EMBO/sd-character-level-ner huggingface dataset:
Benchmarking and performance assessment of complex-centric proteome profiling.Figure 2Benchmark of CCprofiler algorithm and error model in reference to a manually curated reference set of signals displays conservative decoy-based FDR control and high sensitivity, recalling 91 % of high quality co-elution signals at 5% target-decoy-derived FDR Assessment of complex identification performance based on the recovery of CORUM complexes. A CORUM complex is considered as recovered when more than 50 % of its annotated subunits are reported within one complex module in the respective dataset. CORUM complex recovery is compared between our complex-centric analysis strategy using StringDB connectivity information priors (This study (CC StringDB)), the complexes reported by Havugimana et al. (2012), the complexes reported by Larance et al. (2016), and complexes detected by complex-centric analysis of the native SEC-MS data of Larance et al. (2016) using StringDB as prior connectivity information Comparison of SWATH-MS-based quantification to DDA-MS-based strategies (MS1 XIC and spectral counting) with regard to consistency (as judged based upon protein-level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections) and precision (judged based on the correlation between sibling peptide SEC chromatograms) of quantification and overall performance in error-controlled complex-centric query of CORUM complexes in the respective protein level chromatogram sets (also see Appendix Figure S3C). TP, true positive assignments according to error model, P, all positives.
Figure 3Representative Western blot (A) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg. quantification (B) of Parkin levels in spinal cord homogenates. Protein levels are normalized by β-actin. Results are expressed as mean ± SEM and relative to Non Tg control at 30 days; n= 8 (4 males and 4 females). Paired Wilcoxon's test at 90 days, **p= 0.0078; paired Student's t test at 120 days, **p= 0.0054. Paired Wilcoxon's test for 30 and 60 days showed no statistically significant differences. Parkin levels are decreased in SOD1-G93A at 90 and 120 days compared to Non Tg. C) qPCR (fold change) of Parkin mRNA normalized by β-actin mRNA. Results are expressed as mean ± SEM and fold change relative to Non Tg at 30 days of age; n= 6 (3 males and 3 females) for 30 and 120 days, n= 4 (2 males and 2 females) for 60 days and n= 5 (3 males and 2 females) for 90 days. No statistically significant differences were found by paired Student's t test (90 days) and Wilcoxon's test (30, 60 and 120 days). D, E) Representative Western blot (D) and quantification (E) of Parkin levels in spinal cord mitochondria at 120 days. Protein levels are normalized by Complex V. Parkin is reduced in spinal cord mitochondria of SOD1-G93A mice at 120 days. Results are expressed as mean ± SEM and shown as percent of Non Tg; n=8 (4 males and 4 females); **p= 0.0078, by Wilcoxon's t test.
Pore mutations in PC1 or PC2 led to changes in ion permeability of the PC1-CTF/PC2_AA channel.Figure 6A. TOP: a bottom view of a previously reported cryo-EM structure of PC1/PC2 channel (PDB code 6A70) [42], which is missing the NTF of PC1 and the intracellular C-terminal tails from both proteins. S10-S11 of one PC1 subunit and S5-S6 from three PC2 subunits assembled to form the pore. Bottom: the mutated amino acids in this experiment (in purple) are mapped on S10-S11 of one PC1 and S5-S6 of one PC2 subunit in the complex. The dashed grey line indicates the putative ion flow path. Since human PC1 was used for structure determination, mouse amino acids mutated in this study are indicated in parentheses. Due to the low resolution of the PC1 pore region in the structure, side chains are not seen for the three mutated amino acids at the putative selectivity filter region. E4068 (E4078 in human) is not solved in the structure, and it is labeled to indicate its approximate location in the structure. Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test). Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test). Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases. Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.
Figure 3RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Pie chart showing genes regulated by hnRNP-L or CALIC RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated. Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk. Genes regulated in common and in the same direction by hnRNP-L and CALIC. Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score. Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.
The calcium-binding protein caldendrin interacts with a 49 amino-acid region containing the first IQ motif of myosin Va in a Ca2+-dependent manner.Figure 1A Schematic showing overall domain organization of the caldendrin protein, including an unstructured N-terminal part, and four EF-hand binding motifs in the C-terminal part. Green boxes indicate functional EF-hands, blue indicates cryptic EF-hand. The dashed line indicates where the Nt and Ct constructs were separated. Co-immunoprecipitation experiments from extracts of HEK293T cells, co-expressing full-length GFP-myosin Va (myoVa fl) and indicated caldendrin-tagRFP (cald) constructs, show that caldendrin interacts with myoVa in a Ca2+-dependent manner via its N-terminus (Nt), and not via its C-terminus (Ct). Tubulin was used as a loading control for the input. MW = molecular weight marker. B Pull down assay indicates that recombinant purified GFP-myoVa and untagged caldendrin (cald wt) directly bind to each other in a Ca2+-dependent manner. Note that there is no binding observed for a Ca2+-binding mutant of caldendrin (cald mut / Left panel). Right panel: neither wt nor mut caldendrin bind to GFP-coupled beads. C-E Mapping of the binding region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP and GFP-myoVa fragments expressed in HEK293T cells. Arrows in D indicate the size of the respective myosin fragment. F Summary of the results from C - E. Caldendrin binds specifically to a fragment containing IQ-motif 1 (amino acids 742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.
Figure 2A. Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214) or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control. B. HeLa cells were initially transfected with indicated siRNAs, followed by renilla luciferase (RL) reporter constructs; RL-control (no let-7a binding site in the 3'UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3'-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test. C. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test. D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3'-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.
