EMBO/sd-character-level-ner huggingface dataset:
Figure 7Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2 or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots. Student's t-test; pMYRF: ***P=0.00026; MBP; **P=0.0036. Data are presented as mean ±SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF. Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 μm. Quantitative analysis of the pMYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=0.00022. Data are mean ±SEM. Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 μm. Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40X) in each section were examined. n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=4.43x10-5. Data are mean ±SEM. A proposed model for the protective effect of LAQ in HD mice. LAQ increases myelin protein expression by reducing the expression of PRKG2, leading to reduced phosphorylation of MYRF and its dissociation from mutant HTT and therefore increasing its transcriptional activity to express myelin associated genes.
Figure 10(A) To generate MITOL-3Flag knock-in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9-based gene editing. Insertion of the 3Flag-tag was verified by immunoblotting with an anti-Flag antibody. Asterisk indicates a cross-reacting band. (B) The MITOL subcellular localization was observed in MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Endogenous MITOL (detectable with an anti-Flag antibody) overlapped with Hsp60 under steady-state conditions, whereas 3 hours of valinomycin (10 µM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm. (C) Correlation statistics for the localization of endogenous MITOL-3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (D) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS-873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 µm. (E) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (F) Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin were treated with valinomycin for 3 hours, the collected cell lysates were immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were blotted using anti-Flag and anti-ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL-3Flag.
Regulation of ROS and peroxidated LDs by ADAM17 and components of the TNF pathway.Figure 8Cleavage of full length TNF/Eiger by ADAM17 leads to the production of soluble TNF/Eiger, which can engage the TNF receptor, Grindelwald. TNF signalling inhibits the production of mitochondrial ROS inside pigmented glial cells, through unknown mechanisms. In the absence of TNF signalling, glial generated ROS combines with ROS from neighbouring photoreceptor neurons to trigger production of LDs. Upon their later dispersal, the combination of fatty acids and ROS leads to high levels of toxic peroxidated lipids, which are toxic to the glia. Finally, glial degeneration is followed by neuronal degeneration.
Figure 2Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape. Left: Rigid body fit of the mouse ID2 atomic structure (3S4W) into the human IUbD2Ub-dsDNA cryoEM map. Right: Flexible fit of the mouse ID2 atomic model into the human IUbD2Ub-dsDNA cryoEM map (iMODFIT). The C-termini "arms" of FANCD2 and FANCI close in the flexible fitting. A tube-like volume in the IUbD2Ub-DNA cryoEM map, which is not occupied by the ID2 iMODFIT structure, is present just beneath the Arm ID2 interface and likely represents bound DNA (solid density).
Figure 6A Immunoblot analysis of RBM47 and IFNAR1 in the lungs, livers, and spleens of RBM47+/+ (WT) and RBM47+/- mice. Each band represents tissue samples from one mouse. B Analysis of viral mRNA from blood, lung, brain, and spleen samples of RBM47+/+ and RBM47+/- mice (n = 6 per group) infected with VSV (1× 108 pfu), and the results were normalized to those of mouse β-actin. Each dot represents data from one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). C Viral titers in serum, lung, brain, and spleen samples of RBM47+/+ and RBM47+/- mice at day 1 post VSV infection.The viral titer data are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). D Micrographs of hematoxylin-and-eosin-stained lung sections from mice treated with PBS or VSV (day 1 post infection). Scale bar, 50 μm. E IFN-β protein levels in the serum of RBM47+/+ and RBM47+/- mice determined using IFN-β ELISA kit.Data are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). F qPCR analysis of IFNAR1, IFIT1, and Cig5 mRNA in the blood samples of infected mice. The relative expression levels of genes in WT mice were set to "1.". The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). G RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
Figure 4A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM). C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).
Figure 6A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds. B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2. C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).
Figure 2NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group). (B) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group). (C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (D) Body temperature of AdRiKO and control mice housed at 22°C (n=11 (control), n=9 (AdRiKO)). (E) Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n=8/group). (F) Body temperature loss upon cold exposure of AdRiKO and control mice (n=20 (control), n=17 (AdRiKO)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Figure 3(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.
