EMBO/sd-character-level-ner huggingface dataset:
Neuronal mislocalisation and migration defects found in individuals with ECE2 variants are recapitulated in iPSC-derived in vitro models.Figure 1Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles. ECE2 expression on RNA level. In situ hybridisation for ECE2 RNA in 50d old cerebral organoids (COs) shows higher signal in the cortical plate-like zone (CP') (Scalebar=100 µm). Ventricle-like lumen in COs is marked as V'. ECE2 expression on protein level. Immunohistochemistry (IHC) for ECE2 in 50d old COs shows accumulation in the CP' and at the apical surface (Scalebar=50µm). Ventricle-like lumen in COs is marked as V'. Scheme showing the electroporation of DNA into ventricles of COs and the organisation of different cell types within the germinal zone. DNA is injected into the ventricle-like lumen and taken up by aRG via their apical processes. 7 dpe, the transfected construct can additionally be found in IPs and neurons upon differentiation of transfected aRG (green) (VZ' = ventricular zone, SVZ' = subventricular zone, IZ' = intermediate zone, CP' = cortical plate, aRG' = apical radial glia, bRG' = basal radial glia, IP' = intermediate progenitor). COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). Ventricle-like lumen in COs is marked as V'. Graph depicting the number of ectopically located NEUN+ cells in CTRL and KD electroporated COs using two different micro-RNAs targeting ECE2. Data shown as box plot (mean = red line, median = black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of ventricles analysed; ***P < 0.001 in Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pairwise Multiple Comparison). Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42]. Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test).
Figure 6A, Schematic representation of viral injection and sleep deprivation (SD) experiments. Zeitgeber time 0 (ZT0) corresponding to lights ON. Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (B) Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice) Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (C) Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice) Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (D) Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). mEPSC (E) mean amplitude of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05. F, mEPSC (F) frequency of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05. G, Representative examples of the current-voltage relationship of evoked EPSC amplitude recorded from S (top panel) and SD (bottom panel) mice. H, Summary bar graphs showing rectification index of control (S n=12 cells/5 mice, and SD n=10 cells/5 mice), Fxr1P overexpressing (S n=9 cells/4 mice, and SD n=14 cells/4 mice) and Gsk3 sKO (S n=9 cells/3 mice, and SD n=9 cells/3 mice) neurons. One way ANOVA with Bonferroni's Multiple Comparison *p < 0.05.
Figure 5A. Western blot for Ctrl and Ifnar1-KO MC38 cells, analyzed with Ifnar1 and Gapdh antibodies. B. Ctrl or Ifnar1-KO MC38 cells were treated with PBS or recombinant Ifnβ for 4 hours. The RNA expression levels of indicated ISGs were analyzed using qRT-PCR. N=3. Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (C) Pictures of tumors and spleens from a representative experiment. Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (D) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (E) Tumor and spleen weights at the endpoint for (D), with each dot representing a mouse. Ctrl tumor data in (C-E) are the same as those in Figure 4B-4D. Schematics (F) of the experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS.  experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (G) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (H) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (G). experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (I) Tumor and spleen weights at the endpoint for (H), with each dot representing a mouse.
Figure 5Western blot analysis of CD68, CLEC7A, TREM2 and ApoE in total brain lysates from 4.5-month-old mice with the given genotype. Asterisk indicate unspecific band. (n = 3 biological replicates per genotype). Quantification of A. Protein expression was normalized to levels in wild type animals. Expression of CD68 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (TH = Thalamus). Expression of CLEC7A and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Expression of TREM2 and the microglial marker IBA1 in sagittal brain sections. Representative images of indicated brain regions (MY = medulla). Scale bar indicates 25 µm.
Figure 5Branching analysis of LPS-activated microglia by Monocle 2 leads to 9 distinct clusters in a two-dimensional state space inferred by generalized regression modelling (see Materials and Methods) showing the major difference of "subset LPS" (in yellow) compared to the other clusters corresponding to "main LPS" (in red). Monocle estimated a pseudotime for each cell along the inferred cell trajectory within the state space showing a delayed activation pattern of "subset LPS" compared to the other fractions. Pseudotime dynamics of inflammatory (Ccl12, Ccl2, Gpr84) and homeostatic (Mef2c, P2ry12, Siglech) genes in dependence on inferred cell states.
