EMBO/sd-character-level-ner huggingface dataset:
γ-H2AX nuclear foci formation in different cell cycle phases.Figure 3A, B Percentages of γ-H2AX nuclear foci remaining in CENP-F-negative (A) and CENP-F-positive (B) cells at the indicated time-points after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated controls, are the mean ± SD of three independent experiments. ***P < 0.001; *P < 0.05 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.C Immunofluorescence micrographs of γ-H2AX foci in CENP-F-positive and negative fibroblasts at 2 h after irradiation. Arrow heads indicate the CENP-F-positive cells.
Figure 5EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Vascularization evaluated by H&E staining of pulmonary arteries (C). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C) Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D). EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).
Figure 4(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3). (B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3). (C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D). (E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top and bottom panels show 5 sequential frames of trimeric Spike/hCLEC4G and trimeric Spike/hCD209 complexes, acquired at a rate of 303 ms/frame. Association and dissociation events between hCLEC4G or hCD209 and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike and trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.
Figure 2A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002. C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.
a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.figf4(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells. (b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units. (b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units. (d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells. (e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).
Figure 5(A) Schematic of SRPK1 signaling pathway that regulates subcellular SRSF1 localization, which in turns can impact export and translation of repeat RNAs in the cytoplasm. SRPK1 may also act directly on protein translation pathways (dotted arrow). Known pharmacological compounds that inhibit SRPK1 can disrupt this pathway. (B) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer. (C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9 biological replicates) following treatment with DMSO and SRPIN340. (D) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. (E) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
Luc-CTL assay design used for identification of immune-checkpoint molecules.Figure 1Assay principle: RNAi was performed with luciferase-expressing cells that were challenged with or without CTLs and bi-specific antibody. Before readout, cell supernatant was removed and the remaining intact cells were lysed to measure the residual-cell-associated luciferase. To identify immune-checkpoint regulators, the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated. siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs; hence, the difference between luciferase measurements will be > 0. Luc-CTL assay performed at different T cell to MCF7 cell ratio with PBMC-derived CD8+ T cells and anti-EpCAM x CD3 bi-specific antibody (○). Anti-CD19 × CD3 bi-specific Ab (▪) was used as a specificity control since CD19 is a B-lymphocyte-specific antigen and therefore this bsAb fails to cross-link tumor to T cells. Lower luciferase intensity indicates higher lysis. Error bars denote ± SEM. Luc-CTL assay was performed with MCF7 cells transfected with control or PD-L1-specific siRNAs and cocultured with or without CTLs and bsAb. For each condition, the luciferase activity of PD-L1-siRNA-treated cells was normalized to that of the control siRNA; n = 8. Error bars denote ± SEM. Comparison between the Luc-CTL assay (▪) and the classical chromium (Cr)-release assay (○) with MCF7 as target cells and survivin-specific T cells as effector cells at varying effector to target (E:T) ratios. Error bars denote ± SEM.
Figure 1A. Overview of the Trafficking Phosphoproteomics Approaches (TPAs). B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D D. Confluence over time of BT20, T47D, and BT549 transfected with HA-FGFR2b stimulated with FGF7 (dark green) or FGF10 (burgundy). Data represent the mean ± SD of N=3 compared to FGF10. P value = <0.05 *, <0.01 **, <0.001*** (Student's t-test). E. Hierarchical clustering of the phosphorylated sites differentially quantified T47D stimulated with FGF7 (green) or FGF10 (burgundy). Specific clusters are highlighted with black lines. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively. F. Enriched terms in the selected clusters of E. G G. Network of phosphorylated proteins belonging to "protein localization" in FGF10 clusters, based on STRING, visualized in Cytoscape, and color-coded based on cell components. The squared shape represents phosphorylated proteins found in the database DISEASES. The recycling adaptors TTP and RCP are highlighted in grey. H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. I. The presence (total), internalization (internalized), and recycling (cell surface) of transfected HA-FGFR2b in BT549 upon stimulation were quantified as in (Francavilla et al., 2016) and in the section "Quantification of the Recycling assay". Values represent the median ± SD of N=3. Representative pictures upon FGF10 stimulation are shown in H.
IP3R1 controls the migration persistence of DCs through the Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK) A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.Figure 6A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7. B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis. D, E Analysis of shScramble (gray)‐ and shIP3R(1,3) C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.
Figure 3A. Pie charts representing, on a feature-by-feature basis, the relative distribution of COG categories among the gene deletion strains associated with s ≥ 3. The enriched COG categories are labeled and highlighted with an exploded pie sector. The under-represented COG categories are further highlighted by an asterisk. Enrichments and under-representations with an associated (FDR-corrected) q-value < 0.05 were considered significant. Only morphological and growth features with at least one enriched or under-represented COG category are represented. B. Same as A but for s ≤ -3.
E.Figure 3A.) Construct used to express the E. coli DPCK in the apicoplast. Appended are the N-terminal signal sequence and transit peptide from ACP (api) and a C-terminal mCherry tag. B.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide. C.) Genotyping PCR confirming the deletion of the endogenous DPCK, with amplification demonstrating integration at the Δ5' and Δ3' loci and lack of wild type parasites (as indicated by the failure to amplify at the wild type 5' and 3' loci) as compared to the parental control. D.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). E.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide. F.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the apicoplast organellar genome.
Protective R522 M-MOP and microglia display increased depletion of PI(4,5)P2 without corresponding compensation from other PIP speciesA-C.Figure 7A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5) P2 (A), PI(3,4) P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)
Figure 9In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, the interaction between p14ARF and PRMT1 is reinforced and the C-terminal NLS/NoLS of p14ARF is methylated by PRMT1. This stress-induced arginine methylation promotes the release of p14ARF from NPM and nucleolar sequestration, leading to p53-independent apoptosis.
Figure 5A The distribution of CHAF1B in the presence of CHAF1A (upper panel) and in the absence of endogenous CHAF1A (lower panel). B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A. C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A. The distribution of SUV39H1 in the presence or absence of CHAF1A. The distribution of SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A. G The distribution of SUMO paralogs in the absence of CHAF1A.
Figure 5(A) HTM-dependent quantification of EdU, γH2AX, H3S10P and MPM-2 levels per individual nucleus in U2OS cells treated with 50 μM P22077 (USP7i) for the indicated times or with DMSO as a control. Quantification data derive from 3 independent experiments. Data represent mean values ± SEM. N>2000 cells analysed per condition. (B) Flow cytometry profiles illustrating the levels of γH2AX and MPM-2 in U2OS cells in control conditions (Control, black) and after treatment with 50 μM P22077 for the indicated times (USP7i, red). Experiments were repeated 3 times and one representative result is shown. (C) Flow cytometry profiles representing the correlation between γH2AX and MPM-2 or histone H3S10P signals in HCT-116 cells treated with DMSO (Control) or 50 μM P22077 for 8 h (USP7i). (D) Flow cytometry profiles representing the levels of γH2AX in HCT-116 cells in control conditions or after treatment with 50 μM P22077 (USP7i) for 4 hrs alone or in combination with 10 μM RO3306 (USP7i + CDK1i) for 4 hrs. DNA content was measured with Propidium Iodide (PI). (E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.
MPK4 and PAT1 interact in ArabidopsisMPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants.Figure 1MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP. Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures. Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies. Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.