Figure 4A. MTT assay for cell proliferation in MDA-MB-231 cells infected with lentiviruses carrying control shRNA (shSCR) or shRNA against hTrmt13 (sh-1, sh-2). Error bars represent mean ± SD for three independent experiments. B. Transwell migration assays in MDA-MB-231 cells. Control with siRNA scramble (NC), treated with siRNA against hTrmt13 (si-1, si-2) and stably expressing wild-type hTrmt13 (si-1+WT) or enzymatic inactive hTrmt13 mutant (si-1+E463A). Representative images (left panel) and statistical analysis (middle panel) are shown. Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for four independent experiments. C. Transwell migration assays in HCC38 cells infected with scrambled siRNA (NC) or siRNA against hTrmt13 (si-1, si-2). Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments. D. Western blot of the expression of Vimentin, N-cadherin, and E-cadherin in MDA-MB-231 cells with the indicated treatments. E. Volcano plot of differential expressed genes analysis results for RNA-seq data comparing hTrmt13 knockdown MDA-MB-231 cells (N=2) to control cells (N=2), the dots for significant regulated genes (FDR corrected p-value < 0.05) were colored red, the dots and labels of interesting genes with oncogenic implications have been colored in blue or red. F. Functional categories of downregulated genes in hTrmt13 knockdown MDA-MB-231 cells showing the p-value for the enrichment of biological process GO term. G. GSEA plot showing an interesting gene set from MSigDB were enriched for gene down-regulated in hTrmt13 knockdown MDA-MB-231 cells (hTrmt13 KD) for POOLA_INVASIVE_BREAST_CANCER_UP and SARRIO_EPITHELIAL_MESENCHYMAL_TRANSITION_UP (extracted from GSE47203). H. Heatmap of expression level for highlighted genes and core enriched genes in EMT gene set.
Figure 3(A) Left Panel: Representative ABR to supramaximal click stimuli revealed stable response at the characteristic peaks I-V in 3-week old WT pigs at 100 dB SPL, but not in age-matched USH1C pigs up to 120 dB SPL (left) (1 WT and 2 USH1C pigs, both ears, 2 traces each). Middle Panel: Hearing deficits measured by ABR were more pronounced at higher frequencies in 8-week old USH1C pigs. Differences between WT and heterozygous USH1C+/- (het) were not significant (t-test) for the respective frequencies, but a 2-sided ANOVA test indicated a <0.01 significance (**) overall. p-values for differences between USH1C pigs and WT or heterozygous pigs were <0.0001 (****) at all tested frequencies and <0.01 (**) for a click test. Data points represent mean values (MV) ± standard deviation (SD). Right Panel: Behavior tests supported impaired hearing in USH1C pigs. In their first days of life, reaction times of sleeping piglets upon calling by the mother sow were measured. Piglets waking up immediately entered the box within 15 seconds (Video S7). If not waking up within 60 seconds, piglets were stimulated by nudging (#). Mean values are indicated. (B-E) In the eye, representative results were gained from 2 WT and 2 USH1C pigs at 12 months of age. For the limited number of animals, no statistical evaluation was performed. (B) A retinal dysfunction is evident in the multifocal electroretinogram (mfERG) of the visual streak in USH1C pigs to WT. Yellow/red colors indicate higher response amplitudes (1-1.5 µV), while blue/green colors indicate lower response amplitudes (0-1 µV) reflecting cone photoreceptor activity in the tested area. (C) Upper Panel: The average sum potential from the tested area highlights a delayed and lower peak amplitude in the visual streak in USH1C vs WT pigs. Traces indicate the average of all 61 hexagonal segments of single mfERG recordings. Lower Panel: Localization of the mfERG recordings. (D) Ganzfeld (gf) ERG recordings in USH1C pigs vs WT demonstrate a significant reduction in the scotopic standard flash responses and responses after photopic standard flash stimuli. Left panel (top to bottom): Dark adapted single flash recordings following a low light (0.03cd*s/m2, rod only) or a brighter (3cd*s/m2, rod and cone) stimulus. The oscillatory potentials shown at the bottom are extracted from the above standard flash recording. Right panel (top to bottom): Response after a light adapted single flash at 3cd*s/m2 intensity and responses following a light adapted flicker (same intensity) at 30Hz. (E) The retinal architecture as demonstrated by optical coherence tomography remained fairly intact, indicating a large therapeutic window.
