EMBO/sd-character-level-ner huggingface dataset:
Figure 4Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum. Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (B) Total cortical plaque area (normalized to total sample area (*10−2), stained with antibody NT-12. Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (C) Formic acid-soluble Aβ40 in forebrain. (D) Formic acid-soluble Aβ42 in forebrain. Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (E) Double-immunofluorescence stained brain sections with NT12 (against amyloid-β, green) and Iba1 (against microglia, red) or GFAP (astrocytes, red) for the different treatment groups as indicated. Scale bar: 500 µm. Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (F) Quantification of total Iba1 positive microglia (normalized to total area (*10−2), only a subset of samples analyzed. Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (G) Quantification of total GFAP positive astrocyte area (normalized to total area (*10−2). Data were analyzed with Dunnett's multiple comparison test.
Bioinformatic analysis of Matrin3‐regulated splicing eventsIntron lengths flanking exons regulated by Matrin3, PTB or control exons.Figure 4A Intron lengths flanking exons regulated by Matrin3, PTB or control exons. Kruskal-Wallis rank‐sum test was used to test for significant changes (Matr3 P‐value = 3.947e−15, PTB P‐value = 0.3896). ***P 0.001. B Diagram of Matrin3‐repressed cassette exons with enriched RBP motifs for human proteins (Ray et al, ) shown above and the number of enriched pentamers shown below in each of 7 locations. The RBP motifs are shown with their consensus binding site logo (Ray et al, ) and the respective motif enrichment score (odds ratio). The significant enriched k‐mers are shown in Supplementary Table S4, and all enriched RBP motifs (multiple species) are shown in Supplementary Table S5.
Figure 7(A) Experimental set-up used to assay transfer through conditioned medium (upper scheme) and cell-to-cell contacts (lower scheme) in neurons. For the CM experiments, donor neurons are loaded with α-synucleinfibrils for 16 hours, washed 3 times with PBS and then fresh medium is added and left to condition for 72 hours. CM is then collected and transferred to acceptor neurons (10 div) for 72 hours. In the cell-to-cell contact impairment (No contact) experiment the donor (already loaded) and newly dissected acceptor (already labelled) neurons are plated on different coverslips placed in the same Petri dish. In both experiments, the percentage of cells containing α-synucleinATTO-550puncta, and the number and mean size of α-synucleinpuncta per cell are quantified using the ICY software after 72 hours. (B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm. (C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student's t-test. Data show mean ± s.e.m from 3 independent experiments.(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments).. (E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.
Figure 2C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP. C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF. C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF. C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight. C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.
Figure 4A, B Schematic diagram of Drosophila (D. melanogaster) CYLD (dCYLD) and human (H. sapiens) CYLD (A), and a multiple amino acid sequence alignment of a catalytic region of the ubiquitin carboxyl-terminal hydrolase (UCH) domain in different species (B). dCYLD is 639 amino acids in length, and contains one cytoskeletal-associated protein-glycine-conserved (CAP-Gly) domain (Red) and the UCH domain (Green) (A). Human CYLD is 965 amino acids in length and contains three CAP-Gly domains, an unstructured phosphorylation region (Phos-site (Pink); specific for CYLD chordata species), and a UCH domain (Green). A conserved active Cys (C284 in Drosophila) is indicated by * (B). C In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band. D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL. E Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.
Figure 2(A) Schematic diagram of strategy employed to C-terminally tag PABPC1 in PABPC4KO HeLa cells using CRISPR-Cas9 mediated homology directed repair (HDR). Adapted from (Sheridan & Bentley, 2016). Homology arms flanked repair cassettes containing a DHFR sequence, preceded by a HA tag and separated from an antibiotic resistance gene (denoted as AbR and either Puro or Hygro) by a T2A cleavage site followed by a SV40 signal. (B) Western blot confirmation of homozygous PABPC1 tagging with DHFR and degron activity. Successful homozygous tagging of PABPC1 is indicated by the higher molecular weight migration (lane 2) as compared to untagged PABPC1 (lane 1). Degron activity was assessed by culturing PABPDHFR cells in the absence of TMP for 4 or 8 hours and assessing PABPC1 levels by western blotting. (C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm. (D) Western blot analysis of lysates derived from cells imaged in (C).
Figure 2CSF T-tau (panel A), Ng (panel B) and NFL (panel C) in different combinations of clinical diagnosis and A pathology (CN Aβ-, n=69; CN Aβ+, n=40; MCI Aβ-, n=50; MCI Aβ+, n=137; AD Aβ-, n=8; AD Aβ+, n=85). See Table 5 for comparisons between groups. Biomarker levels are standardized to z-scores and shown in boxplots (indicating median and interquartile ranges; whiskers are defined as quartiles 1 and 3 +/- 1.5 * interquartile range, respectively). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Figure 4Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy). Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation. Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.
