Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

First record of Pseudoterranova decipiens in sprat (Sprattus sprattus) from the Baltic Sea.

A dispersion of Anisakidae nematodes (particularly Contracaecum osculatum) among marine organisms in the Baltic Sea has been reported over the last decade. This is in line with an increase in the number of grey seal that act as final host for Contracaecum osculatum and Pseudoterranova sp., and are thus indispensable for the completion of their life cycles. Most attention has been paid to zoonotic nematode species, like Pseudoterranova sp., which have been noted in commercially important fish in the area. Little is known about the spread and transmission of Pseudoterranova sp. in the Baltic Sea. The aim of this study was to investigate whether sprat may play a role as a transport host for this Anisakidae. Samples were collected in three areas of the southern Baltic Sea (south and east of Bornholm, Slupsk Farrow and the Gulf of Gdansk) during a research cruise in August 2019. A visual inspection of the viscera of 556 sprats was conducted. Parasites were identified using anatomomorphological and molecular methods. Nematodes were recorded only in sprat caught southeast of Bornholm (prevalence 2.7%; intensity of infection 1-4; abundance 0.05). Molecular identification revealed the presence of Pseudoterranova decipiens. This is the first report of P. decipiens in sprat from the Baltic Sea. Sprat is likely a transmitter of P. decipiens in the Baltic Sea food web.

Drug efficacy on zoonotic nematodes of the Anisakidae family: new metabolic data.

In the Anisakidae family, there are nematodes, most of which are parasitic for important commercial fish species. Both public health risks and socio-economic problems are attributed to these parasites. Despite these concerns, knowledge of the metabolism of these parasites remains unknown. Therefore, the main objective of this study was to investigate the receptors of drugs and oxidative metabolic status of two Anisakidae species, Pseudoterranova decipiens (s. s.) and Contracaecum osculatum (s. s.), under the influence of anthelminthic drugs, ivermectin (IVM) and pyrantel (PYR), at different concentrations: 1.56, 3.125 and 6.25 µg mL−1 of culture medium for 3, 6, 9, 12 and 72 h. The mRNA expressions of the γ-aminobutyric acid receptor, acetylcholine receptor subunits, adenosine triphosphate-binding cassette transporters and antioxidative enzymes were determined. The total antioxidant capacity and glutathione S-transferase activity were also examined. To the best of the authors' knowledge, this is the first time that IVM and PYR have been tested against these parasitic nematodes.

Investigating the phenotypic and genetic associations between personality traits and suicidal behavior across major mental health diagnoses.

Personality traits influence risk for suicidal behavior. We examined phenotype- and genotype-level associations between the Big Five personality traits and suicidal ideation and attempt in major depressive, bipolar and schizoaffective disorder, and schizophrenia patients (N = 3012) using fixed- and random-effects inverse variance-weighted meta-analyses. Suicidal ideations were more likely to be reported by patients with higher neuroticism and lower extraversion phenotypic scores, but showed no significant association with polygenic load for these personality traits. Our findings provide new insights into the association between personality and suicidal behavior across mental illnesses and suggest that the genetic component of personality traits is unlikely to have strong causal effects on suicidal behavior.

In situ evidence of the role of Crangon crangon in infection of cod Gadus morhua with nematode parasite Hysterothylacium aduncum in the Baltic Sea.

Cod was one of the most important fish species in the Baltic Sea, but its condition is deteriorating for several reasons, including an increasing parasite burden. The aim of this study was to determine the source of infection of Baltic cod with parasites by examination of invertebrates found in situ in the cod stomach. A total of 1681 cod were sampled during four research cruises in the southern Baltic Sea in 2012, 2013 and 2014 and the composition of their diet was analysed. Each prey item from cod stomach was identified to the lowest possible taxonomic level and a parasitological analysis of all invertebrates collected was performed. Crangon crangon, Saduria entomon and Mysis mixta were the most commonly represented invertebrates among food items. Hysterothylacium aduncum was found only in C. crangon. This host­parasite system is reported here for the first time in situ in the stomach of cod from the Baltic Sea, confirming the role of C. crangon in cod infection with H. aduncum.

Expression Biomarkers of Pharmacological Treatment Outcomes in Women with Unipolar and Bipolar Depression.

