EMBO/sd-character-level-ner huggingface dataset:
Ectopically expressed aluRNA accumulates in the nucleolus together with associated genomic lociCLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene).Figure 7CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red). The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain. MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.
Kinetochore-microtubule attachment stability is required to confine MTOCs at spindle poles(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A.Figure 3(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm. (B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume). (C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments). (D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle , was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments).
Model for regulation of metabolism at the pyruvate branch point Fermentative conditions.Figure 6A Fermentative conditions. The low‐activity MPCFERM (composed of Mpc1 and Mpc2) is competing for cytosolic pyruvate produced through glycolysis with pyruvate decarboxylase (PDC), the first enzyme of the alcoholic fermentation pathway. Consequently, the majority of pyruvate remains in the cytosol is decarboxylated by PDC and is finally transformed into ethanol. Low‐level transport of pyruvate into mitochondria is required for the biosynthesis of leucine and valine. In addition, mitochondrial pyruvate oxidation may be blocked by a hypothetical inactivation of pyruvate dehydrogenase (PDH) by phosphorylation of the E1 subunit under these conditions.B Respiratory conditions. The high‐activity MPCOX (composed of Mpc1 and Mpc3) is expressed while PDC is downregulated. This allows the efficient uptake of pyruvate into mitochondria. Inside the organelle, pyruvate is converted to acetyl‐CoA by PDH, which enters the TCA cycle and thus finally fuels ATP production in the respiratory chain. The subunit exchange in the MPC thus very likely participates in the Crabtree effect in Saccharomyces cerevisiae.
a, Effect of oligomycin (Oli) and DMSO (Ctrl) on spherogenic potential of +/−KRas cells (n = 8); representative calcein staining.f3a, Effect of oligomycin (Oli) and DMSO (Ctrl) on spherogenic potential of +/−KRas cells (n = 8); representative calcein staining. b, Kaplan-Meier overall survival after KRas reactivation in mice bearing regressed tumours treated for 2 weeks with oligomycin or vehicle. c, Effects of oligomycin and DMSO on spherogenic potential of cells treated (AB) or not with AZD8330 plus BEZ235 in p53−/− and Ink4a/Arf−/− backgrounds (n = 3). d, ECAR response of human tumour spheres treated (AZD+BEZ) and untreated (Ctrl) to glucose, oligomycin and 2DG (n = 3). e, Effect of oligomycin and DMSO on spherogenic potential of human treated and untreated tumour cells (n = 3). Data are mean ± s.d.
Figure 2A Description of the systems chronopharmacology model for NHP. (i) The translocation of PF-670 from plasma to the SCN and its inhibition of PER phosphorylation by CK1δ/ε in NHP were accurately modelled to reproduce the PF-670 exposure and its effect in NHPs (ii) The dependence of light-induced Per1/2 gene transcription via CREB on circadian time (gating) and light duration (adaptation) are included in the model The estimated 10 pairs of gating and adaptation allow for the model to successfully reproduce the light-induced phase shift in NHPs and humans (iii) The model simulates the combined effect of PF-670 (i) and light (ii) on PER2 rhythms in the SCN of NHPs. B The model accurately reproduces the phase delay of NHPs induced by 3-day LD dosing at ZT11. Here, the PF-670-induced phase delay from which the vehicle-induced phase shift is subtracted was used (n=8; mean±SEM Each orange line is an individual simulation result obtained with each pair of gating and adaptation in (Aii) and their average is represented by the gray line. C Double-plotted actogram of NHPs' activity for 16 days. The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm (i.e. LD is delayed by 2 h). D The model successfully predicts the phase shift induced by 2 h jet lag (Days 8-10) and its recovery after jet lag (Days 11-13) (n=7; mean±SEM E NHPs were treated with 10 mpk PF-670 for 3 consecutive days at ZT4 in LD 12:12, which are highlighted as the red line. F The phase delay induced by 3-day LD dosing at ZT4 (n=8; mean±SEM; E) is accurately predicted by the model.
