Relationships and Mendelian Randomization of Gut Microbe-Derived Metabolites with Metabolic Syndrome Traits in the METSIM Cohort.

The role of gut microbe-derived metabolites in the development of metabolic syndrome (MetS) remains unclear. This study aimed to evaluate the associations of gut microbe-derived metabolites and MetS traits in the cross-sectional Metabolic Syndrome In Men (METSIM) study. The sample included 10,194 randomly related men (age 57.65 ± 7.12 years) from Eastern Finland. Levels of 35 metabolites were tested for associations with 13 MetS traits using lasso and stepwise regression. Significant associations were observed between multiple MetS traits and 32 metabolites, three of which exhibited particularly robust associations. N-acetyltryptophan was positively associated with Homeostatic Model Assessment for Insulin Resistant (HOMA-IR) (ß = 0.02, p = 0.033), body mass index (BMI) (ß = 0.025, p = 1.3 × 10-16), low-density lipoprotein cholesterol (LDL-C) (ß = 0.034, p = 5.8 × 10-10), triglyceride (0.087, p = 1.3 × 10-16), systolic (ß = 0.012, p = 2.5 × 10-6) and diastolic blood pressure (ß = 0.011, p = 3.4 × 10-6). In addition, 3-(4-hydroxyphenyl) lactate yielded the strongest positive associations among all metabolites, for example, with HOMA-IR (ß = 0.23, p = 4.4 × 10-33), and BMI (ß = 0.097, p = 5.1 × 10-52). By comparison, 3-aminoisobutyrate was inversely associated with HOMA-IR (ß = -0.19, p = 3.8 × 10-51) and triglycerides (ß = -0.12, p = 5.9 × 10-36). Mendelian randomization analyses did not provide evidence that the observed associations with these three metabolites represented causal relationships. We identified significant associations between several gut microbiota-derived metabolites and MetS traits, consistent with the notion that gut microbes influence metabolic homeostasis, beyond traditional risk factors.

Genetic analysis of activity, brain and behavioral associations in extended families with heavy genetic loading for bipolar disorder.

BACKGROUND: Disturbed sleep and activity are prominent features of bipolar disorder type I (BP-I). However, the relationship of sleep and activity characteristics to brain structure and behavior in euthymic BP-I patients and their non-BP-I relatives is unknown. Additionally, underlying genetic relationships between these traits have not been investigated. METHODS: Relationships between sleep and activity phenotypes, assessed using actigraphy, with structural neuroimaging (brain) and cognitive and temperament (behavior) phenotypes were investigated in 558 euthymic individuals from multi-generational pedigrees including at least one member with BP-I. Genetic correlations between actigraphy-brain and actigraphy-behavior associations were assessed, and bivariate linkage analysis was conducted for trait pairs with evidence of shared genetic influences. RESULTS: More physical activity and longer awake time were significantly associated with increased brain volumes and cortical thickness, better performance on neurocognitive measures of long-term memory and executive function, and less extreme scores on measures of temperament (impulsivity, cyclothymia). These associations did not differ between BP-I patients and their non-BP-I relatives. For nine activity-brain or activity-behavior pairs there was evidence for shared genetic influence (genetic correlations); of these pairs, a suggestive bivariate quantitative trait locus on chromosome 7 for wake duration and verbal working memory was identified. CONCLUSIONS: Our findings indicate that increased physical activity and more adequate sleep are associated with increased brain size, better cognitive function and more stable temperament in BP-I patients and their non-BP-I relatives. Additionally, we found evidence for pleiotropy of several actigraphy-behavior and actigraphy-brain phenotypes, suggesting a shared genetic basis for these traits.

The Genetic Architecture of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

BACKGROUND & AIMS: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics. METHODS: Chronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen. RESULTS: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis. CONCLUSIONS: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.

Genome-wide mapping of brain phenotypes in extended pedigrees with strong genetic loading for bipolar disorder.

Bipolar disorder is a highly heritable illness, associated with alterations of brain structure. As such, identification of genes influencing inter-individual differences in brain morphology may help elucidate the underlying pathophysiology of bipolar disorder (BP). To identify quantitative trait loci (QTL) that contribute to phenotypic variance of brain structure, structural neuroimages were acquired from family members (n = 527) of extended pedigrees heavily loaded for bipolar disorder ascertained from genetically isolated populations in Latin America. Genome-wide linkage and association analysis were conducted on the subset of heritable brain traits that showed significant evidence of association with bipolar disorder (n = 24) to map QTL influencing regional measures of brain volume and cortical thickness. Two chromosomal regions showed significant evidence of linkage; a QTL on chromosome 1p influencing corpus callosum volume and a region on chromosome 7p linked to cortical volume. Association analysis within the two QTLs identified three SNPs correlated with the brain measures.

