EMBO/sd-character-level-ner huggingface dataset:
D hydrogels of different stiffnesses.Figure 3(A) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test. (B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm. (C) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test. (D) Sporozoite on planar uncoated hydrogels. (E) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test. (G) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test. (H) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.
Metformin ameliorates the cognitive deficits in arsenic-exposed mice.Figure 5A Test procedure of NORT (left panel) and the bar graph showing the percent of exploration time for familiar (F) and novel (N) objects of the offspring mice (right panel). B-F MWMT. Training and testing protocol (B). The time needed to reach the hidden platform during the acquisition phase (C), the percent of time spent in the target quadrant on the probe test day 8 (D), the time needed to reach the reversal hidden platform during the reversal phase of each trial (E), and the percent of time spent in the reversal target quadrant on the reversal probe test day 10 (F) were shown for each group of mice. G, H 5meC enrichment analysis of the IAP (G), and Xist, H19 loci (H) in the hippocampus of offspring mice from each group, as measured by MeDIP-qPCR. Each of the biological replicate was run in three technical replicates and means were shown as cycles in the graph. I mRNA levels of the 12 selected DEGs assessed by RT-qPCR.
Figure 1(A) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4). (B) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates. (C) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test). (D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+cells; CD1d and CCR6 expression in NCR-ILC3s and CD1d and CD4 expression in NCR-CCR6+ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates. (E) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).
Figure 7(A) Schematic representation of the genomic region upstream of the Tcf7l1 transcriptional start site (TSS). (B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028. (C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026. (D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. (E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.
in the AT-ND transitionA.Figure 2A. Normalized expression levels of Ccnd1 in each cluster (Fig 1C) as indicated by violin plots. B. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001. C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2). G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).
Vasculotide and Ang1 treatments counteract thrombin induction of trans-endothelium macromolecular permeability and tumor cell migration in vitro.Figure 1A-E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A-C) and dermal HMVECs (D-E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10-20 ng/mL VT and 200-400 ng/mL Ang1, are estimated molar equivalents (0.71-1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B-C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, 'extravasated' CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3-5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.
Figure 4A Workflow used for the generation of the co-recruitment correlation network (CN). Left: Normalized intensities of recruitment of two identified partners (denoted Xi and Xj) to the considered bait as a function of time. Center left: Normalized intensities of recruitment for preys Xi and Xj are plotted against each other (Rij: Pearson correlation value; pij corresponding p value). Center right: Correlation matrix (Rij) between all pairs of identified interactors partitioned into different clusters. Right: Portion of the obtained correlation network after filtering according to pij values. B, C Representation of the CBL (B) CNs. Nodes represent specific interacting partners and edges connect pairs of nodes for which the p-value associated with the Pearson correlation coefficient exceeded a set threshold. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. B, C Representation of the CBLB (C) CNs. Nodes represent specific interacting partners and edges connect pairs of nodes for which the p-value associated with the Pearson correlation coefficient exceeded a set threshold. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. D, E Venn diagrams showing overlaps between protein-protein interactions deduced from the CBL (D) or CBLB (E) CNs and those reported in the BioGRID database for Homo sapiens and Mus musculus. Two sets of analysis are shown according to Pearson correlation coefficient values (R<0.8 or R>0.8). Histograms below the Venn diagrams compare the probabilities to predict existing interactions with a random network (Random), the complete CN or the high confidence CN (R>0.8).
Figure 7 Transcription factors (TFs) interact with DNA via the DNA binding domain (DBD, blue triangle) and to their interaction partners (grey) via their transactivation domains (TAD, green rectangle). The enrichment for negatively charged residues leads to a local extended conformation of the TAD via intra-chain repulsions, providing the appropriate context for aromatic residues to be exposed and to bind to their interaction partners in hydrophobic binding pocket (circular inset). The aromatic residues could fit the pocket in a stochastic manner, binding in different configurations. This would result in the formation of a "fuzzy" complex. In this case, the negative charges could furthermore contribute to the affinity of binding given that the TAD-interaction surfaces often expose positively charged patches. The absence of positively charged residues, compositional bias and particular spacing of negatively charged and aromatic residues could hence be considered as giving rise to a collection of short mini motifs that collectively contributes to TAD functionality
a–c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3.figf3 (a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3. d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization.  (e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a-c) or × 63 (d,e). Results in a-e are representative of three independent experiments. (f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples).  (g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments.  (i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).
