Cellular Long-chain Fatty Acids Research Articles

Prevotella illustrans sp. nov., derived from human oropharyngeal abscess puncture fluid.

An obligately anaerobic strain, designated as A2931T, was isolated from oropharyngeal abscess puncture fluid of a patient sampled during routine care at a hospital and further characterized both phenotypically, biochemically and genotypically. This Gram-negative rod-shaped bacterium was moderately saccharolytic and proteolytic. Phylogenetic analyses of full-length 16S rRNA gene and whole-genome sequences revealed it to be best placed in the genus Prevotella, but to be only comparatively distantly related to recognized species, with the closest relationship to Prevotella baroniae (average nucleotide identity and digital DNA-DNA hybridization values both well below the generally accepted thresholds). Strain A2931T had a genomic DNA G+C content of 47.7 mol%. Its most abundant cellular long-chain fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. Taken together, this polyphasic data suggests strain A2931T to represent a novel species within the genus Prevotella, for which the name Prevotella illustrans sp. nov. is proposed. The type strain is A2931T (=DSM 108028T=CCOS 1232T=CCUG 72806T). Interestingly, we found strain A2931T to correspond to the oral taxon Prevotella HMT-820 in the Human Oral Microbiome Database, as supported by overall genome relatedness index analyses >99 %. Thus, our work not only closes one of the gaps of knowledge about hitherto unnamed species isolated from humans, but also will facilitate identification of this taxon both in the clinical microbiology context and in research alike.

Prevotella vespertina sp. nov., isolated from an abscess of a hospital patient.

A strain of an obligately anaerobic, Gram-stain-negative rod-shaped bacterium is described by phenotypical, biochemical and genotypical characterization. This strain A2879T was isolated from an abscess swab of a patient sampled during routine care at hospital. Phylogenetic analyses (full-length 16S rRNA gene and whole-genome sequence) revealed the strain to belong to the genus Prevotella, but to be distant from recognized species, with the closest relationship to Prevotella veroralis. Unambiguous identification also proved possible by MALDI-TOF MS. The genomic DNA G+C content was 41.5 mol%. Strain A2879T was moderately saccharolytic and proteolytic. The most abundant cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. In view of these data, strain A2879T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella vespertina sp. nov. is proposed. The type strain is A2879T (=DSM 108027T=CCOS 1233T=CCUG72808T). As this strain has been isolated from a clinical sample, it is considered relevant for human medicine and health in general, and in particular for the fields of clinical microbiology and infectious diseases. This description will enable routine and research laboratories alike to easily identify the novel taxon, allowing its role in the context of human health and disease or microbiota to be further elucidated.

AMPK facilitates intestinal long-chain fatty acid uptake by manipulating CD36 expression and translocation.

Cellular long-chain fatty acids' (LCFAs) uptake is a crucial physiological process that regulates cellular energy homeostasis. AMPK has been shown to modulate LCFAs uptake in several kinds of cells, but whether it exerts an impact on intestinal LCFAs uptake is not quite clear. In the current study, we found that AMPK reinforced LCFAs uptake in intestinal epithelial cells (IECs). Moreover, intestinal epithelium-specific AMPK deletion impaired intestinal LCFAs absorption and protected mice from high-fat diet-induced obesity. Mechanistically, we discovered that AMPK deletion reduced the CD36 protein level by upregulating Parkin-mediated polyubiquitination of CD36 in IECs. Furthermore, our results revealed that AMPK affected PARK2 (gene name of Parkin) mRNA stability in a YTHDF2-dependent manner through FTO-dependent demethylation of N6 -methyladenosine (m6 A). Besides, AMPK promoted the translocation of CD36 to the plasma membrane in IECs, but the inhibition of AKT signaling suppressed this effect, which also halted the accelerated fatty acid uptake induced by AMPK. These results suggest that AMPK facilitates the intestinal LCFAs uptake by upregulating CD36 protein abundance and promoting its membrane translocation simultaneously. Such findings shed light on the role of AMPK in the regulation of intestinal LCFAs uptake.

Hydrogeniiclostidium mannosilyticum gen. nov., sp. nov. isolated from human faeces.

