The pleiotropic approach to coronavirus disease-19 pathogenesis: The impact of liver diseases associated host genetic variants.

Coronavirus disease-2019 (COVID-19) is a novel multisystemic viral disease caused pandemic. The disease impact involves liver and associated systems. Undoubtedly, host genetic background influences the predisposition and prediction of infection. Variants among human populations might increase susceptibility or protect against severe outcomes. In this manner, rs738409 variant of patatin-like phospholipase domain-containing protein 3 gene appears to be protective in some populations in spite of its aggravating effect on non-alcoholic fatty liver diseases (NAFLDs) and steatohepatitis. DRB1*15:01 allele of human leukocyte antigen is associated with protective effect in European and Japanese populations. DRB1*03:01 contrarily increases the susceptibility of severe COVID-19 infection in European populations. rs1260326 in glucokinase regulatory protein gene, rs112875651 in tribbles homolog 1 gene, rs429358 in apolipoprotein 1, and rs58542926 in transmembrane 6 superfamily 2 alleles are found related with NAFLD and obesity; thus, hypercoagulability and severe COVID-19 outcomes. In chronic or acute liver diseases, comorbid syndromes are the key factors to explain increased severity. There might not be a direct association between the variant and severe COVID-19 infection. As it is concluded, there are genes and variants known and unknown yet to be studied to reveal the association with disease severity.

Changing trends in the etiology of liver transplantation in Turkiye: A multicenter study.

Background and Aim: This study aimed to identify the indications for liver transplantation (LT) based on underlying etiology and to characterize the patients who underwent LT. Materials and Methods: We conducted a multicenter cross-sectional observational study across 11 tertiary centers in Turkiye from 2010 to 2020. The study included 5,080 adult patients. Results: The mean age of patients was 50.3±15.2 years, with a predominance of female patients (70%). Chronic viral hepatitis (46%) was the leading etiological factor, with Hepatitis B virus infection at 35%, followed by cryptogenic cirrhosis (24%), Hepatitis C virus infection (8%), and alcohol-related liver disease (ALD) (6%). Post-2015, there was a significant increase in both the number of liver transplants and the proportion of living donor liver transplants (p<0.001). A comparative analysis of patient characteristics before and after 2015 showed a significant decline in viral hepatitis-related LT (p<0.001), whereas fatty liver disease-related LT significantly increased (p<0.001). Conclusion: Chronic viral hepatitis continues to be the primary indication for LT in Turkiye. However, the proportions of non-alcoholic fatty liver disease (NAFLD) and ALD-related LT have seen an upward trend over the years.

Chronotype preference, sleep quality, and night-eating behaviors in patients with metabolic dysfunction-associated steatotic liver disease: Assessing the relationship with disease severity and fibrosis.

Background and Aim: Our primary objective is to examine the variance in chronotype, night-eating patterns, and sleep quality in patients with biopsy-proven metabolic dysfunction-associated steatotic liver disease. In addition, we aim to establish a correlation between these variables and the severity of the disease and fibrosis. Materials and Methods: Patients who were following up with biopsy-proven metabolic dysfunction associated steatotic liver disease (MASLD) were included in the study. Histologically severe disease is characterized by a Steatosis, Activity, and Fibrosis activity score of ≥3 or the presence of advanced fibrosis (≥F3). Participants who met the inclusion criteria were given the Morningness and Evening Questionnaire (MEQ), the Pittsburgh Sleep Quality Index, and the Night Eating Questionnaire to complete. Results: A total of 93 patients were included in this study. According to the MEQ, 48 patients were morning type (51.6%), and 42 (45.2%) were neither type. Sleep quality was determined to be inferior in the non-morningness group (p=0.002). A significantly higher proportion of patients with nocturnal eating syndrome had a non-morningness chronotype preference (n=22, 23.7%), compared to those with a morningness chronotype (n=9, 9.7%) (p=0.001). In the multivariate analysis, both age and poor sleep quality had significant impacts on advanced fibrosis, with odds ratios of 1.11 and 3.81, respectively. Conclusion: Despite the non-morningness chronotype demonstrating poorer sleep quality and a higher prevalence of night-eating behavior, our findings revealed no statistically significant differences in terms of sleep quality, nocturnal eating habits, or chronotype preferences among patients with varying degrees of MASLD severity. On the other hand, advanced fibrosis was significantly impacted by poor sleep quality.