Figure 7(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot. (B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test). (C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines. (D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines. (E) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test). (F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).
Beclin‐1 is required for proper mitotic progression.Figure 1(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1. (B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release. (C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively). (E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.
a–c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti–IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.figf3(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20. (d) Analysis of survival of indicated mice. (e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection. (f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).
Figure 3(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patientsfibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates.
Motor neuron loss in FusΔNLS/ΔNLS mice.Figure 6(A) Representative light microscopy images of spinal cord sections of Fus+/+, FusΔNLS/+, FusΔNLS/ΔNLS and Fus-/- mice at birth stained with cresyl violet (Nissl, A1, A3 A5, A7), or anti-choline acetyl transferase (ChAT, A2, A4 A6, A8).(B-C) Quantification of ChAT+ motor neurons (B) and Nissl+ motor neurons (defined as Nissl positive cells with a soma area >80µm2) (C) per spinal cordventral horn in FusΔNLS/ΔNLS mice. For Nissl+ N=8 Fus+/+, N=5 FusΔNLS/+, N=7 FusΔNLS/ΔNLS and for ChAT+ N=7 per genotype, (**) p<0.01 vs Fus+/+, (##) p<0.01 vs Fus∆NLS/+ ; One way ANOVA followed by Tukey post hoc test.(D-E) Quantification of ChAT+ (D) and Nissl+ (E) motor neurons per spinal cordventral horn in Fus-/- mice. N=6 per genotype, no significant differences were found, by Student's unpaired t-test.
AβO-induced peripheral glucose intolerance and hypothalamic insulin resistance are mediated by TNF-α signaling and hypothalamic ER stress.Figure 7Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice that received i.c.v. injections of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA (when used, TUDCA was administered in 5 i.c.v. injections of 5 μg TUDCA each, before and after oligomer injection; see Materials and Methods. Control groups received injections of saline). Glucose tolerance test (GTT) was performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 15 Veh; 15 AβOs; 10 Veh + TUDCA; 16 AβOs + TUDCA). Data are representative of two independent experiments with similar results. Left panel: **P = 0.0048, **P = 0.003, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0384, one-way ANOVA followed by Bonferroni post hoc test. Plasma noradrenaline (NA) levels measured 7 days after i.c.v. injection of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA in mice (n = 7 animals/group). Data are representative of two independent experiments with similar results. *P = 0.0071, one-way ANOVA followed by Bonferroni post hoc test. Glucose tolerance test (2 g glucose/kg body weight, i.p.) in TNFR1−/− mice or wild-type littermates performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 8 WT + Veh; 7 WT + AβOs; 7 TNFR−/− + Veh; 8 TNFR−/− + AβOs). Left panel: **P = 0.0049, ***P < 0.0001, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0001, one-way ANOVA followed by Bonferroni post hoc test. Western blot analysis of phospho-IKKβ (D; n = 6 WT + Veh; 6 WT + AβOs; 4 TNFR−/− + Veh; 6 TNFR−/− + AβOs) and IRS-1pSer636 levels (E; n = 6 WT + Veh; 5 WT + AβOs; 6 TNFR−/− + Veh; 5 TNFR−/− + AβOs) in the hypothalamus of wild-type (WT) or TNFR1−/− mice 10 days after i.c.v. injection of vehicle or AβOs. Representative images from Western blot experiments were always run on the same gels but represent noncontiguous lanes. In (D), *P = 0.0088, Student's t-test; in (E), *P = 0.0428, one-way ANOVA followed by Bonferroni post hoc test. Adult Swiss mice pre-treated with minocycline or PBS received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (F; n = 5 Veh; 6 AβOs; 5 Veh + Mino; 4 AβOs + Mino) and NPY (G; n = 14 Veh; 13 AβOs; 8 Veh + Mino; 9 AβOs + Mino) were analyzed 7 days after injection. In (F), *P = 0.0097, one-way ANOVA followed by Bonferroni post hoc test; in (G), *P = 0.0219, one-way ANOVA followed by Bonferroni post hoc test. Glucose tolerance test (GTT) in APP/PS1 mice before and after i.c.v. injection of infliximab (0.2 μg daily for 4 days). Bar graph represents areas under the curves (AUC) in the time course plots (n = 9 animals/group). Left panel: *P = 0.0177, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0327, paired t-test.
Figure 6T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m. T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m Representative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.