EMBO/sd-character-level-ner huggingface dataset:
(E and F) Quantification of sibling clones size in the wing pouch. In (D), sibling wild-type clones are labelled with 2xGFP and 2xRFP. In (E), unlabelled clones are homozygous for the yki2-only-A allele whereas sibling clones (RFP/RFP) are wild type. In both graphs, clones pairs are sorted according to RFP/RFP clones size.
A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).
(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.
B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.
F, Cells were treated for 1 day in 5 % LPDS followed by 50 μg/ml LDL loading for the indicated times. Representative confocal images of cells triple labeled with intracellular accessible cholesterol (D4H), NPC1-GFP and internalized integrin β1. Dashed lines indicate cell outlines. G, Quantification of D4H area positive for NPC1 or internalized integrin β1 as in F. H, Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a peripheral region toward the leading edge (exemplary box shown as inset in F). Mean ± SD, n = 27-30 cells pooled from 2 independent experiments. Student's t-test.
Proposed model of how palmitoylation of Golga7b regulates plasma membrane localisation of DHHC5
(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.
PIF4, IAA29 and ATHB2 mRNA levels in SDs in WT (data: black lines, filled squares; simulation: black lines).
a, DARPP-32 (D32) immunostaining shows that striatal neuron cultures consist mainly of D32-positive medium spiny neurons (MSNs). 40x magnification, scale bar = 50 µm. MAP2 marks neurons, Hoechst (Hoe) stains DNA. n = 3 cultures from independent mouse litters.
F. Schematic representation of the experimental paradigm. Concentrations used: 4μM coMO; 2μM MOs-3p + 2μM co-MO; 2μM MOs-3p + 2μM MOs-TUBB3; 200ng/mL Sema3A. Abbreviations: co-MO, control morpholino; TUBB3, tubulin beta 3 class III.
(D) Venn diagram and showing the shared 263 CREB-controlled genes significantly altered by treatment with K+ channel blockers; the bar plot represents the significantly altered genes involved in neuronal activity and autophagy obtained by DESeq2 analysis.
(C) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress.
D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.
(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.
BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.
C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).
C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.
(B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.
E ) Fork stability (average IdU/CldU ratio) of Mybl2+/+ ESC and Mybl2Δ/Δ ESCs treated with the indicated inhibitors alone or in combination. At least 240 ratios were calculated for non-ATM inhibitor treated groups, and a minimm of 130 for ATM inhibitor treated groups. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using Mann Whitney U test (**** p< 0.0001). Data for Mybl2+/+ ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor, was collected from two independent experiments. For the rest of the conditions, a minimum of three independent repeats was performed.
High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.
B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.
A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.
C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.
Under normal cell growth conditions, the pre-ribosomal complex formed by RPL5, RPL11 and 5S rRNA is incorporated into nascent ribosomes (Left panel). When nucleotide levels become limiting for 47S rRNA synthesis, ribosome biogenesis is impaired, which converts the RPL5/RPL11/5S rRNA complex into the IRBC complex, which is able to bind and inhibit MDM2, releasing p53, leading to p21 induction and G1 arrest (Middle panel). However, a more severe drop in nucleotide levels leads to the degradation of p21 by the proteasome, impairing IRBC-dependent G1 arrest and allowing S phase entry despite reduced nucleotide pools. Cells are therefore exposed to replicative stress, sensed by the ATR-Chk1 pathway responsible for p53 S15 phosphorylation. Chronic replicative stress leads to DNA-damage and eventually genomic instability, key events in tumor progression (Right panel).
(C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01.
(c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001).
(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.
(e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments; n, total number of death events; ***P0.0001.
Quantitative analysis of hepatic bud area (prox1 staining) in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. n>10, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m
DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.
(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.
C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments.
D. GST-Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST-Rab18 was pre-loaded with GTPγS or GDP, or stripped off nucleotide by EDTA. Lysates from 293T cells were subjected to GST-Rab18 pull-down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII, but not TRAPPIII complex was co-isolated with GST-Rab18, most strongly with Rab18 in nucleotide-free state.
(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.
Genome organization of SARS and SARS-like CoVs including Tor2, CoVZC45 and WHCV determined here.
(A) Heat map showing log2 fold change of expression of ISGs in A549-ACE2 cells across 8, 16 and 24 h time points upon IFN-α treatment compared to non-treated controls. The common upregulated genes (≥ 3-fold; padj < 0.05) in IFN-induced versus non-induced conditions at all time points were categorized as ISGs.
Representative confocal images of Crebh in the liver cryosections of 8-week-old mice (zoomed out versions in Figure EV3F). Note that a fraction of hepatocytes is binucleated. Yellow arrows represent Crebh staining inside in the hepatocyte nucleus.