Figure 7A. Experimental scheme for the knock-in (KI) mice production and characterization. The single-stranded DNA donor-mediated repair of the Cas9-induced Rabl2 DNA breaks is expected to introduce the Q80L mutation in fertilized eggs or early embryos. Neonatal F1 mice were obtained through the caesarean section at E18.5. Genotyping was performed by directly sequencing the PCR products that covered the expected Cas9-cleavage region of Rabl2. B. Representative sequencing results for Rabl2KI/KI mice, shown with corresponding sequences for wildtype Rabl2 and the 3' end of the sgRNA. The TTG codon for the Q80L mutation is underlined. C. Viability of neonatal mice in the day of birth after the caesarean section at E18.5. +, wildtype allele of Rabl2; KI, knock-in allele with Q80L mutation; mt, allele with Cas9-induced mutation; x, ambiguous allele. Refer to Fig EV5 for additional genotyping results. As these "heterozygous" mice might contain chimeric ones, the putative genotypes are used only for simplicity. D. Morphologically normal and duplex kidneys of Rabl2KI/KI mice. The whole kidneys were from a single mouse. The histological sections, stained with hematoxylin and eosin, were from different mice. Arrows point to the constriction of the duplex kidneys. E. Renal ducts of Rabl2KI/KI kidney displayed normal ciliary formation as compared to wildtype kidney. Nuclear DNA was stained with DAPI. F, G. Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome (Bbs5) and its cargo GPCR (Smo). mEPCs cultured from Rabl2KI/KI and wildtype neonatal mice were fixed at day 7 post serum starvation. Anti-Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L. Ac-tub or Arl13b served as ciliary marker. Representative micrographs of multicilia (F) and primary cilia (G) are shown. The white arrows point to positions of cilia. H. A model delineating the switch role of ciliary Rabl2 in the BBSome-mediated GPCR turnover: (1) GTP hydrolysis induced presumably by a GTPase-activating protein (GAP) dissociates Rabl2 from IFT-B to enable a tight BBSome-IFT-B interaction in the retrograde IFT machinery for concomitant TZ passage; (2) Without the GTP hydrolysis, Rabl2-GTP (or Rabl2Q80L) binds persistently to IFT-B, resulting in shedding of the BBSome and its cargos from the IFT machinery in the TZ passing process and accordingly their ciliary retention.
Fig.Figure 1Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT. (C) illustration of the intracellular carbon transition in metabolic tissues after [U-13C]glucose administration. (D,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).
Figure 6A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right. B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.
GEMC1 is required for female fertility and multiciliogenesis.Figure 3A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;amp;E staining. Scale bars = 50 µm (top panels). C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).
Figure 1ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins. Cryo-EM density of the post-SC highlights the archaeal ribosomal protein eS21 and ABCE1. Domain architecture of ABCE1 including the mutation sites is shown below. Molecular model of the archaeal post-SC, domain colors as in B.
Figure 2A HEK293T cells with stable knockdown of RNF123 were generated using shRNA. Western blot was performed with anti-RNF123, anti-VISA (as a control), and anti-GAPDH antibodies after infection with five lentiviruses encoding a shRNA control (GFPsh) and four shRNAs targeting RNF123 (RNF123 sh #1, #2, #3, and #4) in HEK293T cells. B, C Knockdown of RNF123 facilitated the activation of IFN-β and NF-κB promoters induced by SeV and EMCV but not by cGAS and STING. Stable HEK293T cells with GFPsh or RNF123 shRNAs (2×105) were transfected with the indicated reporter plasmid (100 ng), pRL-TK Renilla luciferase plasmid (100 ng), cGAS (50 ng), and STING (50 ng), or empty vector (100 ng). At 12 h after transfection, they were infected for 12 h with SeV or EMCV or left uninfected, followed by analysis as described in the legend of Fig 1A. D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and RT-PCR (D) were performed using the indicated primers. D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and Q-PCR (E) were performed using the indicated primers. F, G Time-course of potentiation of SeV-induced IFN-β transcription (F) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated, and RT-PCR was performed using the indicated primers (F). F, G Time-course of potentiation of SeV-induced IFN-β secretion (G) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated. Bioassays of type-I interferon in the supernatants of HEK293T cells were performed, and the results are presented relative to untreated control cells (G). H HeLa cells with stable knockdown of RNF123 were generated by shRNA. Western blot was performed as described in Fig 2A. I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and RT-PCR (I) was performed using the indicated primers. I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and Q-PCR (J) was performed using the indicated primers. K Knockdown of RNF123 suppresses NDV-eGFP replication. HEK293T cells (2×105) were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot with GFP antibody. L Knockdown of RNF123 inhibits EMCV replication. HeLa cells (2×105) were infected with EMCV for 0, 12, or 18 h.