A strain of obligately anaerobic, spore-forming, Gram-positive rods was isolated from child faeces and characterized both phenotypically and genotypically. Phylogenetic analysis based on 16S rRNA gene and whole genome sequencing revealed the strain to represent a member of the family Ruminococcaceae distant from described species and genera. The strain was moderately saccharolytic with mannose as the preferred substrate and produced lactic acid, acetic acid and H2 as the end products. The major cellular long-chain fatty acids were C16 : 0 and C16 : 0 aldehyde. The genomic DNA G+C content was 52.3 mol%. On the basis of chemotaxonomic and genomic properties it was concluded that the strain represents a novel species in a new genus within the family Ruminococcaceae, for which the name Hydrogeniiclostidium mannosilyticum gen. nov., sp. nov. is proposed. The type strain of Hydrogeniiclostidium mannosilyticum is ASD2818T (=VKM B-3268T=JCM 33295T).

Prevotella brunnea sp. nov., isolated from a wound of a patient.

A strain of an obligately anaerobic, Gram-stain-negative rod-shaped bacterium is described by phenotypical, biochemical and genotypical characterization. Strain A2672T was isolated from a wound of a patient sampled during routine care at hospital. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to belong to the genus Prevotella, but to be distant from known species, with the closest relationship to Prevotella corporis. The genomic DNA G+C content was 44.0 mol%. Strain A2672T was moderately saccharolytic and proteolytic. The most abundant cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. In view of these characteristics as well as whole-genome sequence analysis, strain A2672T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella brunnea sp. nov. is proposed. The type strain is A2672T (=DSM 108033T=CCOS 1231T=CCUG 72809T).

Prevotella rara sp. nov., isolated from human faeces.

A strain of obligately anaerobic, Gram-stain-negative rods was isolated from human faeces and characterized both phenotypically and genotypically. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed the strain to represent a member of the genus Prevotella, distant from the species with validly published names, with the closest relationship to Prevotella oryzae. The strain was moderately saccharolytic and proteolytic. The predominant menaquinones were MK-13 and MK-12. The major cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The genomic DNA G+C content was 45.7 mol%. On the basis of chemotaxonomic and genotypic properties, it was concluded that the strain represent a novel species within the genus Prevotella, for which the name Prevotellarara sp. nov. is proposed. The type strain of Prevotellarara is 109T (=VKM B-2992T=DSM 105141T).

Prevotella colorans sp. nov., isolated from a human wound.

A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).

Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting.

Description of Alloprevotella rava gen. nov., sp. nov., isolated from the human oral cavity, and reclassification of Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov.

Five strains of anaerobic, gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that these strains represented a novel group within the family Prevotellaceae, and the most closely related species was Prevotella tannerae. P. tannerae and the novel taxon are deeply branched from the genus Prevotella, with sequence identities to the type strain of the type species of Prevotella, Prevotella melaninogenica, of 82.2 and 85.6 %, respectively. The novel genus Alloprevotella gen. nov. is proposed to accommodate the novel species Alloprevotella rava gen. nov., sp. nov. and the previously named Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov. The type species is Alloprevotella tannerae. The type strain of Alloprevotella rava is 81/4-12(T) ( = DSM 22548(T) = CCUG 58091(T)) and the type strain of Alloprevotella tannerae is ATCC 51259(T) = CCUG 34292(T) = CIP 104476(T) = NCTC 13073(T). Alloprevotella rava is weakly to moderately saccharolytic and produces moderate amounts of acetic acid and major amounts of succinic acid as end products of fermentation. Strains are sensitive to 20 % bile and hydrolyse gelatin. The principal cellular long-chain fatty acids are anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C17 : 0 and iso-C17 : 0 3-OH. The G+C content of the DNA of the type strain is 47 mol%.

Prevotella fusca sp. nov. and Prevotella scopos sp. nov., isolated from the human oral cavity

Two strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to belong to two separate taxa. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that the strains were both related to, but distinct from, the type strain of Prevotella melaninogenica. Two novel species, Prevotella fusca sp. nov. and Prevotella scopos sp. nov., are proposed to accommodate these strains. Both strains were saccharolytic and produced acetic and succinic acids, with lesser amounts of lactic and isovaleric acids, as end products of fermentation, and both were sensitive to 20 % bile. The principal cellular long-chain fatty acids of both strains were ai-C(15 : 0), 3-OH i-C(17 : 0), 3-OH C(16 : 0), i-C(15 : 0) and C(16 : 0). The DNA G+C contents of the type strains of Prevotella fusca (W1435(T) = DSM 22504(T) = CCUG 57946(T)) and Prevotella scopos (W2052(T) = DSM 22613(T ) = CCUG 57945(T)) were 43 and 41 mol%, respectively. The two species could be differentiated by gelatin hydrolysis, cellobiose and ribose fermentation, and production of β-glucosidase.

