EMBO/soda-nel huggingface dataset:
Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases (2020)
Figure 2

Figure 2:

GRA15 enhances IFNγ-induced PVM decoration with autophagy-related proteins and endo-lysosomal-mediated vacuole destruction in HFFs

A-G) HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (A) Total ubiquitin (n=3 for RH, n=5 for Pru and n=3 for PruΔgra15), (B) K63-linked ubiquitin (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), (C) p62 (n=3 for RH, n=10 for Pru and n=7 for PruΔgra15, n=4 for Pru Δgra15+GRA15), (D) NDP52 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), (E) LC3B (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15, n=3 for Pru Δgra15+GRA15), (F) GABARAP (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) and (G) LAMP1 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.

H) The number of PVs per 20X objective field was counted and compared between IFNγ-stimulated and IFNγ + bafilomycin A1 (100 nM) treated HFFs 24 h p.i. with Pru strain. Images from at least 6 fields were taken for each condition (n=3).

Data information: Each dot represents one experiment. Each time at least 100 different vacuoles were scored and analyzed. Statistical analysis was done by Two-Way ANOVA followed with Tukey's multiple comparison test (A-G) and One-way ANOVA for (H). Data are represented as mean ± SEM.
Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases (2020)
Figure 3

Figure 3:

PVM decoration with autophagy adaptors correlates with p62 but not ubiquitination

A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection. Cells were washed and subsequently infected with Pru parasites (MOI=3) for 3 h. The percentage of vacuoles that stained positive for total ubiquitin, p62 or LC3B was determined (n=3).

B-F) For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, p62 and/or LC3B/GABARAP was calculated.

Data information: Statistical analysis was done by One-way ANOVA followed with Tukey's multiple comparison test. Data are represented as mean ± SEM.
Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases (2020)
Figure 5

Figure 5:

GRA15 enhanced susceptibility of type II strains to IFNγ-mediated killing in MEFs correlates with enhanced recruitment of IRGB6, GBPs, Ubiquitin, and LC3B.

MEFs were stimulated with IFNγ for 24 h (100 U/ml).

A-F) IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (A) IRGB6, (B) GBPs, (C) total ubiquitin, (D) K63-linked ubiquitin, (E) K48-linked ubiquitin and (F) LC3B. For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.

G) MEFs were stimulated with IFNγ for 24 h (100 U/ml) or left unstimulated and subsequently infected with Pru, PruΔgra15 and GRA15 complemented parasites for 24 h for measuring parasite growth by luciferase assay. All experiments were performed 3 independent times.

H) Plaque numbers were measured for Pru (n=12), PruΔgra15 (n=12) and GRA15 complemented parasites (n=6).

I-J) MEFs were stimulated with IFNγ for 24 h (100 U/ml) or left unstimulated and subsequently infected with RH or RH expressing type II GRA15. Plaque numbers were counted (n=3), and areas were measured 4 days p.i. (n=3).

Data information: Statistical analysis was done by One-way ANOVA followed with Tukey's multiple comparison test (A-H), except for (G, J) for which Two-way ANOVA followed with Tukey's multiple comparison test was performed whereas for (I) two sample student's t test was performed. Data are represented as mean ± SEM.
Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases (2020)
Figure 6

Figure 6:

GRA15 enhanced susceptibility of the type II Pru strain in IFNγ-stimulated MEFs is dependent on TRAF6

A) Immunoprecipitations and Western blots were performed on MEFs stimulated or not with IFNγ (100 U/ml) and infected with an RH strain expressing type II GRA15-HA and as a control RH expressing GRA45-HA. The blots using antibodies against TRAF6 and TRAF2 were made after stripping the first blot. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively.

B) IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 and RH for 3 h and subsequently stained for TRAF6. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. Experiments were performed 3 times.

C) IRGB6 staining on PVM after infection of wild-type or Traf6 -/- MEFs for 3 h with Pru, PruΔgra15 and RH. On the right-hand side, a representative fluorescent image is shown of IRGB6 coating on the PVM of both wild-type and Traf6 -/- MEF. Scale bar is 10 µm. At least 100 different vacuoles were observed and analyzed for each experiment (n=3).

D) Parasite growth was measured 24 h p.i. using luciferase readout from unstimulated and IFNγ-stimulated wild-type, NFκB p65-/- and Traf6 -/- MEFs. Growth was compared between Pru, PruΔgra15 and PruΔgra15+GRA15 (complemented) strains. Reading from unstimulated cells was considered as 100% and percentage growth in IFNγ-stimulated cells was expressed relative to unstimulated cells. Experiments were performed three times with each MEF type.

E) Immunoprecipitation and Western blot were performed in MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut and as a control RH expressing GRA45-HA. Left panel and right panel were run on a single gel; vertical white lines indicate excision of irrelevant lanes. Full length blots are in the source data for this figure. The antibodies used against TRAF2 and TRAF6 were purchased from Cell Signaling Technology and Abnova, respectively.

