Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Conformational differences between linear alpha (2----8)-linked homosialooligosaccharides and the epitope of the group B meningococcal polysaccharide

The alpha-(2----8)-linked sialic acid oligosaccharides (NeuAc)n exhibit an unusual degree of heterogeneity in the conformation of their linkages. This was diagnosed by observation in their 13C NMR spectra of an equivalent and unique heterogeneity in the chemical shifts of their anomeric carbons and subsequently confirmed by more comprehensive 1H and 13C NMR studies. In these studies both one-dimensional and two-dimensional experiments were carried out on the trisaccharide (NeuAc)3 and colominic acid. In addition to the unambiguous assignment of the signals in the spectra, these experiments demonstrated that both linkages of (NeuAc)3 differed in conformation from each other and from the inner linkages of colominic acid. The NMR data indicate that these conformational differences extend to both terminal disaccharides of oligosaccharides larger than (NeuAc)5, a result that has considerable physical and biological significance. In the context of the group B meningococcal polysaccharide, it provides an explanation for the conformational epitope of the group B meningococcal polysaccharide, which was proposed on the evidence that (NeuAc)10, larger than the optimum size of an antibody site, was the smallest oligosaccharide able to bind to group B polysaccharide specific antibodies. Because the two terminal disaccharides of (NeuAc)10 differ in conformation to its inner residues, the immunologically functional part of (NeuAc)10 resides in its inner six residues. This number of residues is now consistent with the maximum size of an antibody site.     

Electrospray ionization mass spectrometry of oligosaccharides derived from fucoidan of Ascophyllum nodosum

Algal fucoidan is a complex sulfated polysaccharide whose structural characterization requires powerful spectroscopic methodologies. While most of the structural investigations reported so far have been performed using NMR as the main spectroscopic method, we report herein data obtained by negative electrospray ionization mass spectrometry. MS analysis has been carried out on oligosaccharides obtained by partial hydrolysis of fucoidan from the brown algae Ascophyllum nodosum. Oligosaccharide mixtures were fractionated by size exclusion chromatography, which allowed the analysis of oligomers ranging from monosaccharide to pentasaccharide. Monosaccharides were detected as monosulfated as well as disulfated forms. Besides, part of the oligosaccharides exhibited a high content of sulfate, evidencing that fucoidan contains disulfated fucosyl units. Fragmentation experiments yielded characteristic fragment ions indicating that the fucose units are mainly 2-O-sulfated. This study demonstrates that highly sulfated oligosaccharides from fucoidan can be analyzed by ESIMS which gives additional information about the structure of this highly complex polysaccharide.     

Structural elucidation of the nonclassical secondary cell wall polysaccharide from Bacillus cereus ATCC 10987. Comparison with the polysaccharides from Bacillus anthracis and B. cereus type strain ATCC 14579 reveals both unique and common structural features

Nonclassical secondary cell wall polysaccharides constitute a major cell wall structure in the Bacillus cereus group of bacteria. The structure of the secondary cell wall polysaccharide from Bacillus cereus ATCC 10987, a strain that is closely related to Bacillus anthracis, was determined. This polysaccharide was released from the cell wall with aqueous hydrogen fluoride (HF) and purified by gel filtration chromatography. The purified polysaccharide, HF-PS, was characterized by glycosyl composition and linkage analyses, mass spectrometry, and one- and two-dimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a -->6)-alpha-GalNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1--> trisaccharide that is substituted with beta-Gal at O3 of the alpha-GalNAc residue and nonstoichiometrically acetylated at O3 of the N-acetylmannosamine (ManNAc) residue. Comparison of this structure with that of the B. anthracis HF-PS and with structural data obtained for the HF-PS from B. cereus type strain ATCC 14579 revealed that each HF-PS had the same general structural theme consisting of three HexNAc and one Hex residues. A common structural feature in the HF-PSs from B. cereus ATCC 10987 and B. anthracis was the presence of a repeating unit consisting of a HexNAc(3) trisaccharide backbone in which two of the three HexNAc residues are GlcNAc and ManNAc and the third can be either GlcNAc or GalNAc. The implications of these results with regard to the possible functions of the HF-PSs are discussed.     