Prevotella saccharolytica sp. nov., isolated from the human oral cavity.

Two strains of anaerobic, Gram-stain-negative bacilli isolated from the human oral cavity (D033B-12-2(T) and D080A-01) were subjected to a comprehensive range of phenotypic and genotypic tests and were found to be distinct from any previously described species. 16S rRNA gene sequence analysis revealed that the strains were related most closely to the type strain of Prevotella marshii (93.5 % sequence identity). The novel strains were saccharolytic and produced acetic acid and succinic acid as end products of fermentation. The principal cellular long-chain fatty acids were C₁₆ :₀), iso-C₁₄:₀, C₁₄:₀, anteiso-C₁₅:₀, iso-C₁₆ :₀ and C₁₆:₀) 3-OH. The G+C content of the DNA of strain D033B-12-2(T) was 44 mol%. Strains D033B-12-2(T) and D080A-01 are considered to represent a single novel species of the genus Prevotella, for which the name Prevotella saccharolytica sp. nov. is proposed. The type strain is D033B-12-2(T) (=DSM 22473(T) =CCUG 57944(T)).

AMPK-mediated increase in myocardial long-chain fatty acid uptake critically depends on sarcolemmal CD36

CD36, also named fatty acid translocase, has been identified as a putative membrane transporter for long-chain fatty acids (LCFA). In the heart, contraction-induced 5′ AMP-activated protein kinase (AMPK) signaling regulates cellular LCFA uptake through translocation of CD36 and possibly of other LCFA transporters from intracellular storage compartments to the sarcolemma. In this study, isolated cardiomyocytes from CD36 +/+- and CD36 −/− mice were used to investigate to what extent basal and AMPK-mediated LCFA uptake are CD36-dependent. Basal LCFA uptake was not altered in CD36 −/− cardiomyocytes, most likely resulting from a (1.8-fold) compensatory upregulation of fatty acid-transport protein-1. The stimulatory effect of contraction-mimetic stimuli, oligomycin (2.5-fold) and dipyridamole (1.6-fold), on LCFA uptake into CD36 +/+ cardiomyocytes was almost completely lost in CD36 −/− cardiomyocytes, despite that AMPK signaling was fully intact. CD36 is almost entirely responsible for AMPK-mediated stimulation of LCFA uptake in cardiomyocytes, indicating a pivotal role for CD36 in mediating changes in cardiac LCFA fluxes.

New concepts of cellular fatty acid uptake: role of fatty acid transport proteins and of caveolae.

Efficient uptake and channelling of long-chain fatty acids (LCFA) are critical cell functions. Evidence is emerging that proteins are important mediators of LCFA-trafficking into cells and various proteins have been suggested to be involved in this process. Amongst these proteins is a family of membrane-associated proteins termed fatty acid transport proteins (FATP). So far six members of this family, designated FATP 1-6, have been characterized. FATP 1, 2 and 6 show a highly-conserved AMP-binding region that participates in the activation of very-long-chain fatty acids (VLCFA) to form their acyl-CoA derivatives. The mechanisms by which FATP mediate LCFA uptake are not well understood, but several studies provide evidence that uptake of LCFA across cellular membranes is closely linked to acyl-CoA synthetase activity. It is proposed that FATP indirectly enhance LCFA uptake by activating VLCFA to their CoA esters, which are required to maintain the typical structure of lipid rafts in cellular membranes. Recent work has shown that the structural integrity of lipid rafts is essential for cellular LCFA uptake. This effect might be exerted by proteins, e.g. caveolin-1 and FAT/CD36, that use lipid rafts as platforms and bind or transport LCFA. The proposed molecular mechanisms await further experimental investigation.

Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations

Because of the importance of long-chain fatty acids (LCFAs) as a myocardial energy substrate, myocardial LCFA metabolism has been of particular interest for the understanding of cardiac pathophysiology. Recently, by using radiolabeled LCFA analogues, myocardial LCFA metabolism has been clinically evaluated, which revealed a total defect of myocardial LCFA accumulation in a small number of subjects. The mechanism for the cellular LCFA uptake process is still disputable, but recent results suggest that fatty acid translocase (FAT)/CD36 is a transporter in the heart. In the present study, we analyzed mutations and protein expression of the FAT/CD36 gene in 47 patients who showed total lack of the accumulation of a radiolabeled LCFA analogue in the heart. All the patients carried two mutations in the FAT/CD36 gene, and expression of the FAT/CD36 protein was not detected on either platelet or monocyte membranes. Our results showed the link between mutations of the FAT/CD36 gene and a defect in the accumulation of LCFAs in the human heart.—Tanaka, T., T. Nakata, T. Oka, T. Ogawa, F. Okamoto, Y. Kusaka, K. Sohmiya, K. Shimamoto, and K. Itakura. Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations. J. Lipid Res. 2001. 42: 751–759.

Reliable identification of Prevotella and Butyrivibrio spp. from rumen by fatty acid methyl ester profiles.

Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera, Prevotella and Butyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzed Prevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The strains of Prevotella could be well identified on species level by the characteristic ratios among major fatty acids and by acids unique fatty for each species. The 45 Butyrivibrio strains were grouped into 4 major and 2 minor groups according to FAMEs profiles. The major fatty acids for the bulk of the Butyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups corresponded to those based on 16S rDNA sequences.

Dysgonomonas gen. nov. to accommodate Dysgonomonas gadei sp. nov., an organism isolated from a human gall bladder, and Dysgonomonas capnocytophagoides (formerly CDC group DF-3).

Results of a polyphasic taxonomic study on an unknown Gram-negative, facultatively anaerobic, coccobacillus-shaped organism isolated from an infected human gall bladder are presented. Phenotypic and molecular taxonomic studies revealed the organism to be close to, but distinct from, organisms designated CDC (Centers for Disease Control and Prevention) group DF-3. The unknown bacterium was readily distinguished from reference strains of Bacteroides, Prevotella, Porphyromonas and related taxa by 16S rRNA gene sequencing, biochemical tests, analysis of cellular long-chain fatty acids and electrophoretic analysis of whole-cell proteins. Based on the results of the present study, it is proposed that the unknown bacterium be classified in a new genus, Dysgonomonas, as Dysgonomonas gadei sp. nov. (type strain CCUG 42882T = CIP 106420T). In addition, a new species, Dysgonomonas capnocytophagoides sp. nov., is proposed to accommodate strains previously belonging to CDC group DF-3. The type species of the genus Dysgonomonas is Dysgonomonas gadei.

The Distribution of the ω3- and ω6-Series of Cellular Long-chain Fatty Acids in Fungi

Summary Stable long-chain fatty acid profiles were produced when 245 fungi (some associated with disease) were analysed for fatty acid composition during late growth or stationary growth phases. Fungi of the Protoctista studied i.e. Allomyces were all characterized by the presence of the omega 6 (ω6) series of fatty acids and include C18 and C20 polyunsaturated fatty acids (PUFA's). The disease associated zygomycetous fungi studied, also produced the ω6 series of fatty acids, although most representatives were characterized by the presence of ω6 C18 PUFA's. Disease associated fungi belonging to the Deuteromycotina did not produce the ω6 series of PUFA's. Some were characterized by 18:3 omega 3 (ω3) and others by the production of fatty acids up to 18:2. Some yeasts did not produce ω3- or ω6 series of PUFA's. These results further confirm the general pattern observed in fungi where the “ancestral” protoctistan fungi are characterized by the presence of the ω6 series of C18 and C20 PUFA's, the zygomycetous fungi by the presence of mainly ω6 C18 PUFA's and the Dikaryomycota by the presence of ω3 and not ω6 PUFA's. Consequently, this phenotypic characteristic may also have value in discriminating between higher fungal taxa.