Analysis of microbiota profile and nutritional status in male professional football players.

BACKGROUND: The interest in the effect of gut microbiota on athlete health has increased in recent years. Available data indicate a relationship between gut microbiota composition and physical activity, suggesting that changes in the microbiota may contribute to the host's physical performance. Studies show that leaky gut syndrome is highly correlated with upper respiratory infections and gastrointestinal disorders in endurance sports. This study aims to reveal the relationship between microbiota profiles, and the nutritional status of football players who perform endurance exercises. METHODS: Twenty male professional football players playing in one of the Turkish Football Federation Second League clubs participated in the study. Fecal samples were collected and stored at -86 °C, and the fecal microbiota was analyzed through 16s rRNA gene sequencing. The body composition of the football players was measured using a bioelectrical impedance analyzer. In addition, the 3-day food intake of the participants was recorded with the help of a dietitian. RESULTS: In the microbiota of football players, four phyla, 10 genera, and four species with densities above 1% were found. Body fat percentage was observed to be negatively correlated with the species of Faecalibacterium prausnitzii and Bacteroides vulgatus and the genus of Faecalibacterium (P<0.05). Considering the nutritional status, the fat intake was found to be positively correlated with Actinobacteria and Blautia coccoides; energy and fiber intake with Prevotella and Prevotella copri (P<0.05). In addition, there was a negative correlation between carbohydrate intake and Faecalibacterium (P<0.05). CONCLUSIONS: Our study is the first to reveal the microbiota profile of professional Turkish football players. It was found that football players' nutritional status and anthropometric measurements of are significantly related to phylum, genus and species ranks in the microbiota. These results support the bidirectional interaction between microbiota and sports. The relationship between microbiota and sports health/performance is thought to be further clarified with future studies.

SARS-CoV-2 vaccination and risk of severe COVID-19 outcomes in patients with autoimmune hepatitis.

BACKGROUND: Data regarding outcome of Coronavirus disease 2019 (COVID-19) in vaccinated patients with autoimmune hepatitis (AIH) are lacking. We evaluated the outcome of COVID-19 in AIH patients who received at least one dose of Pfizer- BioNTech (BNT162b2), Moderna (mRNA-1273) or AstraZeneca (ChAdOx1-S) vaccine. PATIENTS AND METHODS: We performed a retrospective study on AIH patients with COVID-19. The outcomes of AIH patients who had acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection after at least one dose of COVID-19 vaccine were compared to unvaccinated patients with AIH. COVID-19 outcome was classified according to clinical state during the disease course as: (i) no hospitalization, (ii) hospitalization without oxygen supplementation, (iii) hospitalization with oxygen supplementation by nasal cannula or mask, (iv) intensive care unit (ICU) admission with non-invasive mechanical ventilation, (v) ICU admission with invasive mechanical ventilation or (vi) death, and data was analyzed using ordinal logistic regression. RESULTS: We included 413 (258 unvaccinated and 155 vaccinated) patients (81%, female) with a median age of 52 (range: 17-85) years at COVID-19 diagnosis. The rates of hospitalization were (36.4% vs. 14.2%), need for any supplemental oxygen (29.5% vs. 9%) and mortality (7% vs. 0.6%) in unvaccinated and vaccinated AIH patients with COVID-19. Having received at least one dose of SARS-CoV-2 vaccine was associated with a significantly lower risk of worse COVID-19 severity, after adjusting for age, sex, comorbidities and presence of cirrhosis (adjusted odds ratio [aOR] 0.18, 95% confidence interval [CI], 0.10-0.31). Overall, vaccination against SARS-CoV-2 was associated with a significantly lower risk of mortality from COVID-19 (aOR 0.20, 95% CI 0.11-0.35). CONCLUSIONS: SARS-CoV-2 vaccination significantly reduced the risk of COVID-19 severity and mortality in patients with AIH.

Effects of immunosuppressive drugs on COVID-19 severity in patients with autoimmune hepatitis.

