=17) and T47D (n=14) and ILC, MM134 (n=38) and SUM44 cells (n=47). Black dotted line shows take rate 95%. C Graph showing in vivo growth of the different cell line xenografts. Curves represent mean log-transformed luminescence ± SEM of individual glands; MCF7 (n=17), T47D (n=10), MM134 (n=12), and SUM44 (n=12). Statistical testing for interactions group cell growth/ time relative to MCF-7 was carried out by applying mixed-effect linear models. P-values are associated to likelihood-ratio test statistics of the ANOVA function in R. D Box plot showing log10 (radiance fold change) of individual glands 5 months after injection of MCF7, T47D, MM134, and SUM44 cells; n=17, 14, 38, 47, respectively. Vertical Lines outside the box end at maximum and minimum values, upper and lower borders of the box represent lower and upper quartiles, and line inside the box identifies the median. Statistical significance of difference to MCF7 xenografts determined by unpaired, Student's t-test, two-tailed, ***p < 0.001, n.s. not significant. P-values have been corrected for multiple testing by FDR. E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm. I Ex vivo luminescence images of bones, brain, lungs and liver from MM134 engrafted mice 1 month post injection (top) and ovaries (bottom) 12 months post injection. Scale bar, 1 cm. J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis. Number of mice analyzed MM134 and SUM44 for 30-180: N=5, 10, 180-270 days: N=9, 5, 270-360 days: N=14, 20, respectively. Dashed line indicates background levels.
Endothelial cell-activating cytokines produced by Tie2− tumor cells may influence the anti-permeability efficacy of Vasculotide and Ang1.Figure 4A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)-that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells-and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2-3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.C Representative fluorescent images (10×) of 'extravasated' CMTPX-labeled TCs (red) fixed on membrane undersides. D LM2-4luc produced the highest levels of VEGF and IL-8, but lower levels of IL-6, compared to other TC types; ***P < 0.001 and *P < 0.05 (unpaired t-tests).
The 32 β-strands forming an eight-bladed β-propeller are numbered as in Supplementary Note.figf2The 32 β-strands forming an eight-bladed β-propeller are numbered as in Supplementary Note. The location of the variant amino acid T300A in strand β3, corresponding to rs2241880, is marked in yellow.