INTRODUCTION: This study aimed to find the expression biomarkers of pharmacological treatment response in a naturalistic hospital setting. Through gene expression profiling, we were able to find differentially-expressed genes (DEGs) in unipolar (UD) and bipolar (BD) depressed women. METHODS: We performed gene expression profiling in hospitalized women with unipolar (n=24) and bipolar depression (n=32) who achieved clinical improvement after pharmacological treatment (without any restriction). To identify DEGs in peripheral blood mononuclear cells (PBMCs), we used the SurePrint G3 Microarray and GeneSpring software. RESULTS: After pharmacological treatment, UD and BD varied in the number of regulated genes and ontological pathways. Also, the pathways of neurogenesis and synaptic transmission were significantly up-regulated. Our research focused on DEGs with a minimum fold change (FC) of more than 2. For both types of depression, 2 up-regulated genes, OPRM1 and CELF4 (p=0.013), were significantly associated with treatment response (defined as a 50% reduction on the Hamilton Depression Rating Scale [HDRS]). We also uncovered the SHANK3 (p=0.001) gene that is unique for UD and found that the RASGRF1 (p=0.010) gene may be a potential specific biomarker of treatment response for BD. CONCLUSION: Based on transcriptomic profiling, we identified potential expression biomarkers of treatment outcomes for UD and BD. We also proved that the Ras-GEF pathway associated with long-term memory, female stress response, and treatment response modulation in animal studies impacts treatment efficacy in patients with BD. Further studies focused on the outlined genes may help provide predictive markers of treatment outcomes in UD and BD.

Transcriptome Changes in Three Brain Regions during Chronic Lithium Administration in the Rat Models of Mania and Depression.

Lithium has been the most important mood stabilizer used for the treatment of bipolar disorder and prophylaxis of manic and depressive episodes. Despite long use in clinical practice, the exact molecular mechanisms of lithium are still not well identified. Previous experimental studies produced inconsistent results due to different duration of lithium treatment and using animals without manic-like or depressive-like symptoms. Therefore, we aimed to analyze the gene expression profile in three brain regions (amygdala, frontal cortex and hippocampus) in the rat model of mania and depression during chronic lithium administration (2 and 4 weeks). Behavioral changes were verified by the forced swim test, open field test and elevated maze test. After the experiment, nucleic acid was extracted from the frontal cortex, hippocampus and amygdala. Gene expression profile was done using SurePrint G3 Rat Gene Expression whole transcriptome microarrays. Data were analyzed using Gene Spring 14.9 software. We found that chronic lithium treatment significantly influenced gene expression profile in both mania and depression models. In manic rats, chronic lithium treatment significantly influenced the expression of the genes enriched in olfactory and taste transduction pathway and long non-coding RNAs in all three brain regions. We report here for the first time that genes regulating olfactory and taste receptor pathways and long non-coding RNAs may be targeted by chronic lithium treatment in the animal model of mania.

First evidence of the presence of Anisakis simplex in Crangon crangon and Contracaecum osculatum in Gammarus sp. by in situ examination of the stomach contents of cod (Gadus morhua) from the southern Baltic Sea.

Cod (Gadus morhua), an important fish species in the Baltic Sea, is the paratenic host for many parasite species, including the zoonotic nematodes, Anisakis sp. and Contracaecum osculatum. We aimed to identify which invertebrate species (found in situ in the fish stomach) are responsible for infection of cod with zoonotic nematodes. We found that Crangon crangon and Gammarus sp., both invertebrate prey species of cod, were infected with Anisakis simplex and C. osculatum, respectively. These host-parasite systems are reported here for the first time, implicating C. crangon and Gammarus sp. as sources of infection of Baltic cod with zoonotic nematodes.

How effective is freezing at killing Anisakis simplex, Pseudoterranova krabbei, and P. decipiens larvae? An experimental evaluation of time-temperature conditions.