BACKGROUND: We investigated associations between baseline use of immunosuppressive drugs and severity of Coronavirus Disease 2019 (COVID-19) in autoimmune hepatitis (AIH). PATIENTS AND METHODS: Data of AIH patients with laboratory confirmed COVID-19 were retrospectively collected from 15 countries. The outcomes of AIH patients who were on immunosuppression at the time of COVID-19 were compared to patients who were not on AIH medication. The clinical courses of COVID-19 were classified as (i)-no hospitalization, (ii)-hospitalization without oxygen supplementation, (iii)-hospitalization with oxygen supplementation by nasal cannula or mask, (iv)-intensive care unit (ICU) admission with non-invasive mechanical ventilation, (v)-ICU admission with invasive mechanical ventilation or (vi)-death and analysed using ordinal logistic regression. RESULTS: We included 254 AIH patients (79.5%, female) with a median age of 50 (range, 17-85) years. At the onset of COVID-19, 234 patients (92.1%) were on treatment with glucocorticoids (n = 156), thiopurines (n = 151), mycophenolate mofetil (n = 22) or tacrolimus (n = 16), alone or in combinations. Overall, 94 (37%) patients were hospitalized and 18 (7.1%) patients died. Use of systemic glucocorticoids (adjusted odds ratio [aOR] 4.73, 95% CI 1.12-25.89) and thiopurines (aOR 4.78, 95% CI 1.33-23.50) for AIH was associated with worse COVID-19 severity, after adjusting for age-sex, comorbidities and presence of cirrhosis. Baseline treatment with mycophenolate mofetil (aOR 3.56, 95% CI 0.76-20.56) and tacrolimus (aOR 4.09, 95% CI 0.69-27.00) were also associated with more severe COVID-19 courses in a smaller subset of treated patients. CONCLUSION: Baseline treatment with systemic glucocorticoids or thiopurines prior to the onset of COVID-19 was significantly associated with COVID-19 severity in patients with AIH.

Accuracy of Fibrosis-4 index and non-alcoholic fatty liver disease fibrosis scores in metabolic (dysfunction) associated fatty liver disease according to body mass index: failure in the prediction of advanced fibrosis in lean and morbidly obese individuals.

BACKGROUND AND AIM: While non-invasive scores are increasingly being used to screen for advanced fibrosis in metabolic (dysfunction) associated fatty liver disease (MAFLD), the effect of BMI on their clinical utility remains uncertain. This study assessed the usefulness of the Fibrosis-4 index (FIB-4) and the non-alcoholic fatty liver disease fibrosis score (NFS) in lean, overweight, obese, severely obese, and morbidly obese patients with biopsy-proven MAFLD. METHODS: A total of 560 patients (28 lean, 174 overweight, 229 obese, 89 severely obese, 40 morbidly obese) were included. Diagnostic performances and optimal cut-off values for FIB-4 and NFS were calculated using receiver operating characteristic (ROC) curve analysis. RESULTS: In both lean and morbidly obese patients with MAFLD, both FIB-4 and NFS failed to discriminate advanced fibrosis. Conversely, both scores showed acceptable diagnostic performances in exclusion of advanced fibrosis in overweight, obese, and severely obese patients. FIB-4 was able to exclude advanced fibrosis with the highest diagnostic accuracy in the subgroup of overweight patients (area under the ROC curve: 0.829, 95% confidence interval: 0.738-0.919). CONCLUSION: FIB-4 and NFS can confidently be used to exclude advanced fibrosis in overweight, obese, and severely obese patients. However, they do not appear clinically useful in lean and morbidly obese patients.

Outcome of COVID-19 in Patients With Autoimmune Hepatitis: An International Multicenter Study.

