Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis.

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.

Protein N-glycosylation in the bronchoalveolar space differs between never-smokers and long-term smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n = 5) and long-term smokers (LTS) with (LTS+, n = 4) and without COPD (LTS-, n = 8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

Identification of Novel Glycans in the Mucus Layer of Shark and Skate Skin.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage. While the mucus layers of various bony fish species have been investigated, the composition and glycan profiles of shark skin mucus remain relatively unexplored. In this pilot study, we aimed to explore the structure and composition of shark skin mucus through histological analysis and glycan profiling. Histological examination of skin samples from Atlantic spiny dogfish (Squalus acanthias) sharks and chain catsharks (Scyliorhinus retifer) revealed distinct mucin-producing cells and a mucus layer, indicating the presence of a functional mucus layer similar to bony fish mucus albeit thinner. Glycan profiling using liquid chromatography-electrospray ionization tandem mass spectrometry unveiled a diverse repertoire of mostly O-glycans in the mucus of the two sharks as well as little skate (Leucoraja erinacea). Elasmobranch glycans differ significantly from bony fish, especially in being more sulfated, and some bear resemblance to human glycans, such as gastric mucin O-glycans and H blood group-type glycans. This study contributes to the concept of shark skin having unique properties and provides a foundation for further research into the functional roles and potential biomedical implications of shark skin mucus glycans.

Mucin O-glycosylation and pathogen binding ability differ between rainbow trout epithelial sites.

Mucins are highly glycosylated proteins that make up the mucus covering internal and external surfaces of fish. Mucin O-glycans regulate pathogen quorum sensing, growth, virulence and attachment to the host. Knowledge on this mucosal defense system can enable alternative treatments to diseases posing a threat to productivity and welfare in aquaculture. Here, we characterize the rainbow trout (Oncorhynchus mykiss) gill, skin, pyloric ceca and distal intestinal mucin O-glycosylation and compare it to known teleost O-glycomes. We identified 54 O-glycans, consisting of up to nine monosaccharide residues. Skin glycans were most acidic, shortest on average and consisted mainly of NeuAcα2-6GalNAc. Glycans from the gills were less acidic with predominantly core 1 and 2 glycans, whereas glycans from pyloric ceca and distal intestine expressed an increased number of core 5 glycans, distinctly decorated with NeuAcα2-8NeuAc- like epitopes. When compared to Atlantic salmon and Arctic charr, trends on the core distribution, average size and overall acidity remained similar, although the epitopes varied. Rainbow trout mucins from gill and intestine bound A. salmonicida and A. hydrophila more efficiently than skin mucins. This is in line with a model where skin mucins with small glycans limit bacterial adhesion to the fish surface whereas the complex intestinal mucin glycans aid in trapping and removing pathogens from the epithelial surface.

Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1α: Interplay between O-linked glycosylation and inflammatory cytokines.

The primary aim of the study was to identify inflammatory markers relevant for osteoarthritis (OA)-related systemic (plasma) and local (synovial fluid, SF) inflammation. From this, we looked for inflammatory markers that coincided with the increased amount of O-linked Tn antigen (GalNAcα1-Ser/Thr) glycan on SF lubricin. Inflammatory markers in plasma and SF in OA patients and controls were measured using a 44-multiplex immunoassay. We found consistently 29 markers detected in both plasma and SF. The difference in their concentration and the low correlation when comparing SF and plasma suggests an independent inflammatory environment in the two biofluids. Only plasma MCP-4 and TARC increased in our patient cohort compared to control plasma. To address the second task, we concluded that plasma markers were irrelevant for a direct connection with SF glycosylation. Hence, we correlated the SF-inflammatory marker concentrations with the level of altered glycosylation of SF-lubricin. We found that the level of SF-IL-8 and SF-MIP-1α and SF-VEGFA in OA patients displayed a positive correlation with the altered lubricin glycosylation. Furthermore, when exposing fibroblast-like synoviocytes from both controls and OA patients to glycovariants of recombinant lubricin, the secretion of IL-8 and MIP-1α and VEGFA were elevated using lubricin with Tn antigens, while lubricin with sialylated and nonsialylated T antigens had less or no measurable effect. These data suggest that truncated glycans of lubricin, as found in OA, promote synovial proinflammatory cytokine production and exacerbate local synovial inflammation.

Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors.

Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.

The O-Glycome of Human Nigrostriatal Tissue and Its Alteration in Parkinson's Disease.