Figure 3(A) MALT1 expression is essential for CARD14sh-induced NF-kB activation. MALT1-deficient HEK293T cells were transfected with NF-kB reporter plasmid and the indicated expression plasmids. Luciferase activity in cell lysates was measured 24 h later. CARD11(L232LI) and TRAF6 transfection were used as MALT1-dependent and MALT1-independent controls, respectively. Values are the mean of triplicates ± s.e. Expression of the transfected plasmids was verified by immunoblotting (lower panel). (B) MALT1 expression is essential for CARD14sh-induced JNK activation. MALT1-deficient HEK293T cells were transfected with FLAG-CARD14sh (30ng) either alone or together with either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A) as indicated. c-Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control. (C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.
Identifying a core TORC1-dependent protein interaction network in regulating rates of protein synthesisIdentification of a TORC1-dependent protein interaction network through a comparative phosphoproteomic studyA - D Summary profile plots illustrating the minimum and maximum range of phosphorylation for the respective phosphorylation sites in wild type (in green) and tor1∆ (in yellow) cells, with lines indicating the median values of the phosphorylation profiles and the shaded areas representing the range of values.Figure 3y A - D Summary profile plots illustrating the minimum and maximum range of phosphorylation for the respective phosphorylation sites in wild type (in green) and tor1∆ (in yellow) cells, with lines indicating the median values of the phosphorylation profiles and the shaded areas representing the range of values. Values are normalised to their respective values at T = 0 minutes in each strain. Panels representing the 43 TORC2-dependent phosphosites that show more than 2-fold change in wild type (A) but within 1.3-fold difference in tor1∆ cells (B). Panels illustrating the 158 phosphosites that show relative phosphorylation changes of more than 2-fold in both wild type (C) and tor1∆ (D) cells . E Interaction network generated from the interactome analysis of the 49 potential TORC1-dependent proteins conserved among fission yeast, budding yeast and human, based on collated annotations from the STRING database. Interactions between proteins were scored based on both functional and physical interactions from experimental, text mining and database sources only. Connections were drawn upon reaching a minimum interaction score of 0.4, and the lines were weighted to indicate the relative strength of the annotated interactions. The resultant network of the 28 connected proteins were functionally categorised based on their GO biological processes as annotated on PomBase, and represented by the following colours respectively: cytoplasmic translation (GO:0002181) and ribosome biogenesis (GO:0042254) in green, mRNA metabolism (GO:0016071) and transcription (GO:0006351) in purple, vesicle-mediated transport (GO:0016192) in yellow and actin cytoskeleton organisation (GO:0030036) in brown. Grey nodes indicate proteins that were not assigned to the highlighted functional categories. Proteins that were identified as common in both phosphoproteomics studies are denoted by the darker hues of the respective colour categories, and the nodes of essential proteins are outlined in white (9/28)
Figure 1A Schematic representation of the enhanced green fluorescent protein (eGFP) intein constructs and of the intein-mediated protein trans-splicing. ITR, inverted terminal repeats; Prom, promoter; 5'eGFP,5'eGFP coding sequence (CDS); n-intein, N-terminal of DnaE intein (star symbol), 3xFlag tag; PolyA, short synthetic polyadenylation signal; c-intein, C terminal of DnaE intein; 3'eGFP, 3' eGFP CDS. B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.
Fig.Figure 6Quantification of PML puncta in P6 SCG neurons transduced with either control non-targeting shRNA or shRNA targeting PML for 3 days, then treated with IFNα (600 IU/ml) for 18 hours. n=60 cells from 3 biological replicates. Quantification of colocalization of vDNA foci detected by click chemistry to PML in primary SCG neurons transduced with shRNA targeting PML for 3 days prior to being infected with HSV-1EdC in the presence or absence of IFNα (150 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates. Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML for 3 days prior to infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates.