BACKGROUND AND AIMS: Data regarding outcome of COVID-19 in patients with autoimmune hepatitis (AIH) are lacking. APPROACH AND RESULTS: We performed a retrospective study on patients with AIH and COVID-19 from 34 centers in Europe and the Americas. We analyzed factors associated with severe COVID-19 outcomes, defined as the need for mechanical ventilation, intensive care admission, and/or death. The outcomes of patients with AIH were compared to a propensity score-matched cohort of patients without AIH but with chronic liver diseases (CLD) and COVID-19. The frequency and clinical significance of new-onset liver injury (alanine aminotransferase > 2 × the upper limit of normal) during COVID-19 was also evaluated. We included 110 patients with AIH (80% female) with a median age of 49 (range, 18-85) years at COVID-19 diagnosis. New-onset liver injury was observed in 37.1% (33/89) of the patients. Use of antivirals was associated with liver injury (P = 0.041; OR, 3.36; 95% CI, 1.05-10.78), while continued immunosuppression during COVID-19 was associated with a lower rate of liver injury (P = 0.009; OR, 0.26; 95% CI, 0.09-0.71). The rates of severe COVID-19 (15.5% versus 20.2%, P = 0.231) and all-cause mortality (10% versus 11.5%, P = 0.852) were not different between AIH and non-AIH CLD. Cirrhosis was an independent predictor of severe COVID-19 in patients with AIH (P < 0.001; OR, 17.46; 95% CI, 4.22-72.13). Continuation of immunosuppression or presence of liver injury during COVID-19 was not associated with severe COVID-19. CONCLUSIONS: This international, multicenter study reveals that patients with AIH were not at risk for worse outcomes with COVID-19 than other causes of CLD. Cirrhosis was the strongest predictor for severe COVID-19 in patients with AIH. Maintenance of immunosuppression during COVID-19 was not associated with increased risk for severe COVID-19 but did lower the risk for new-onset liver injury during COVID-19.

The relationship between polyphenols and miRNAs: A novel therapeutic strategy for metabolic associated fatty liver disease.

Metabolic-associated fatty liver disease (MAFLD) is a public health problem that is increasingly recognized, currently affecting up to a quarter of the world's adult population. Although a biopsy is the current gold standard to diagnose MAFLD, there are potentially serious complications, making it inadequate. Thus far, noninvasive methods have not been able to determine the stage and the subtype of MAFLD. The development and prognosis of MAFLD are modulated by epigenetic factors, including microRNAs (miRNAs), which may be potential biomarkers for MAFLD. Polyphenols, found in many fruits and vegetables, may be useful, as they alter gene expression with epigenetic factors, such as miRNAs. This review presents an overview of the relationship between polyphenols and miRNAs in MAFLD. The literature suggests that miRNAs could be used as a diagnostic method for MAFLD, especially miRNA-122 and miRNA-34a. However, though it has been demonstrated that polyphenols may contribute to improving MAFLD, to our knowledge, no study to date has shown the relationship between polyphenols and miRNAs in MAFLD. The exact mechanisms of polyphenols on miRNAs in MAFLD remain unclear. Future studies may provide hope for diet therapy for MAFLD patients as well as the development of polyphenol-related foods or drugs that target miRNAs to treat MAFLD.

Extrahepatic autoimmune diseases in primary biliary cholangitis: Prevalence and significance for clinical presentation and disease outcome.

BACKGROUND AND AIM: The prevalence and clinical significance of extrahepatic autoimmune diseases (EHAIDs) have not been evaluated in a large cohort of primary biliary cholangitis (PBC). METHODS: The medical records of 1554 patients with PBC from 20 international centers were retrospectively reviewed. Development of decompensated cirrhosis (ascites, variceal bleeding, and/or hepatic encephalopathy) and hepatocellular carcinoma were considered clinical endpoints. RESULTS: A total of 35 different EHAIDs were diagnosed in 440 (28.3%) patients with PBC. Patients with EHAIDs were more often female (92.5% vs 86.1%, P < 0.001) and seropositive for anti-mitochondrial antibodies (88% vs 84%, P = 0.05) and antinuclear antibodies and/or smooth muscle antibodies (53.8% vs 43.6%, P = 0.005). At presentation, patients with EHAIDs had significantly lower levels of alkaline phosphatase (1.76 vs 1.98 × upper limit of normal [ULN], P = 0.006), aspartate aminotransferase (1.29 vs 1.50 × ULN, P < 0.001), and total bilirubin (0.53 vs 0.58 × ULN, P = 0.002). Patients with EHAIDs and without EHAIDs had similar rates of GLOBE high-risk status (12.3% vs 16.1%, P = 0.07) and Paris II response (71.4% vs 69.4%, P = 0.59). Overall, event-free survival was not different in patients with and without EHAIDs (90.8% vs 90.7%, P = 0.53, log rank). Coexistence of each autoimmune thyroid diseases (10.6%), Sjögren disease (8.3%), systemic sclerosis (2.9%), rheumatoid arthritis (2.7%), systemic lupus erythematosus (1.7%), celiac disease (1.7%), psoriasis (1.5%), and inflammatory bowel diseases (1.3%) did not influence the outcome. CONCLUSIONS: Our study confirms that EHAIDs are frequently diagnosed in patients with PBC. The presence of EHAIDs may influence the clinical phenotype of PBC at presentation but has no impact on PBC outcome.