Synthesis of hexa- to tridecasaccharides related to Shigella dysenteriae type 1 for incorporation in experimental vaccines

Hexa- to tridecasaccharides corresponding to the O-specific polysaccharide (O-SP) of the Gram-negative bacterium Shigella dysenteriae type 1 were synthesized in solution phase. The syntheses utilized tetra-, octa-, and dodecasaccharide intermediates that represent one to three contiguous tetrasaccharide repeating units of the O-SP [Synlett2003, 743]. These compounds were glycosylated with mono-, di-, and trisaccharide trichloroacetamidates, which were synthesized in this study. The excellent stereodirecting effect of 4,6-O-benzophenone ketals in glycosylation reactions of 2-azido-2-deoxy-glucopyranosyl donors was demonstrated. The free oligosaccharides were characterized by 1H and 13C NMR spectroscopy and by high-resolution mass spectrometry. The oligosaccharides described herein contain the 5-(methoxycarbonyl)pentyl aglycon for eventual attachment to immunogenic carriers using a recently published protocol [J. Org. Chem.2005, 70, 6987].     

Structural investigation of chondroitin/dermatan sulfate oligosaccharides from human skin fibroblast decorin

Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide, Delta-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group.     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.     

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.     

An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus

We have developed an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. Purified group B streptococcal type III capsular polysaccharide was depolymerized by enzymatic digestion using endo-beta-galactosidase produced by Citrobacter freundii. Following enzymatic digestion, oligosaccharides were fractionated by gel filtration chromatography on Sephadex G-75. An oligosaccharide pool of average Mr = 14,500 (corresponding to 13.6 repeating units of the type III polysaccharide) was used for conjugation to tetanus toxoid. Tetanus toxoid was covalently coupled via a synthetic spacer molecule to the reducing end of the oligosaccharide by reductive amination. The oligosaccharide-tetanus toxoid conjugate elicited type III-specific anticapsular antibodies (measured in enzyme-linked immunosorbent assay) in three out of three rabbits whereas the unconjugated native type III polysaccharide was nonimmunogenic. Antiserum from rabbits vaccinated with the oligosaccharide-protein conjugate protected mice against lethal challenge with live group B streptococci (16 out of 16 mice survived) and opsonized group B streptococci for phagocytosis in vitro. No protection was conferred by preimmune serum nor by serum from rabbits vaccinated with unconjugated native type III polysaccharide. An oligosaccharide-protein conjugate vaccine of this design may prove to be an effective immunogen for protection against group B streptococcal infection in humans. In addition, the approach to vaccine design utilized in these studies will facilitate further definition of the structural parameters that determine immune response to glycoconjugate vaccines.     

Multiantennary group-specific polysaccharide of group B Streptococcus

The group-specific antigen of group B Streptococcus is composed of four different oligosaccharide units of Mw 766 (III), 1277 (II), 1462 (IV), and 1788 (I). The major constituent sugars of the oligosaccharides are alpha-L-rhamnopyranose, alpha-D-galactopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, and D-glucitol except that III does not contain alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues and IV contains no D-glucitol but has one additional beta-L-rhamnopyranosyl residue. The structures of II and III have been previously elucidated [Michon, F., Katzenellenbogen, E., Kasper, D. L., & Jennings, H. J. (1987) Biochemistry 26, 476-486]. In the group B antigen all the oligosaccharides are linked by one type of phosphodiester bond from O6 of the D-glucitol residue of one oligosaccharide to O6 of the alpha-D-galactopyranosyl residue of the next to form a complex and highly branched multiantennary structure. However, despite the heterogeneous nature of its component oligosaccharides, some order has been identified in the biosynthesis of the group B antigen from chemical and enzymatic sequence studies. Because III lacks an alpha-D-galactopyranosyl residue but has a D-glucitol residue, it is situated at the reducing terminus of all the branches of the group B antigen where it is always adjacent to a II moiety. Conversely, IV has an alpha-D-galactopyranosyl residue but has no D-glucitol and is therefore located at the reducing terminus of the group B antigen where it probably functions as a linker molecule between the group B polysaccharide and the cell wall peptidoglycan of the group B streptococcal organisms. Oligosaccharide I contains two alpha-D-galactopyranosyl residues and one D-glucitol residue and thus constitutes the branch point in the group B antigen, whereas II contains one of each of the above residues and therefore is situated in linear interchain positions. The group B antigen is highly branched and probably has a unique multiantennary structure.     