Figure 6Western blot analysis of cathepsin (Cat) D, B, and L in total brain lysates from 4.5-month-old mice with the indicated genotype. Different maturation variants are indicated (p = preform, sc = single chain, hc = heavy chain) (n = 3 biological replicates per genotype). Quantification of proteolytically generated variants of cathepsin D, B, and L normalized to levels in wild type animals from (A). Catalytic activity of cathepsin D, B, and L. Total brain lysates from 4.5-month-old mice were incubated with quenched fluorogenic substrate for Cat D, Cat B and Cat L. The linear increase of fluorescence signal was measured and then normalized to wildtype. Data represent the mean normalized activity ± SD. (n = 3 biological replicates per genotype).
regulates adaptive thermogenesis and contributes to cold tolerance (A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time.Figure 6(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time. Body temperature (B) before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice. BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice. Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E). Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F). (G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm. (H) Schematic model of PGC-1α-NRF1-FUNDC1 axis in mitochondrial quality and quantity control and adaptive thermogenesis.
Figure 9 (A) Proportion of mtDNA cluster following 48 or 96 hours of growth in medium with (+) or without (-) 25 mM glucose (HG), 5 mM glucose (LG), ß-hydroxybutyrate (BHB) or 1 mM pyruvate. At least 50 cells per cell line were counted per condition, n = 2 experiments. Data are expressed as mean ± SEM. Representative images appear below the charts. Scale bar 15 µm  (B) 35S labeling of de novo mitochondrial protein synthesis for 1 h, 72 h after transfection of HeLa cells with either a non-target dsRNA (NT) or LETM1 siR1; KBs (0.1 mM BHB or acetoacetate (AA)) replaced HG (25 mM glucose, 1 mM pyruvate) 9 hours before the labeling reaction. Polypeptide assignments flank the gel image; coomassie stained gels are used as loading controls, and immunoblots indicate the efficiency of LETM1 knockdown  (C) Perceived or actual nutrient availability alters the organization of mitochondrial nucleoprotein complexes. In yeasts, mitochondrial ribosomes are physically linked to mitochondrial nucleoids (Kehrein et al, 2015), and purification studies of a mitochondrial ribosomal protein (Rorbach et al, 2008) and mitochondrial nucleoid proteins (He et al, 2012a) suggest that the same is true of mammalian mitochondria, as illustrated in (a). The current study indicates that changes in nutrient availability (a switch from glucose to ketone body supplemented culture medium) lead to re-organization of mitochondrial nucleoproteins (MNPC) (panel A and Fig 8D and E, and illustrated in (b)). We infer that MNPC disorganization in LETM1 deficiency is the result of a failure to utilize pyruvate - a defect in nutrient sensing - that can be attributed to repression of pyruvate dehydrogenase (PDH) via PDK3 (Fig 8B and EV3A). Because the bulk of PDH appears to be physically associated with MNPC (Fig 8A), this could be mediated by the LETM1 bound to mitochondrial ribosomes/MNPC (Fig 4C). The direct and marked impact of nutrients on MNPCs is further evidenced by the adaptation of WHS cells (LETM1+/-) to KBs (Fig 8E-ii, Fig EV3 and Appendix Figs S8-9); and the demonstration that KBs mitigate the mitochondrial translation impairment caused by LETM1 depletion (Fig 9B). Hence, WHS cells progressively adapt mtDNA organization to the alternative carbon source (c), whereas control cells are not primed for KB utilization and in their case MNPC clustering is followed by cell death (d) (Fig 8C and Movie EV1)
Phosphorylation of EB2 Ser222 is suppressed by NMDAR activity.Figure 3 A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates  D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates
Figure 6(A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used. (B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material. (C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.