Yeast Eicosanoids I. The Distribution and Taxonomic Value of Cellular Fatty Acids and Arachidonic Acid Metabolites in the Dipodascaceae and Related Taxa

Summary The cellular long-chain fatty acids of 34 strains representing the genera Clavispora, Dipodascopsis, Dipodascus, Geotrichum, Lodderomyces and Schwanniomyces were extracted and analyzed as methyl esters by gas chromatography. The fatty acid composition and coenzyme Q systems of these yeasts and others from literature were compared in a coordinated scheme and their taxonomic positions assessed. In contrast to other families of the Endomycetales, the hyphomycete-like genera of the Dipodascaeae showed a remarkable degree of homogeneity regarding fatty acid composition and coenzyme Q system. The hyphomycete-like genera of the Dipodascaceae could be distinguished from the superficially similar Dipodascopsis by the absence of linolenic acid and their ability to grow in the presence of 1mM aspirin. Both species representing Dipodascopsis were able to produce aspirin sensitive 3-hydroxy arachidonic acid (3-HETE) from arachidonic acid while species representing Dipodascus, Galactomyces and Geotrichum were unable to do so. These criteria correlate well with known taxonomic criteria which differentiate between the taxa concerned.

Differentiation between Yeast Species, and Strains within a Species, by Cellular Fatty Acid Analysis 5. A Feasible Technique?

Summary The cellular long-chain fatty acids (FA's) of 58 strains representing 16 yeast species were examined by capillary gas chromatography (GC) on a polar column. Many of the yeast strains selected for examination in this study did not grow, or grew very poorly, under the cultivation parameters applied. This indicated that all yeasts cannot be cultivated under a single set of rigidly standardised cultivation parameters. The variation between relative percentages (RP's) of C18:1,C18:2, and C18:3 in replicate cultivations of the same strain was often unacceptably high. In most cases this variation was reduced to acceptable levels upon recultivation of the organism. This implicated the batch culture process as possible source of the variation. The yeast cellular acid profile (CFAP) is clearly more complex than recognised previously. Some yeasts contained at least four dienoic and two trienoic FA's, whilst other species contained three delta-11 monoenoic FA's. The presence of C17 : 1(8) in all yeasts studied was confirmed by interpretation of picolinyl ester-mass spectra. Minor FA's were again proved to be extremely important when attempting to differentiate between yeast species by means of cellular fatty acid analysis (CFAA). Although data were limited, it seems likely that CFAA will be able to differentiate between a number of Kluyveromyces spp. Torulaspora spp. had unique CFAP's amongst the species studied here. Integration of FA-data from this paper with data from our previous papers indicated some overlap between the CFAP's of Saccharomyces spp. and Kluyveromyces spp. These facts indicate that CFAA is not a generally applicable yeast identification technique. It is, however, an easily applied, reliable method to characterise yeast strains in a variety of species.

Differentiation between Yeast Species, and Strains within a Species, by Cellular Fatty Acid Analysis: 4. Saccharomyces sensu stricto, Hanseniaspora, Saccharomycodes and Wickerhamiella

The cellular long-chain fatty acids (FA’s) of 88 strains representing 12 yeast species were examined by gas Chromatographic analysis on a polar capillary column. Mass spectral evidence for the presence of C16 : 2(9, 12), C17 : 2(8, 11), C17 : 2(9, 12), and C16 : 3(9, 12, 15) in the cellular fatty acid profile (CFAP) of Saccharomyces kluyveri is presented, the latter acid being only the second trienoic FA to be positively identified in yeasts. The mean relative percentages (MRP’s) of 15 fatty acids were used to differentiate between strains and species. Data were compared by visual examination, analysis of variance (ANOVA), calculation of similarity coefficients (So) and stepwise discriminant analysis (SDA). ANOVA failed to differentiate between many strains in various Hanseniaspora spp but So-values indicated that no two strains had exactly equivalent CFAP’s. Saccharomyces kluyveri has a CFAP unique amongst the species of Saccharomyces, but the CFAP’s of Sacch. bay anus, Sacch. cerevisiae, and Sacch. pastorianus do not allow differentiation between these three species. As a group the latter three species clearly have CFAP’s different to all other yeasts included in the study. The CFAP’s of the Hanseniaspora spp differed from all other species studied as did the profiles of Saccharomycodes ludwigii and Wickerhamiella domercqiae. The CFAP’s of the different Hanseniaspora spp were very similar and so precluded differentiation between these species.