Contribution of common and rare variants to bipolar disorder susceptibility in extended pedigrees from population isolates.

Current evidence from case/control studies indicates that genetic risk for psychiatric disorders derives primarily from numerous common variants, each with a small phenotypic impact. The literature describing apparent segregation of bipolar disorder (BP) in numerous multigenerational pedigrees suggests that, in such families, large-effect inherited variants might play a greater role. To identify roles of rare and common variants on BP, we conducted genetic analyses in 26 Colombia and Costa Rica pedigrees ascertained for bipolar disorder 1 (BP1), the most severe and heritable form of BP. In these pedigrees, we performed microarray SNP genotyping of 838 individuals and high-coverage whole-genome sequencing of 449 individuals. We compared polygenic risk scores (PRS), estimated using the latest BP1 genome-wide association study (GWAS) summary statistics, between BP1 individuals and related controls. We also evaluated whether BP1 individuals had a higher burden of rare deleterious single-nucleotide variants (SNVs) and rare copy number variants (CNVs) in a set of genes related to BP1. We found that compared with unaffected relatives, BP1 individuals had higher PRS estimated from BP1 GWAS statistics (P = 0.001 ~ 0.007) and displayed modest increase in burdens of rare deleterious SNVs (P = 0.047) and rare CNVs (P = 0.002 ~ 0.033) in genes related to BP1. We did not observe rare variants segregating in the pedigrees. These results suggest that small-to-moderate effect rare and common variants are more likely to contribute to BP1 risk in these extended pedigrees than a few large-effect rare variants.

Whole blood transcriptome analysis in bipolar disorder reveals strong lithium effect.

BACKGROUND: Bipolar disorder (BD) is a highly heritable mood disorder with complex genetic architecture and poorly understood etiology. Previous transcriptomic BD studies have had inconsistent findings due to issues such as small sample sizes and difficulty in adequately accounting for confounders like medication use. METHODS: We performed a differential expression analysis in a well-characterized BD case-control sample (Nsubjects = 480) by RNA sequencing of whole blood. We further performed co-expression network analysis, functional enrichment, and cell type decomposition, and integrated differentially expressed genes with genetic risk. RESULTS: While we observed widespread differential gene expression patterns between affected and unaffected individuals, these effects were largely linked to lithium treatment at the time of blood draw (FDR < 0.05, Ngenes = 976) rather than BD diagnosis itself (FDR < 0.05, Ngenes = 6). These lithium-associated genes were enriched for cell signaling and immune response functional annotations, among others, and were associated with neutrophil cell-type proportions, which were elevated in lithium users. Neither genes with altered expression in cases nor in lithium users were enriched for BD, schizophrenia, and depression genetic risk based on information from genome-wide association studies, nor was gene expression associated with polygenic risk scores for BD. CONCLUSIONS: These findings suggest that BD is associated with minimal changes in whole blood gene expression independent of medication use but emphasize the importance of accounting for medication use and cell type heterogeneity in psychiatric transcriptomic studies. The results of this study add to mounting evidence of lithium's cell signaling and immune-related mechanisms.

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

Impact of Individual Traits, Saturated Fat, and Protein Source on the Gut Microbiome.

Interindividual variation in the composition of the human gut microbiome was examined in relation to demographic and anthropometric traits, and to changes in dietary saturated fat intake and protein source. One hundred nine healthy men and women aged 21 to 65, with BMIs of 18 to 36, were randomized, after a two-week baseline diet, to high (15% total energy [E])- or low (7%E)-saturated-fat groups and randomly received three diets (four weeks each) in which the protein source (25%E) was mainly red meat (beef, pork) (12%E), white meat (chicken, turkey) (12%E), and nonmeat sources (nuts, beans, soy) (16%E). Taxonomic characterization using 16S ribosomal DNA was performed on fecal samples collected at each diet completion. Interindividual differences in age, body fat (%), height, ethnicity, sex, and alpha diversity (Shannon) were all significant factors, and most samples clustered by participant in the PCoA ordination. The dietary interventions did not significantly alter the overall microbiome community in ordination space, but there was an effect on taxon abundance levels. Saturated fat had a greater effect than protein source on taxon differential abundance, but protein source had a significant effect once the fat influence was removed. Higher alpha diversity predicted lower beta diversity between the experimental and baseline diets, indicating greater resistance to change in people with higher microbiome diversity. Our results suggest that interindividual differences outweighed the influence of these specific dietary changes on the microbiome and that moderate changes in saturated fat level and protein source correspond to modest changes in the microbiome.IMPORTANCE The microbiome has proven to influence health and disease, but how combinations of external factors affect the microbiome is relatively unknown. Diet can cause changes, but this is usually achieved by altering macronutrient ratios and has not focused on dietary protein source or saturated fat intake levels. In addition, each individual's unique microbiome profile can be an important factor during studies, and it has even been shown to affect therapeutic outcomes. We show here that the effects of individual differences outweighed the effect of experimental diets and that protein source is less influential than saturated fat level. This suggests that fat and protein composition, separate from macronutrient ratio and carbohydrate composition, is an important consideration in dietary studies.