Effect of carbon dioxide versus room air insufflation on post-colonoscopic pain: A prospective, randomized, controlled study.

BACKGROUND/AIMS: Room air (RA) and carbon dioxide (CO2) are widely used to insufflate the colon to examine the mucosa in colonoscopy. Pain, discomfort, and bloating can be seen during and after colonoscopy secondary to bowel distention. This study aimed to investigate the effect of CO2 on post-procedure pain sensation (PPPS) in comparison with RA. MATERIALS AND METHODS: Patients were randomly assigned to the RA and CO2 insufflation groups in a 1:1 ratio. The visual analog scale (VAS) was used to measure the pain before and after the colonoscopy. VAS score of 0 was accepted as the absence of pain and above 0 was accepted as the presence of pain. The primary outcome was to investigate the effect of CO2 insufflation on PPPS. Secondary outcomes were to investigate the other contributing factors affecting PPPS and the effect of CO2 on PPPS in patients with inflammatory bowel disease (IBD). RESULTS: A total of 204 patients were enrolled in the study. No significant difference in PPPS was seen between the 2 groups at any point in time after the colonoscopy. Furthermore, there was no significant difference in pain sensation between the CO2 and RA groups in patients with IBD. When we investigated the other contributing factors to pain sensation, body-mass index (BMI) was found to be significant at 30 minutes and BMI and colonoscopy time were found to be significant at 6 hours afterwards. CONCLUSION: We found no favorable effect of CO2 insufflation on PPPS in colonoscopy, including in patients with IBD.

Evaluation of the Association between Programmed Cell Death-1 Gene Polymorphisms and Hepatocellular Carcinoma Susceptibility in Turkish Subjects. A Pilot Study.

BACKGROUND AND AIMS: Programmed cell death-1 (PD-1) has a vital role in regulating T-cell function, and immune escape mechanism of cancer cells. It was shown that there could be a relationship between single nucleotide polymorphisms (SNPs) in the PD-1 gene and susceptibility to hepatocellular carcinoma (HCC) based on various studies. We aimed to investigate the role of three SNPs within the PD-1 gene in susceptibility to HCC in the Turkish population. METHODS: Single nucleotide polymorphisms of PD-1.1, 1.5, and 1.6 were genotyped by using TaqMan Allelic Discrimination Assays in blood samples of 137 HCC and 136 control subjects, matched for age and gender. The genotype, allele and haplotype frequencies were compared in HCC and control groups using logistic regression analysis. RESULTS: Genotype distributions of PD-1.1, PD-1.5 and PD-1.6 SNPs were in Hardy-Weinberg equilibrium. No significant difference was observed in the genotype distribution of PD-1.1, PD-1.5 and PD-1.6 polymorphisms among gender and age-matched HCC (M/F: 96/41; mean age: 61.4 ±11.7 years) and control group (M/F: 94/42; mean age: 61.4±10.1). In the haplotype analysis of PD-1.1/PD-1.5/PD-1.6, no significant difference was found among HCC and control group adjusted for sex and age (all p values>0.1). CONCLUSION: Our findings, firstly reporting the association of PD-1.5 polymorphism with HCC, and PD-1.1 and PD-1.6 with HCC in the Turkish population, suggest that PD-1 polymorphisms are not predisposing factors for HCC development. Future studies with larger sample sizes and different ethnic populations are required to validate our findings.

The utility of noninvasive scores in non-alcoholic fatty liver disease patients with normal and elevated serum transaminases.