F-J) Immunofluorescence analysis of TRAF6, IRGB6, GBP1-5, p62 and ubiquitin was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).

K) Plaque assays were performed with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=4).

Data information: Statistical analysis was done by two samples student's t test (F-J) and One-way ANOVA followed with Tukey's multiple comparison test for (B and K) and Two-way ANOVA for (C and D). Data are represented as mean ± SEM.

Expanded view Figure legends
Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2 (2020)
Figure 4

Figure 4. H2A.B residues dictate nucleosome resistance to MNase digestion.

A Top: Sequence alignment of human H2A.B and H2A. Residues not conserved in H2A.B and H2A are colored in red. Secondary structures are indicated above. H2A.B N-terminal domain (NTD) and the docking domain (DD) are indicated. H2A.B residues subject to mutagenesis analyses are indicated as boxes. Bottom: Schematic view of mutants used for MNase analysis. The H2A and H2A.B sequences are indicated by blue and gray, respectively.

B,C Effects of H2A mutations (B) and H2A.B mutations (C) on nucleosome resistance to MNase digestion. MNase digested nucleosomal DNA are analyzed by 10% Native-PAGE. All MNase digestion experiments are repeated twice.

D,E Graph presentation of effects of H2A mutations (D) and H2A.B mutations (E) on nucleosome resistance to MNase digestion. MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA. All experiments are repeated twice. Data are mean ± SD, n = 2.

F Structural comparison of H2A.B α2-α3 helices with their H2A counterparts. Structures of H2A-H2B dimer in the canonical nucleosome (yellow, PDB: 3LZ0) and H2A.B-H2B dimer in the H2A.B-NCP (orange) were superimposed for comparison. The dashed lines indicate the ~15 degrees kink of C-terminal region of H2A.B α2 helix.
Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2 (2020)
Figure 5

Figure 5. Effect of H2A.B and H2A.Z.2.2 ROF on modulating histone octamer assembly.

A Schematic view of canonical and variant H2A used for octamer assembly analysis. The H2A.B, H2A, and H2A.Z.2.2 sequences are indicated by gray, blue, and orange, respectively. The regulating-octamer-folding (ROF) sequences from different H2A histones are displayed for comparison.

B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants.

C Close-up view of structure of H2A C-terminal regions shown in cartoon representation. Histones H2A, H3 and ROF region in H2A-NCP (PDB: 3LZ0) are colored in yellow, blue and red, respectively.

D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.
Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2 (2020)
Figure 6

Figure 6. H2A.Z.2.2 ROF modulates nucleosome dynamics and facilitates histone replacement.

A Comparison of the cryo-EM structure of H2A.Z.2.2-NCP with the structure of canonical nucleosome (PDB: 3LZ0) in disc view. Histone octamers containing H2A.Z.2.2 and canonical H2A are superimposed for comparison. The 125 bp DNA of Z.2.2-NCP in green color is shown in surface mode. The 145 bp DNA of canonical nucleosome is colored in grey. Histones H2A.B, H2B, H3 and DNA at SHL 6 through SHL 7 are highlighted to show the structural differences between two nucleosomes. The Z.2.2-NCP histones are colored in orange, warmpink, and cyan. Their counterparts in canonical nucleosome are colored in yellow, red, and blue. Histone H4 is colored in grey.

B Close-up view of the structures of Z.2.2 C-terminal regions (top) and H2A C-terminal regions (bottom). Black and white arrows indicate last residue at the C-terminus of Z.2.2 (G106) and H2A (K118) that are observable in the structures.

C Comparison of the cryo-EM structure of Z.2.2-NCP with the crystal structure of canonical nucleosome (PDB: 3LZ0) in gyre view. Arrows indicate the direction of Z.2.2-NCP DNA movement during nucleosome gaping transition. H2B N-tail refers to H2B residues 26-32 which are exclusively observed in the canonical nucleosome structure. Histones H2B and DNA in Z.2.2-NCP are colored in warmpink and green, their counterparts in canonical nucleosome are colored in red and grey.

D SDS-PAGE analysis of SWR1 complex purified from S. cerevisiae.

E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.

Table 1. Cryo-EM data collection, refinement and validation statistics.
Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters (2022)
Figure 5

Figure 5: Antigen-induced micro-clusters (Ag-MC) localize to and move on the ridge network.

A. IgM-BCR cluster distribution upon Ag stimulation. Ramos B cells in the Matrigel were stimulated with 50 ng/ml NIP-15-BSA Ag for at least 15 minutes. Ag-MC on the B cell surface were segmented and overlaid with the ridge network (including microvilli) with a 1.25x and 3.0x local intensity cut-off for cluster detection. Scale bar = 5 µm.