(D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).
Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.
Medial confocal planes from z-stacks from (a) and (b) demonstrating attachment of the FM 4-64-stained cell plate with the peripheral plasma membrane, defining t0. Scale bars, 10 µm.
(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).
(H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.
(C) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.
B-C, Analysis of the number (B) and the total area (C) of the GFP-Fbl1 foci in P19 cells expressing the constructs indicated for a period of 360 minutes. Band width represents SEM and grey areas represent time points with significant differences between the control and the Trnp1 expressing conditions. The ∆1-16Trnp1 expression does not show any statistical difference compared to the control condition.
(C) Neutrophil decrease model with low dose treatment. After 4 hr of CPA injection, mice were treated with regimens for 3 days. On day 2, mice were infected with 6 x 106 CFU of S. aureus via intravenous injection for up to 16 hr before euthanasia. Control mice without CPA. N, numbers of mice.
A. Protein-protein interaction network of physical interactors of OFD1, praja2 and TBC1D31. Node size represents the degree of association of the proteins (grey nodes) associated with the input proteins set (black nodes). Edge size is representative of the strength of the mapped interaction between the two connecting nodes.
Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.
K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.
C. Statistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT and KO B cells after four days of stimulation with anti-CD40 + IL-4. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4).
Sequence alignment of the MICU1 protein from nine vertebrate species. The Akt consensus phosphorylation motif, R-X-R-X-X-S/T, is marked in yellow
D: Relative expression of ELDR in OSCC cell lines compared to normal oral keratinocytes (NOK) analyzed by qRT-PCR. 18S was used as internal control. Data were analyzed by Student's t test. Small bars indicate standard error (**, p<0.01; *** p<0.001). n = 4 biological replicates. Data are represented as the mean ± SD,
C. LincGET depleted embryos arrest at the 2-cell stage. LNA was injected at phCG 25 hours and photos were taken at phCG 114 hours at the late blastocyst stage. Embryos injected with Control-LNA can develop to the late blastocyst stage, while LincGET depleted embryos arrest at the 2-cell stage. Scale bar, 100 μm. At least three experimental replicates were performed for each LNA injection (Table 1).
(h) IB3-1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA.
Control and si-IRGM transfected HT-29 cells uninfected or infected with CHIKV and qRT-PCR analysis were performed with (I) MX1
(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm
(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates.
(I&amp;amp;J) Free-glucose levels during β-cell regeneration in control (bactin:mCherry) and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) larvae (I), as well as in Tg(ins:kaede);Tg(ins:CFP-NTR) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3-4 dpf to ablate their β cells, and monitored their free-glucose levels at 3-7 dpf. Free-glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1-protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β-cell ablation. n=24 larvae (four pools of six larvae) per data point.
G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).
C Two types of nucleosome positioning: face-to-face, at ~6-nm spacing, and edge-to-edge, at ~11-nm spacing. The image was made based on the structural information published in (Luger et al., 1997).D The modeled scattering profile of an extended dinucleosome structure based on its atomic coordinate (for details, see Materials and Methods). Note that the modeled profile is similar to that of the nucleosomal arrays in 0 mM MgCl2 (A).E Two structural models of 12-mer 30-nm fibers: solenoid (left) and zigzag (right) as a top and side view. The models were constructed using MolScript (Kraulis, 1991).F The scattering profiles of the solenoid (red line) and zigzag (blue line) 30-nm fibers were made from their atomic coordinates computationally. Note that the 30-40 nm peak is prominent in both fiber models.
(d) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).
C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005.
Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.
(c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).
Clustering analysis of the normalized RNA-seq counts on the X chromosome (chrX) for all the duplicates of Xist FL, Xist ΔA and Xist ΔB+C in DOX and noDOX conditions.
F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).
(C) The ratio of compartment sizes (P area divided by A area) is plotted. The 14 genes were knocked down using multiple (2 or 3) RNAi lines per gene. The x-axis indicates RNAi case IDs that represent the genes knocked down and the RNAi line type (KK, GD, or TP as TRiP). Detailed information is provided in Dataset EV6. The horizontal line indicates the size ratio in the negative control cases. Wilcoxon rank sum tests evaluated the statistical significance against controls (*:p <0.05, ***:p<0.001, n.s.: not significant, cont.: control cases).
C, D Representative Western blots for β-catenin detected in nuclear (Nuc) and cytoplasmic (Cyt) protein lysates. Lamin A/C and Actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 3 independent experiments). (C) Spheroids were cultured in stem, enterocyte (EP4i) or WAE (dmPGE2) media. (D) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021.
F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.
F Quantification of the number of costained REST+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).
Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F-G (n=3 per group). Hsp90 , loading controls for Western blot analysis.