SPHK2 and HAT inhibitors prevent the DHS-induced cardiac DNA damage in vitro: (A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.Figure 4(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs. (C) Bar graphs representing the percentage of γH2A.X+ hCMs in the indicated conditions (# cells quantified per replicate ~200). (D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition). (F) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (G) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels. (H) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (I) HAT inhibition prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) on human cardiomyocytes. (J) Quantification of γH2A.X+ hCMs nuclei represented as percentage bar graph. (K) Curcumin-mediated HAT inhibition lead to inactivation of p21 in hCMs. (L) Violin plots depicting the distributions of the grey-scale nuclear intensity of p21 stained nuclei. (When not specified, the experiments were conducted in at least 3 biological replicates).
Figure 6 [A] (This study) In phosphate (Pi)-sufficient plants, diacetyl (DA) partially suppresses the immune system including the production of reactive oxygen species (ROS), primes salicylic acid (SA) accumulation, and increases the competitiveness of GB03 against pathogens, resulting in enhanced symbiont colonization without compromised resistance to the pathogen Pst DC3000. As a result of the mutualistic association, plant growth is promoted. In P-deficient plants, DA simultaneously elevates the levels of salicylic acid (SA) and jasmonic acid (JA), resulting in strong activation of phytohormone-mediated immunity and consequently plant hypersensitivity to P deficiency. As a result, plants display stress symptoms.  [B] (Adapted from Hiruma et al., 2016) Pi-deficient plants allow symbiosis with certain fungi because endophytic fungi can transfer phosphate to plants, whereas Pi-sufficient plants need no fungi-assisted Pi uptake and so they deploys Trp-derived secondary metabolites to defend against fungi invasion.
Figure 6(A) Immunofluorescence microscopy analysis of the endoplasmic reticulum (ER) network in control and subject fibroblasts. The ER was stained in red (KDEL), mitochondria in green (TOMM20). Scale bars: 20 µm. (B) The organization of the reticular ER network was classified as class I (normal), class II (mildly disorganized) or class III (highly disorganized). Scale bars: 10 µm. (C) Quantification of the control and subject ER network organization. In each condition, 30 cells were analysed in three independent experiments. Error bars represent mean +/- SD and p-values were calculated using a t-test. (D) Immunofluorescence microscopy analysis of contacts sites between ER and mitochondria in control and subject fibroblasts. ER was stained in red (KDEL), mitochondria in green (TOMM20). Contact sites are shown by arrowheads. Scale bars: 10 µm.
Figure 2(A) ZBTB7B's domain structure marked with the mutations in BTB and ZF domain used in this study. (B) Two representative biological functions of ZBTB7B: 1) changing target gene expression; 2) recruiting target proteins for ubiquitination. CUL3: Cullin3, E2: ubiquitin-conjugating enzyme, SUB: substrate. (C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA. (D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA. (E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled. (F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score. (G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test. (K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test. (L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.
Figure 3C57BL/6J male mice were injected with asparaginase (ASNase) or PBS every three days for 2 weeks. Mice were exposed to 4˚C for 6 h before sacrifice for gene expression analysis. Serum asparagine and aspartic acid concentration in mice of (A). n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. Rectal temperature in mice of (A) during cold exposure. n=5 mice each group. BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A). Serum triglyceride levels in mice of (A). n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).