Rapamycin, but not FG4592, treatment sustain OXPHOS function in presence of IMT1.Figure 3A 35S-labelling of newly synthesized mitochondrial-encoded proteins of RKO cells treated either with DMSO, IMT1, rapamycin and FG4592 alone or in combination with IMT1. Left: pulse labelling; right: 24 hours chase in normal medium. Putative mitochondrial proteins are reported on the right-hand side of the gel, molecular weights on the left. Coomassie staining shows equal protein loading; the image is representative of n=4 independent experiments. B Densiometric quantification of the ND5, COX1, ND4 band from n=4 35S-labelling experiments. Data are expressed as mean values ± SD. Statistical significance was calculated with one-way ANOVA test. Paired comparisons: IMT1 vs. IMT1 + RAPA pulse: *p=0,015; IMT1 vs IMT1 + RAPA chase: *p=0,046; IMT1 vs IMT1 + FG4592 pulse and chase: non-significant. C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control. D Densiometric quantification of COX2 protein levels from n=4 experiments. Data are expressed as mean values ± SD. Statistical significance was calculated with one-way ANOVA. IMT1 vs IMT1 + RAPA: *p=0,016; IMT1 vs IMT1 + FG4592: non-significant. E Bioenergetic profiles measured with Seahorse flux analyzer showing the contribution of OCR and ECAR to cellular bioenergetics upon the aforementioned treatments. Data represent the mean ± SEM of n=3 independent experiments. F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.
LIF/STAT3 signalling promotes proliferation and mitochondrial transcription.Figure 1(A) Proliferation assay of Stat3+/+ and Stat3-/- cells cultured in N2B27-based 2i media either in the presence or absence of LIF. Cells were seeded and scored for four consecutive days. Scores were normalized to day1. Mean and s.d. of three technical replicates. See also Appendix Figure S1A. (B) Cell cycle analysis of Stat3+/+ cells cultured in 2i media without LIF or with LIF for several passages and Stat3-/- cells in 2i with LIF. 48hours after plating cells were detached, treated with Propidium Iodide and analysed by flow cytometry. Top: Cells cultured in presence of LIF showed an increment in S/G2 and a decrease in G1. Mean and s.e.m. of three technical replicates. Unpaired t-test: * P<0.05. Bottom: representative plots. (C) Scatter plot showing RNA-seq data from Stat3+/+ cells cultured in 2i and stimulated with LIF for 1h (Martello et al, 2013). Absolute expression is shown in RPKM. Green dots indicate known LIF targets that serve as positive controls. Mitochondrial encoded transcripts are represented as orange dots. Only genes with FC>1.7 and a p-value <0.05 are shown. (D) Heatmap showing mean normalised expression of 13 mitochondrial transcripts encoding 4 subunits of the mitochondrial respiratory chain. RNA-seq data are from Stat3+/+ and Stat3-/- cells were expanded in 2i media without LIF and treated with LIF for 1hour or 24 hours. (E) Gene expression analysis by RT-qPCR of Stat3+/+ (blue) and Stat3-/- (red) cells cultured in 2i and treated with LIF for 1 hour, 4 hours or 4 days. Data are normalized to unstimulated 2i cultures. Beta-actin served as an internal control. Mean and s.e.m. of three independent experiments. Unpaired t-test: * P<0.05 ** P<0.01 *** P<0.001. See also Appendix Figure S1B.
Potential conversion of TseTBg homologs of TA system into T6SS effectors in B.Figure 7(A) The genomic loci containing TseTBg (toxin) and TsiTBg (antitoxin) homologs in different B. pseudomallei strains. (B) In B. pseudomallei 3000015237 isolate MX2014, two IS3 family transposable elements that carry two of the T6SS related genes (PAAR and DUF4123) are present upstream to the TA genes. However, in B. pseudomallei Nau24B-3 and vgh16R strains, the IS3 element which flank the TA and T6SS related genes, have been excised and a new ORF encoding hypothetical protein (H.P) has evolved at its place. (C) The excision of IS3 family transposase has created operonic fusion of TA and T6SS related genes. Moreover, a new ORF of 474 bp that encode a H.P (hypothetical protein) has evolved at the locus, through adoption of 3' end of the DUF4123 and 5' end of the toxin gene.
Figure 6A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation. B Quantitative real-time PCR analysis of ATX1 and FLC transcript levels in Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG plants. The transcript levels were normalized to that of TUBULIN. Data are mean ± s.d. of three independent biological replicates, n=3. Flowering time phenotype analysis of lines Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. Flowering time phenotype analysis of lines The percentages of plants with visible buds and the bolting rates were calculated on day 21 and day 24 after plant germination, respectively. Three independent biological repeats were conducted, and data from a total of 71 plants were used for statistical analysis. Data are mean ± s.d., (**), Statistical significance (two-tailed t-test) with P < 0.01. F Working model of SEC activation of ATX1 to regulate FLC transcription by O-GlcNAc modification.
Figure 4 Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down. (B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).