IRF7 is necessary for IFN-I production in pDC, but low protein levels are sufficient to promote this function during MCMV infection (A) IRF7 protein expression in pDC isolated from Irf7-/- (pink, bottom), C57BL/6 (WT, black, top) and Ifnar1-/- (red, middle) uninfected (empty histograms, Ø, left) vs MCMV-infected mice (filled histograms, MCMV, right).Figure 5 (A) IRF7 protein expression in pDC isolated from Irf7-/- (pink, bottom), C57BL/6 (WT, black, top) and Ifnar1-/- (red, middle) uninfected (empty histograms, Ø, left) vs MCMV-infected mice (filled histograms, MCMV, right). For each condition histograms are representative of one out of 4-6 mice from 2 independent experiments  (B) IRF7 protein expression in pDC (left), cDC (middle) and conventional monocytes (cMo, right) isolated from uninfected (Ø, empty) or MCMV-infected (MCMV, filled) C57BL/6 (black) or Ifnar1-/- (red) mice. Data are shown as net IRF7 MFI, after subtraction of the MFI obtained using isotype control mAbs. Data shown (mean±SEM) are pooled from 2 independent experiments with 5-6 mice for each group  (C-E) The expression of CD86 (C) or of indicated cytokines (D-E) was analyzed, as described in Fig 2. Black, CTR MBMC; pink, Irf7-TST MBMC. Mice were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group
a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h.figf4(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading. (b-d) RT-qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h). n = 3 independent experiments; error bars represent s.e.m. (e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown. (f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e. (g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.
Figure 2A Comparison of the cryo-EM structure of H2A.B-NCP with the structure of canonical nucleosome (PDB: 3LZ0) in disc view. Histone octamers containing H2A.B and canonical H2A were superimposed for comparison. The ~103 bp DNA of H2A.B-NCP in green color is shown in surface mode. The 145 bp DNA of canonical nucleosome is colored in grey. Histones H2A.B, H2B, H3 and DNA at SHL 5 through SHL 7 are highlighted to show the structural differences between two nucleosomes. Histones H2A.B, H2B and H3 in H2A.B-NCP are colored in orange, warmpink, and cyan. Their counterparts in canonical nucleosome are colored in yellow, red, and blue. Histone H4 is colored in grey. B Close-up view of the structures of H2A.B C-terminal regions (top) and H2A C-terminal regions (bottom). Black and white arrows indicate the last residue at the C-terminus of H2A.B (V109) and H2A (K118) that are observable in the structures. C Comparison of the cryo-EM structure of H2A.B-NCP with the structure of canonical nucleosome in gyre view. Arrows indicate the direction of H2A.B-NCP DNA movement during nucleosome gaping transition. The dashed lines indicate the ~10 degrees rotation of H2A.B DNA at SHL 5. H2B N-tail refers to H2B residues 26-32 which are exclusively observed in the canonical nucleosome structure. Histones H2B in H2A.B-NCP are colored in warmpink, Their counterparts in canonical nucleosome are colored in red. H2A.B-NCP DNA in green is shown in surface mode for clear comparison. D Close-up view of H2A.B-H2B dimer in the structural comparison. The secondary structure of H2A.B-H2B dimer are indicated. The dashed lines highlight the tilt of H2A.B α2-helix.
Figure 12(A) TAG levels in exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated controls as determined by UPLC-MS/MS, [n=4 experimental replicates on pools of 25 planarians per group per experiment; data were normally distributed (B) Schematic representation of the regime of RNAi treatment in combination with PA/PC (750 μM each, 160 nl per worm). PA/PC was administered 3 hours before injecting dsRNA against Smed-exoc3 or against gfp as a control. (C) RT-qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment schedule (D) Representative images of regenerating planarians that were injected with gfp-RNAi, Smed-β-catenin-1-RNAi or Smed-β-catenin-1-RNAi in combination with PA/PC injection Heads and tails were cut on day-10, images were taken on day-17 after amputation. Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. (E) Images of body plan maintenance in non-injured planarians that were injected with gfp-RNAi, exoc3-RNAi or exoc3-RNAi in combination with PA/PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. (F) RT-qPCR analysis of marker genes of early stem cell differentiation (prog-1 and prog-2) and late stem cell differentiation (Smed-odc1) in planarians exposed to the indicated treatment regiments on day-38 of the experiment as indicated in panel-B [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test].
Figure 5(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients. (B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase (C) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).