B. IgM-BCR intensity enrichment within the segmented clusters at different surface features for unstimulated (US) and Ag-stimulated cells. Intensity enrichment was measured by dividing the average voxel intensity in the IgM-BCR clusters (µci) by the average voxel intensity of the non-clustered ("diffuse") IgM-BCR residing within the different surface feature masks (µdi). Data points and the error bars represent mean ± SD.

C, D. Sum projections of the skeletonized ridge network and microvilli (C) and segmented IgM-BCR clusters (D) in Ag-stimulated cells with a low and high staticity score (duration: 37.5 s). Scale bar = 5 µm.

E. Color coding of the segmented IgM-BCR clusters in (D) according to their assigned surface feature. Green clusters correspond to shallow invaginations (SI), yellow to ridges plus nodes (R + N), purple to microvilli (MV) and microvilli tips (MT).

F. Close-up of the encircled region in (E) displaying IgM-BCR localization, sum projections of the ridge network and segmented clusters. Ridge network lacking Ag-MC are not stabilized. Scale bar = 1 µm.

G, H. Staticity scores for different surface features (R+N, MV) (G) and for segmented IgM-BCR clusters assigned to different surface features (H) in unstimulated and Ag-stimulated cells. Violin plot solid lines represent the median and dotted lines represent the quartiles.

I. Time course images of an Ag-MC undergoing displacement on ridges. Scale bar = 1 µm.

Data information: n = 7 (30-time frames/cell) for unstimulated cells from 4 independent experiments and n = 8 (10 - 50-time frames/cell) for Ag-stimulated cells from 4 independent experiments. Two-tailed, unpaired student t-test was used in (B), (G), (H), except for R+N of (H), where non-parametric Mann-Whitney U test was used. p < 0.05 *, p < 0.01 **, p < 0.001 ***. Comparison in (B) between US and Ag for SI, p = 0.0006; at R, p = 0.0039; at N, p = 0.0107. Comparison in (G) between US and Ag at R+N, p = 0.003; at MV, p = 0.012. Comparison in (H) between US and Ag at SI, p = 0.001; R+N, p = 0.0006; at MV, p = 0.0299.
Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters (2022)
Figure 6

Figure 6: LatA-induced surface elevations anchor IgM-BCR.

A. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment. Scale bar = 5 µm.

B. Topographical features of the Ramos B cell surface in a cell treated with 2 µM LatA for 15 - 30 min. LatA-treated cells display disorganized and polarized elevations. The cell is morphologically separated into smooth (green) and elevated (yellow) regions. Scale bar = 5 µm.

C. Time frames from a LatA-treated cell in which stable elevations undergo merging. Scale bar = 1 µm.

D. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). Scale bars = 5 µm.

E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow. Scale bar = 1 µm.

F. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells.

G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x).

H. Staticity scores for segmented IgM-BCR clusters on smooth and elevated regions in LatA and LatA+Ag cells.

I. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region. Scale bar = 5 µm.

Data information: n = 6 (50-time frames/cell) for LatA cells from 3 independent experiments and n = 7 (50 time-frames/cell) for LatA+Ag cells from 2 independent experiments. Violin plot solid lines represent the median and dotted lines represent the quartiles in (F) and (H). Two-tailed, paired student t-test was used in (G) and two-tailed, unpaired student t-test was used in (F), (H). p < 0.05 *. Comparison in (F) between LatA and LatA+Ag, p = 0.0467. Comparison in (G) between smooth and elevated in LatA+Ag for 1x cut-off, p = 0.0260; for 3x cut-off, p = 0.0124. Comparison in (H) between smooth and elevated in LatA+Ag, p = 0.0143.

Expanded View Figure Legends
F-domain valency determines outcome of signaling through the angiopoietin pathway (2021)
Figure 2

Figure 2. F-domain valency determines level of activation of pAKT phosphorylation.

A-L) Serum starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F-domains for 15 minutes and analyzed by immunoblotting for pAKT (S473), pERK1/2 (T202/Y204), and pFAK (Y397) phosphorylation. A, E, I) pAKT, B, F, J) pERK1/2, and C, G, K) pFAK phosphorylation levels are quantified and normalized to Icos-cage signaling levels. Appendix Figure S1B shows the detection of two bands expected for pERK1/2. Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F-domain: D) helical bundles, H) trimeric and tetrameric homo-oligomers and tetrahedral nanocages, L) Icosahedral nanocages and trimeric subunit.

M-P) Dose-response curves, EC50, and EMAX for pAKT and pERK1/2 activation. The curves, EC50 and EMAX values are calculated using Prism, GraphPad. N, P) Darker red color indicate lower EC50 and higher EMAX values.

Q, R) Quantification (Q) and representative gel (R) for Tie2 knockdown using 20 nM of siRNA before starvation and F-domain scaffold at 18 nM of F-domain or PBS stimulation.