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.     

Structural analysis of the O-glycosidically linked core-region oligosaccharides of human meconium glycoproteins which express oncofoetal antigens

Glycoproteins were extracted from meconium samples of group O neonates of secretor type by pronase digestion followed by precipitation in 67% aqueous ethanol and separated into Ii antigen enriched and depleted fractions by affinity chromatography. The latter fraction strongly expressed the oncofoetal antigens recognised by natural antibodies in mouse sera and the hybridoma antibody FC 10.2, and this activity was enhanced after mild acid hydrolysis to remove sialic acid and fucose residues. Oligosaccharides were released from the mild-acid-treated fraction by base-borohydride degradation and purified by gel permeation chromatography on Bio-Gel P4 and high performance liquid chromatography on octadecylsilyl and aminopropylsilyl columns. The major oligosaccharides were characterised by fast atom bombardment and electron impact mass spectrometry, combined gas-liquid chromatography/mass spectrometry and 500-MHz proton NMR spectroscopy. Their structures, in order of abundance, were: (Formula: see text).     

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic (d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, ¹H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens

The structural domains of the Shigella flexneri variant Y O-antigen epitopes 3,4 have defied definition, despite knowledge of the structure of the linear polysaccharide chain of the LPS molecule. The dual epitope designation of group antigen 3,4 is based on absorption data using polyvalent rabbit antisera. Five monoclonal antibodies specific for the Y antigen, generated after immunization of BALB/c mice or LOU/C rats, were selected on the basis of ELISA by using well-characterized S. flexneri Y LPS and chemically defined glycoconjugates. Chemically defined LPS from all S. flexneri serogroups, synthetic oligosaccharides, and saccharides obtained by phage Sf6-mediated hydrolysis of the O-polysaccharide were used either as free haptens or glycoconjugates in Farr assays and ELISA titrations. Two different patterns of antibody specificities were seen: two monoclonal antibodies had combining sites recognizing the terminal nonreducing end of the O-polysaccharide complementary to the tetrasaccharide repeating unit; and three antibodies bound to intrachain determinants and had larger combining sites, possibly accommodating at least an octasaccharide. The precise specificity of these two general types of antibodies indicate that variant Y polysaccharide generates more than two O-factors.     

Structure of the oligosaccharide portion of human hepatitis B surface antigen

The hepatitis B surface antigen, which constitutes the currently available vaccine, is the empty envelope of the hepatitis B virus. We investigated the carbohydrate structures of the envelope glycoproteins. The intact oligosaccharides were enzymatically released from the coat glycoproteins using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and isolated by gel permeation chromatography. Cesium ion liquid secondary ion mass spectra of the intact, underivatized oligosaccharides showed molecular weights of 1932, 2078, and 2223. The mixture included partially and totally sialylated structures, a fraction (approximately 8%) of which were substituted with a single terminal fucose residue; no desialylated oligosaccharides were detected. The reducing termini of the oligomers were derivatized by reduction of the Schiff base formed using p-aminobenzoic acid ethyl ester, and fragmentation patterns identical to those produced from standard biantennary complex oligosaccharides were obtained. Methylation linkage analysis of the oligosaccharides showed that the carbohydrate composition and the mannose branching patterns also resembled those of a biantennary oligosaccharide. The results of this study indicate that glycosylation of the hepatitis B surface antigen, which takes place in the liver, is typical of other serum glycoproteins made in the liver; and this analytical strategy, including cesium ion liquid secondary ion mass spectrometry, is an effective approach for the structural analysis of complex carbohydrates available in only the 1-10 micrograms sample size range.     

Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.     

Oligosaccharides from feces of preterm infants fed on breast milk

Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.     

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.