Identifying fenofibrate responsive CpG sites.

As part of GAW20, we analyzed the familiality and variability of methylation to identify cytosine-phosphate-guanine (CpG) sites responsive to treatment with fenofibrate. Methylation was measured at approximately 450,000 sites in pedigree members, prior to and after 3 weeks of treatment. Initially, we aimed to identify responsive sites by analyzing the pre- and posttreatment methylation changes within individuals, but these data exhibited a confounding treatment/batch effect. We applied an alternative indirect approach by searching for CpG sites whose methylation levels exhibit a genetic response to the drug. We reasoned that these sites would exhibit highly familial and variable methylation levels posttreatment, but not pretreatment. Using a 0.1% threshold, posttreatment sibling correlation (scor) and standard deviation (SD) distributions share 16 outliers, while the corresponding pretreatment distributions share none. Comparing the pre- and posttreatment CpG outliers, 36 (8%) of SD distributions, and 449/450 (nearly 100%) of scor distributions differ. Combined, these results identify methylation sites within the KIAA1804 and ANAPC2 genes. Each gene also has a highly significant methylation quantitative trait locus (meQTL) (KIAA1804: p < 1e-200; ANAPC2: p < 3e-248), indicating that methylation levels at these CpG sites are driven by meQTL and fenofibrate.

GAW20: methods and strategies for the new frontiers of epigenetics and pharmacogenomics.

GAW20 provided a platform for developing and evaluating statistical methods to analyze human lipid-related phenotypes, DNA methylation, and single-nucleotide markers in a study involving a pharmaceutical intervention. In this article, we present an overview of the data sets and the contributions analyzing these data. The data, donated by the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) investigators, included data from 188 families (N = 1105) which included genome-wide DNA methylation data before and after a 3-week treatment with fenofibrate, single-nucleotide polymorphisms, metabolic syndrome components before and after treatment, and a variety of covariates. The contributions from individual research groups were extensively discussed prior, during, and after the Workshop in groups based on discussion themes, before being submitted for publication.

Detecting responses to treatment with fenofibrate in pedigrees.

BACKGROUND: Fenofibrate (Fb) is a known treatment for elevated triglyceride (TG) levels. The Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study was designed to investigate potential contributors to the effects of Fb on TG levels. Here, we summarize the analyses of 8 papers whose authors had access to the GOLDN data and were grouped together because they pursued investigations into Fb treatment responses as part of GAW20. These papers report explorations of a variety of genetics, epigenetics, and study design questions. Data regarding treatment with 160 mg of micronized Fb per day for 3 weeks included pretreatment and posttreatment TG and methylation levels (ML) at approximately 450,000 epigenetic markers (cytosine-phosphate-guanine [CpG] sites). In addition, approximately 1 million single-nucleotide polymorphisms (SNPs) were genotyped or imputed in each of the study participants, drawn from 188 pedigrees. RESULTS: The analyses of a variety of subsets of the GOLDN data used a number of analytic approaches such as linear mixed models, a kernel score test, penalized regression, and artificial neural networks. CONCLUSIONS: Results indicate that (a) CpG ML are responsive to Fb; (b) CpG ML should be included in models predicting the TG level responses to Fb;

Author Correction: Integration of human adipocyte chromosomal interactions with adipose gene expression prioritizes obesity-related genes from GWAS.

In the original version of this Article, Supplementary Table 10 contained incorrect primer sequences for the mobility shift assay for SNP rs4776984. These errors have now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype.

Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.