Figure 4A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe. B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants. C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity. D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells. E) Model for Ssu72 regulation of telomere replication in fission yeast. See text for details.
Figure 2CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A); typical action potentials (APs) in controls (B, top panel) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac mice the distribution of the membrane potential and firing cells in the two groups (C, ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior). CRH-Cre male mice were injected with either AAV-Flex-GFP or AAV-EF1a-Flex-EGFP-P2A-mNachBac to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing GFP or NachBac (GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn), c-Fos expression (red in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p<0.0001: fasting versus refed; **p=0.0026: fasting versus fasting/NachBac; **p<0.0001: fasting/NachBac versus refed/NachBac) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; **p<0.0001: fasting versus fasting/NachBac; p=0.3423: fasting/NachBac versus refed/NachBac), n=3-4 each, data presented as mean +/- SEM, two way ANOVA tests. CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (G), and comparison of responsiveness to water spray between NachBac and control mice (H). (I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.
Figure 2(A, B) Schematic illustrations of the design of inducible "Tet-On" ESC lines expressing Hoxa5 or Hoxc8 under the doxycycline (Dox)-regulated promoter. In the presence of Dox, the reverse tTA (rtTA) activator is recruited to the TRE (Tetracycline Response Element), thereby initiating transcription of the downstream gene. (C, E) Immunostaining reveals expression of Hoxa5 and Hoxc8 upon induction of exogenous Hoxc8:V5 (iHoxc8:V5) or Hoxa5:V5 (iHoxa5:V5), respectively. (D, F) Quantification of data from (C) and (E) (mean ± SD, N≥3 EBs from three independent experiments, *p < 0.01, Student's t-tests). Scale bars in (C) and (E) represent 50 μm.
Concomitant loss of BIM or PUMA delays the fatal kidney disease caused by the combination of γ-irradiation and inducible deletion of BCL-XL.Figure 5(A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (C) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment. (D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras
Figure 1(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29). (B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification. (C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups. (D) Schematic strategy of Npm1 conditional knockout. Excision of exons 1-6 from the Npm1 allele by Cre-recombinase produces an Npm1-knockout allele. Hypomorphic allele (top), NEO-free allele (middle, Flox) and excised allele are shown. The genomic sequence is depicted as a black line, with black boxes representing exons 1-11. Grey box, red triangles and arrows represent an Frt (blue)-flanked neomycin resistance cassette (NEO), the loxP site, and genotyping primers, respectively. (E) Schema of mating strategy and numbers of viable pups at weaning bearing the 6 possible genotypes for Vav1-Cre. A Chi-square test determined any difference between observed proportions and expected proportions. (F) Schematic representation of Vec and Vav1 expression during embryogenesis. (G) Schema of mating strategy and numbers of viable pups at weaning bearing the 6 possible genotypes for Vec-Cre. A Chi-square test determined any difference between observed proportions and expected proportions.
Transient binding of HSFA2 governs sustained H3K4 methylation after HS.Figure 7Figure 7: Transient binding of HSFA2 governs sustained H3K4 methylation after HS.Top panel: Transient binding of HSFA2 after HS induces sustained induction of H3K4me2 (light blue) and H3K4me3 (dark blue) through direct or indirect recruitment of chromatin modifying factors. H3K4me3 and H3K4me2 levels remain elevated after H3K9ac (orange) and HSFA2 binding (red) have decreased and hence mark recent transcriptional activity of the locus, leading to sustained induction and transcriptional memory upon recurring stress. Bottom panel: In hsfa2, H3K4me2 and H3K4me3 remain low while the H3K9ac levels are only slightly changed compared to wild type. Profiles were drawn based on the data obtained for the APX2 locus.
Figure 3A. Mitochondrial non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. * P=0.021 with two-tailed unpaired T-test.B. Cytosolic non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. Two-tailed unpaired T-test was performed. C. Baseline ejection fraction of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.D. Baseline fractional shortening of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed. E. Lipid peroxidation products in hearts of TG and NTG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed. F. Relative expression of antioxidant genes in NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed. All data are expressed as mean ± SEM. N.S. = not signigicant.
Figure 4(A) Putative binding sites of miR-181a(-5p and -2-3p) in the 3′ UTR region of Map3k2 and Notch2. Potential miR-181a(-5p and -2-3p) gene targets were selected based on predictions obtained in referenced mirDIP v4.1database. The predicted binding regions that were amplified for the luciferase assay are highlighted in grey. (B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples). (C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples). (D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus MAP3K2 (left panel) or NOTCH2 (right panel) expression in γδ T cells isolated from thymic biopsies. Samples were cultured for 11 days with the indicated cytokines (n=6 independent biological samples). (E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).
NM1226 and NM1230 binding epitopes on RBD.Figure 3A Overview of the binding epitopes of NM1226 (light green) on RBD (dark green) and NM1230 (magenta) on RBD (light pink), respectively. B, C The binding interfaces of NM1226 (B) and NM1230 (C) with the RBD are shown as surface representation (upper panels) and in a close-up view as balls and sticks with direct hydrogen bonds and salt bridges indicated as dotted lines (lower panels). All residues involved in contact formation at a distance cut-off of < 4 Å are listed and labeled. A close-up view of both interfaces report a detailed interaction map of both contact areas.
Figure 3A ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test. C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2) p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test. I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.