Background and Aim: Noninvasive scores are developed for the estimation of advanced fibrosis, including parameters in addition to transaminases in non-alcoholic fatty liver disease (NAFLD). In this study, we aimed to investigate the diagnostic performances of Fibrosis-4 (FIB-4) and NAFLD Fibrosis Score (NFS) in the estimation of advanced fibrosis comparing patients with normal and elevated transaminases. Material and Methods: We retrospectively analyzed the prospectively collected data of a total of 407 consecutive patients with biopsy-proven NAFLD. FIB-4 scores of <1.3 and >2.67 or <1.45 and >3.25 indicated a low and high risk for advanced fibrosis, respectively. NFS scores of <-1.455 and >0.676 were used to assess low and high risk for advanced fibrosis, respectiv. Results: FIB-4 cutoffs of <1.3 and <1.45 for low risk of advanced fibrosis had a sensitivity of 70% and 54% in patients with elevated transaminases and 70% and 52% in patients with normal transaminases, respectively. The specificities for the cutoffs of >2.67 and >3.25 were 97% and 98% in patients with elevated transaminases and 99% and 100% in patients with normal transaminases, respectively. Concerning NFS, we found similar results. Conclusion: FIB-4 and NFS showed acceptable diagnostic performance in the exclusion of advanced fibrosis in both populations with normal and elevated transaminases.

The diagnostic utility of fibrosis-4 or nonalcoholic fatty liver disease fibrosis score combined with liver stiffness measurement by fibroscan in assessment of advanced liver fibrosis: a biopsy-proven nonalcoholic fatty liver disease study.

BACKGROUND/AIM: The clinical guidelines recommend the use of nonalcoholic fatty liver disease fibrosis score and fibrosis-4 score for estimating the advanced liver fibrosis in nonalcoholic fatty liver disease. However, these scores are used confidently in eliminating advanced fibrosis, rather than detecting it. Therefore, paired combination with liver stiffness measurement by transient elastography is recommended. In this study, we aimed to validate this combined algorithm in our study population. METHODS: A total of 139 consecutive biopsy-proven nonalcoholic fatty liver disease patients were enrolled in the study. We calculated the noninvasive scores and performed liver stiffness measurement examination for each patient. RESULTS: The optimal cutoff of liver stiffness measurement for advanced fibrosis was 11.0 kPa (area under curve: 0.856) with a sensitivity of 84% and a specificity of 78%. Using the fibrosis-4 score (< 1.45 for low risk of advanced fibrosis and > 3.25 for high risk of advanced fibrosis) in combination with the liver stiffness measurement cutoffs revealed the best diagnostic performance (< 8.8 kPa for low risk of advanced fibrosis and > 10.9 kPa for high risk of advanced fibrosis). This paired combination had the positive predictive value of 0.735 at a sensitivity of 89% and the negative predictive value of 0.932 at a specificity of 82%. CONCLUSION: A paired combination of the fibrosis-4 score and liver stiffness measurement (< 8.8 kPa for exclusion of advanced fibrosis and > 10.9 kPa for inclusion of advanced fibrosis) is able to diagnose the patients with advanced fibrosis with the highest diagnostic accuracy.

Potential clinical variants detected in mitochondrial DNA D-loop hypervariable region I of patients with non-alcoholic steatohepatitis.

PURPOSE: Non-alcoholic steatohepatitis (NASH) is a mitochondrial disease. However, the underlying role of mitochondrial genetics has not yet been completely elucidated. Evaluation of D-loop nucleotide variations with respect to statistical significance and clinical data distribution. METHODS: Genomic DNAs were extracted from the peripheral blood samples of patients with biopsy-proven 150 NASH as well as from 150 healthy individuals to explore the functional D-loop region responsible for the replication and transcription of the mitochondrial genome. DNA sequencing by capillary electrophoresis analysis was performed for the D-loop region of mitochondrial DNA containing the hypervariable region I, and restriction fragment length polymorphism with MnlI analysis was performed for the m.16189 T/C D-loop variant. RESULTS: The m.A16318C variant was detected only in patients with NASH and approached significance level. Based on clinical data, six variants associated with histological subgroups of NASH and NASH-complicated diseases were identified. In patients with NASH, the m.16129 AA genotype was associated with advanced-stage fibrosis; the m.16249 CC genotype was associated with advanced lobular inflammation and advanced-stage histological steatosis; the m.16296 TT genotype was associated with hypothyroidism; the m.16163 GG and m.16294 TT genotypes were associated with metabolic syndrome; and the m.16256 TT+CT genotypes were associated with type II diabetes. In patients with NASH, microRNAs were estimated by targeting the significant variants identified in this study. CONCLUSION: These findings suggest that NASH may be associated with D-loop nucleotide variations and that microRNA-based in vitro and/or in vivo studies may be developed by targeting the D-loop variants.