Repurposing Dantrolene for Long-Term Combination Therapy to Potentiate Antisense-Mediated DMD Exon Skipping in the mdx Mouse.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Transcriptome analysis in whole blood reveals increased microbial diversity in schizophrenia.

The role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain: schizophrenia, amyotrophic lateral sclerosis, and bipolar disorder. By using high-quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity and schizophrenia.

A Strategy for Discovery of Endocrine Interactions with Application to Whole-Body Metabolism.

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions.

Integration of human adipocyte chromosomal interactions with adipose gene expression prioritizes obesity-related genes from GWAS.

Increased adiposity is a hallmark of obesity and overweight, which affect 2.2 billion people world-wide. Understanding the genetic and molecular mechanisms that underlie obesity-related phenotypes can help to improve treatment options and drug development. Here we perform promoter Capture Hi-C in human adipocytes to investigate interactions between gene promoters and distal elements as a transcription-regulating mechanism contributing to these phenotypes. We find that promoter-interacting elements in human adipocytes are enriched for adipose-related transcription factor motifs, such as PPARG and CEBPB, and contribute to heritability of cis-regulated gene expression. We further intersect these data with published genome-wide association studies for BMI and BMI-related metabolic traits to identify the genes that are under genetic cis regulation in human adipocytes via chromosomal interactions. This integrative genomics approach identifies four cis-eQTL-eGene relationships associated with BMI or obesity-related traits, including rs4776984 and MAP2K5, which we further confirm by EMSA, and highlights 38 additional candidate genes.

Diet1, bile acid diarrhea, and FGF15/19: mouse model and human genetic variants.

Diet1 modulates intestinal production of the hormone, fibroblast growth factor (FGF)15, which signals in liver to regulate bile acid synthesis. C57BL/6ByJ mice with a spontaneous Diet1-null mutation are resistant to hypercholesterolemia compared with wild-type C57BL/6J mice through enhanced cholesterol conversion to bile acids. To further characterize the role of Diet1 in metabolism, we generated Diet1-/- mice on the C57BL/6J genetic background. C57BL/6J Diet1-/- mice had elevated bile acid levels, reduced Fgf15 expression, and increased gastrointestinal motility and intestinal luminal water content, which are symptoms of bile acid diarrhea (BAD) in humans. Natural genetic variation in Diet1 mRNA expression levels across 76 inbred mouse strains correlated positively with Ffg15 mRNA and negatively with serum bile acid levels. This led us to investigate the role of DIET1 genetic variation in primary BAD patients. We identified a DIET1 coding variant (rs12256835) that had skewed prevalence between BAD cases and controls. This variant causes an H1721Q amino acid substitution that increases the levels of FGF19 protein secreted from cultured cells. We propose that genetic variation in DIET1 may be a determinant of FGF19 secretion levels, and may affect bile acid metabolism in both physiological and pathological conditions.

Genetic variation and gene expression across multiple tissues and developmental stages in a nonhuman primate.

By analyzing multitissue gene expression and genome-wide genetic variation data in samples from a vervet monkey pedigree, we generated a transcriptome resource and produced the first catalog of expression quantitative trait loci (eQTLs) in a nonhuman primate model. This catalog contains more genome-wide significant eQTLs per sample than comparable human resources and identifies sex- and age-related expression patterns. Findings include a master regulatory locus that likely has a role in immune function and a locus regulating hippocampal long noncoding RNAs (lncRNAs), whose expression correlates with hippocampal volume. This resource will facilitate genetic investigation of quantitative traits, including brain and behavioral phenotypes relevant to neuropsychiatric disorders.

Model-Based Linkage Analysis of a Binary Trait.

Linkage analysis is a statistical genetics method to localize disease and trait genes to specific chromosome regions. The analysis requires pedigrees with members who vary among each other in the trait of interest and who have been genotyped with known genetic markers. Linkage analysis tests whether any of the marker alleles cosegregate with the disease or trait within the pedigree. Evidence of cosegregation is then combined across the families. We describe here the background and methods to conduct a linkage analysis for a binary trait, such as a disease, when the model of the gene contributing to the trait can be formulated. There are a number of statistical genetics software packages that allow you conduct a model-based linkage analysis of a binary trait. We describe in great detail how to run one of the programs, the LODLINK program of the Statistical Analysis for Genetic Epidemiology (S.A.G.E.) package. We provide directions for making the four input files and information on how to access and interpret the output files. We then discuss more complex analyses that can be conducted. We discuss the MLOD program for multipoint linkage analysis, including its relation to LODLINK and the additional file needed. Notes to improve your ability to run the program are included.