S, T) Quantification (S) and representative gel (T) for inhibition of Ang1-dependent pAKT signaling by Ang2, H3, Tet1-A, or Icos1-A; pAKT band intensities were normalized to Ang1-dependent pAKT level). Representative immunoblots for H3, Tet1-A, and Icos1-A competition experiments are in Appendix Figure S1M-O. Quantification of pERK1/2 and pFAK level for the competition assay is in Appendix Figure S1P-Q. Partial valency analysis of icosahedral nanocages is in Appendix Figure S1R-T.

Data information: Each point on all graphs represents a biological replicate, n > 3, bars represent the mean ± SEM. One-way ANOVA with Bonferroni test was used for multiple comparisons to calculate p-values. (****) indicates p<0.0001. p-values are also indicated in Appendix Table S2.
F-domain valency determines outcome of signaling through the angiopoietin pathway (2021)
Figure 3

Figure 3. Designed scaffolds fall into Ang1- or Ang2-like classes depending on valency.

A) Schematic representation of the in vitro scratch cell migration/ wound healing assay.

B) Icos1 at 18 nM F-domain stimulates cell migration relative to PBS/vehicle control. Scale bars are 100 µm.

C) Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 hours of treatment. The change in wound area is normalized to vehicle control. All experiments have at least 3 biological replicates. (*) is the comparison between F-domain scaffolds vs Ang1, and (#) is the comparison between F-domain scaffolds vs Vehicle. (####) indicated p<0.0001.D) Schematic of in vitro tube formation assay; F-domain scaffolds (designed scaffolds) were added at 90 nM F-domain and removed at 24h; vascular stability was analyzed at 48h and 72h timepoints.

E) Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) were quantified using angiogenesis analyzer plugin in ImageJ. Scale bar is100 µm.

F) Effect of F-domain scaffolds on the vascular stability at 48- and 72-hour timepoint were assessed by taking the average of the number of nodes, meshes, and tubes and normalized to vehicle as log2 fold change. All experiments have at least three biological replicates except for Ang2, Tet1, Tet2, akC4, and Icos2 at 72-hour timepoint in tube formation assay that have two biological replicates.

G) Designed F-domain scaffolds fall into Ang1- and Ang2-like classes. Color indicates highest (1) to lowest (0) pAKT, pERK1/2, and pFAK level which were column normalized. Cell migration level and vascular stability (48-hour timepoint) were also column normalized from highest to lowest.

Data information: In C and F, the bars represent the mean ± SEM. One-way ANOVA with Bonferroni test was used for multiple comparisons to calculate p-values. p-values are also indicated in Appendix Table S2.
F-domain valency determines outcome of signaling through the angiopoietin pathway (2021)
Figure 4

Figure 4. Tie2 super-agonist generates large Tie2 clusters and initiates cytoskeleton rearrangement.

Serum starved HUVECs were treated with angiopoietins or F-domain scaffolds at 100 nM of F-domain concentrations for 15 minutes before cell fixation and immunofluorescence stained to visualize FOXO1, Tie2, and α5 integrin.

A-C) Representative images of FOXO1 localization upon scaffolds administration. Scale bars are 10 µm

D) Quantifications of nuclear FOXO1 shown as percent of cells with nuclear FOXO1 stain, 100 cells were counted per sample.

E) Cells were stimulated with scaffolds or angiopoietins and stained with Tie2 antibodies for analyzing plasma membrane Tie2 clusters. Dark gray bars: Quantification shows the percent % of total cells that exhibit large Tie2 clusters on the plasma membrane.

F-G) H8 or H3 treated cells were analyzed under super-resolution microscopy shows actin protrusions with Tie2 clusters at the tip. Lower panels are Imaris reconstructed 3D model of the super-resolution images. Scale bars are 5 µm.

H) Quantification of percent of cells exhibited actin protrusions with Tie2 clusters at the tip. 100 cells were counted per sample.

I) Tie2-integrin colocalizing upon F-domain sheet stimulation at 18 nM of F-domains for 30 minutes before fixation and immunofluorescence stained for Tie2 and α5. Top right: schematic of F-domain sheet labeled with GFP. Imaris reconstructed super-resolution images of F-domain sheet attaching to cell, clustering Tie2, and inducing α5 integrin colocalization. Scale bars are 1 µm.

J) Quantifications of Tie2-integrin colocalization for cells that have F-domain sheet (GFP+) attachment.

K, L) Representatives immunoblot gel (K) and quantifications (L) for α5 integrin knockdown experiments using 20 nM of siRNA.

Data information: Each point on all graphs represents a biological replicate, n > 3, bars represent the mean ± SEM. One-way ANOVA with Bonferroni test was used for multiple comparisons to calculate p-values and t-test were used for H. (****) indicates p < 0.0001.