O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurodegenerative conditions such as Parkinson's disease (PD) and incidental Lewy body disease (ILBD). This work outlines optimizations of a microwave-assisted nonreductive release to limit glycan degradation and employs this methodology to analyze O-glycosylation on the human striatum and substantia nigra tissue in PD, ILBD, and healthy controls, working alongside well-established reductive release approaches. A total of 70 O-glycans were identified, with ILBD presenting significantly decreased levels of mannose-core (p = 0.017) and glucuronylated structures (p = 0.039) in the striatum and PD presenting an increase in sialylation (p < 0.001) and a decrease in sulfation (p = 0.001). Significant increases in sialylation (p = 0.038) in PD were also observed in the substantia nigra. This is the first study to profile the whole nigrostriatal O-glycome in healthy, PD, and ILBD tissues, outlining disease biomarkers alongside benefits of employing orthogonal techniques for O-glycan analysis.

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

Cathepsin g Degrades Both Glycosylated and Unglycosylated Regions of Lubricin, a Synovial Mucin.

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff base in gels, and with proteomics. Full length lubricin (∼300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain (∼250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in all domains of lubricin, including the mucin domain. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the pathological decrease of the lubrication in degenerative joint disease.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.

Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan patterns reveal a regiospecific distribution.

The mouse intestinal mucus is mainly made up by the gel-forming Muc2 mucin and the stomach surface mucus Muc5ac, both extensively O-glycosylated. The oligosaccharide diversity provides a vast library of potential recognition sites for both commensal and pathogenic organisms. The mucin glycans are thus likely very important for the selection and maintenance of a stable intestinal flora. Here we have explored the O-glycan patterns of the mouse gastrointestinal tract mucins. The mucins from the mucus of the distal and proximal colon, ileum, jejunum, duodenum, and stomach of conventionally raised wild-type (C57BL/6) mice were separated by composite gel electrophoresis. The O-linked glycans were released by reductive elimination and structurally characterized by liquid chromatography-mass spectrometry. The mucins glycans were mostly core 2 type [Galß1-3(GlcNAcß1-6)GalNAcol], but also core 1 (Galß1-3GalNAcol). In the stomach about half of the Muc5ac mucin O-glycans were neutral and many monosulfated, but with a low grade of sialylation and fucosylation. Mouse ileum, jejunum, and duodenum had similar glycan patterns dominated by sialylated and sulfated core 2 glycans, but few fucosylated. Colon was on the other hand dominated by highly charged fucosylated glycans. The distal colon is different from the proximal colon because different biosynthetic pathways are utilized, although sialylated and sulfated glycans were highly abundant in both parts. The sulfation was higher in the distal colon, whereas sialic acid was more common in the proximal colon. Many fucosylated glycans were found in both the proximal and distal colon. Thus the mucin O-glycans vary along the mouse gastrointestinal tract.

Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2.

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt(-/-)) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1(-/-)) mice. The Muc2 O-glycans were released by reductive ß-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt(-/-) knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1(-/-) mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species.

Proteomic study of the mucin granulae in an intestinal goblet cell model.

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.

Composition and functional role of the mucus layers in the intestine.

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

Identification and quantification of mucin expression.

The major phenotype of CF is the accumulation of mucus, a phenomenon whose relation to the dysfunctional CFTR is still not fully understood. This means that studies of mucus and its main component, the mucins, are important. Due to the large size and high glycosylation level, such questions need special considerations and methodology. We describe methods for the general quantification of heavily glycosylated proteins as the mucins using dot/slot blot. We also describe the separation of the mucins by gel electrophoresis and the identification with specific antibodies on Western blot and by proteomics.

Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 ß1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Enhanced detection of sialylated and sulfated glycans with negative ion mode nanoliquid chromatography/mass spectrometry at high pH.

Negative ion mode nanoliquid chromatography/mass spectrometry (nano-LC/MS) on porous graphitic carbon columns at pH 11 was studied and compared to capillary LC/MS at pH 8 for the analysis of neutral and acidic glycan alditols. Oligosaccharides were chromatographed with an acetonitrile gradient containing 0.04% ammonium hydroxide and analyzed with a linear ion trap mass spectrometer (LTQ) equipped with a modified nanospray interface. Analysis of acidic N- and O-glycan standards revealed that good quality MS/MS spectra could be obtained when loading 1-3 fmol, a 10-fold increase in sensitivity compared to capillary-LC/MS at pH 8. Analysis of a complex mixture of O-glycans from porcine colonic mucins with nano-LC/MS and MS/MS at high pH revealed 170 oligosaccharides in one analysis, predominantly corresponding to sulfated glycans with up to 11 residues. Analysis of the same sample with capillary-LC/MS showed a lower sensitivity for multiply sulfated glycans. Nano-LC/MS of O-linked oligosaccharides on MUC2 from a human colon biopsy also illustrated that the ionization of oligosaccharides with multiple sialic acid groups was increased compared to those with only one sialic acid residue. Nano-LC/MS at high pH is, thus, a highly sensitive approach for the analysis of acidic oligosaccharides.