Figure 5Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates). Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).
mTOR signalling in TAM-MG promotes immune evasion mechanisms in human glioblastoma.Figure 7(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05. (B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green). (C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2. (D) Heatmap of Pearson correlation analysis comparing ssGSEA enrichment scores of TAM-MG and TAM-BMDM signatures versus signalling pathway signatures, calculated from the TPM of IDH-wild type GBM samples with the signature from group 1. (E) Schematic of the experimental setup to derive patient-matched EPSC and GIC lines. (F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM). (G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm. (I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).
Figure 3 A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test  B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m  C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m
Figure 5A, B Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACA (72 h) and 2R or 66R (24 h), immunoblotted for PKA-Cα and poly-GA (HA-tag) expression. α-Tubulin used as loading control. (B) Quantification of PKA-Cα and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cα: ns siScr 66R vs siScr 2R, ***P = 0.0002 siPRKACA 66R vs siScr 2R, ***P = 0.0003 siPRKACA 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACA 66R vs siScr 2R, **P = 0.0062 siPRKACA 66R vs siScr 66R . C, D Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACB (72 h) and 2R or 66R (24 h), immunoblotted PKA-Cβ and poly-GA (HA tag) expression. α-Tubulin used as loading control. (D) Quantification of PKA-Cβ and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cβ: ns siScr 66R vs siScr 2R, **P = 0.0049 siPRKACB 66R vs siScr 2R, **P = 0.0091 siPRKACB 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACB 66R vs siScr 2R, ***P = 0.0007 siPRKACB 66R vs siScr 66R. E E Co-sedimentation profile of (G4C2) (upper panel) and GAPDH (lower panel) mRNAs along the sucrose gradient fractions of polysomal profiles from control (grey lines) and PRKACA-depleted (orange lines) cells. Data are mean ± SEM from three independent biological replicates. One-tailed t-test, *P<0.05. F Co-sedimentation profile of (G4C2) (upper panel) and GAPDH (lower panel) mRNAs along the sucrose fractions of polysomal profiling from control (grey lines) and PRKACB-depleted (red lines) cells. Data are mean ± SEM from three independent biological replicates. One-tailed t-test, *P<0.05; **P<0.01. ns = not significant
T.Figure 2A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites. B. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.
Figure 1A PF-670 inhibits the phosphorylation of PER by CK1δ/ε (i) and delays the circadian phase, which is counterbalanced by light (ii). Thus, daily dosing leads to continually accumulating phase delay in DD and constant stable phase delay in LD. B Measured free PF-670 exposure in the brain tissue of mice and NHPs. Drug exposure in NHPs after administration of 10 mpk PF-670 (AUC=3.6μMh) is ~7-fold higher than that in mice given 32 mpk PF-670 (AUC=0.5 μMh) (mean±SEM). C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12). E, F Phase delay induced by the 3-day dosing under DD (E) or LD (F). The phase of activity onset in NHPs (10 mpk) is more delayed than that in mice (32 mpk) under DD (E), but not under LD (F) (P=0.03, one-way analysis of variance (ANOVA); n.s., no significant difference). Veh denotes vehicle. The error bars represent mean±SEM. The mouse data are adopted from (Kim et al, 2013). G The quantification of (E) and (F) indicates that light has a stronger attenuating effect on the PF-670-induced phase delay in NHPs than in mice (P=0.036, two-way ANOVA).
Figure 2BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (C) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT, MAPK (ERK, p38, JNK), IκBα, total forms of IκBα. αActin was used as a loading control (left). The densitometric values were calculated from the ratios of protein levels relative to actin (right).
/CXCL12 heterocomplex.Figure 31H-15N HSQC HMGB1 (0.1 mM) spectrum 1H-15N HSQC HMGB1 (0.1 mM) spectrum with 0.2 mM CXCL12. MST measurements of CXCL12 titrated into 50 nM fluorescently labelled HMGB1; the signal increases from ∼930 (free) to ∼950 a.u. (bound) yielding an apparent Kd of 4 ± 0.4 μM. n=3; error bars represent avg ± sd. H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL. MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.