The consumption of raw or inadequately cooked marine fish can lead to several disorders caused by the ingestion of viable anisakid nematodes. Although anisakid larvae can be killed by subzero temperatures, making freezing an important control measure for this potential health hazard, these parasites can survive freezing under some conditions. Therefore, the aim of the present study was to experimentally evaluate the time-temperature conditions needed to kill Anisakis simplex and Pseudoterranova spp. The effectiveness of freezing was tested on two species of fish: cod, Gadus morhua from the North Atlantic, and herring, Clupea harengus membras from the southern Baltic Sea. Samples, which comprised skinless fillets of cod (n = 40) with visible parasites and whole herring (n = 240), were separately frozen at - 15, - 18, or - 20 °C for 24 h, or at - 20 °C for 48 h in the single-compressor freezer and at - 20, - 25, or - 35 °C for 24 h in the double-compressor freezer. After thawing, parasites were stained with malachite green and examined under the microscope for viability. All A. simplex and Pseudoterranova spp. larvae in cod fillets died at a temperature of - 15 °C or lower. However, freezing did not kill all the A. simplex larvae in whole herring: spontaneous movement of these parasites was observed in samples stored in the single-compressor freezer at - 15, - 18, and - 20 °C over 24 h. Our results demonstrate that the freezing procedure must consider both the capability of the freezing device and the nature of the fish product to ensure consumer safety.

Correlation of metabolic parameters, neurotrophin-3, and neurotrophin-4 serum levels in women with schizophrenia and first-onset depression.

BACKGROUND: Neurotrophin-3 (NTF3) and neurotrophin-4 (NTF4) play a crucial role in the neurodevelopment, differentiation, survival, and protection of neurons in different brain regions. Schizophrenia and depression are highly associated with metabolic abnormalities. Longitudinal and cross-sectional comparisons of NTF3 and NTF4 levels, as well as clinical and metabolic parameters, were studied in schizophrenia, first-episode depression, and control groups. MATERIALS AND METHODS: Serum NTF3 and NTF4 levels, body mass index (BMI), fasting serum glucose and lipid profile: cholesterol, triglyceride, high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) were measured at baseline and week 8 in 133 women: 55 patients with schizophrenia (19 with first-episode and 36 chronic), 30 patients with a first-episode depression and 48 healthy controls. The severity of the symptoms was evaluated with the Positive and Negative Syndrome Scale, 17-item Hamilton Depression Rating Scale and the Beck Depression Inventory. RESULTS: Longitudinal and cross-sectional comparisons did not detect any differences in the serum levels of NTF3 and NTF4 between studied groups. NTF3 and NTF4 levels were strongly correlated. Correlation of NTF3 and HDL-C levels at baseline was observed. Significant changes in cholesterol and fasting serum glucose levels in first-episode depression patients during 8 weeks of treatment were detected. Significant differences in BMI and LDL-C levels between schizophrenia and first-episode depression patients were discovered. CONCLUSIONS: To our knowledge, this is the first research which correlates NTF3 and NTF4 with metabolic parameters. Our study does not support the theory that the peripheral levels of NTF3 and NTF4 are disturbed in schizophrenia or first-episode depression.

Psychiatric genetic counseling: A mapping exercise.

Psychiatric genetic counseling (PGC) is gradually developing globally, with countries in various stages of development. In some, PGC is established as a service or as part of research projects while in others, it is just emerging as a concept. In this article, we describe the current global landscape of this genetic counseling specialty and this field's professional development. Drawing on information provided by expert representatives from 16 countries, we highlight the following: (a) current understanding of PGC; (b) availability of services for patients; (c) availability of training; (d) healthcare system disparities and cultural differences impacting practice; and (e) anticipated challenges going forward.

Genes involved in stress response influence lithium efficacy in bipolar patients.

OBJECTIVES: In mood disorders, chronic stimulation with stress results in aberrant regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Lithium was shown to influence HPA axis function. The underlying genetic background as well as environmental context may influence the stress response, and therefore lithium efficacy. The aim of the present study was to analyze if genetic variants located in genes involved in HPA axis regulation affect the response to long-term lithium treatment in bipolar patients. METHODS: We included 93 patients with bipolar disorder (32 males and 61 females), aged 31-80 years. The patients had been treated with lithium carbonate for at least 5 years. The magnitude of the lithium response was assessed using the Alda scale. Genotyping was performed for 28 polymorphisms in the genes encoding the following proteins involved in HPA axis regulation: corticotropin-releasing hormone receptor 1 (CRHR1), arginine vasopressin receptor 1B (AVPR1b), FK506 binding protein (FKBP) 5, FKBP4, BCL2-associated athanogene 1 (BAG1), stress induced phosphoprotein 1 (STIP1), glucocorticoid-induced transcript 1 (GLCC1), dual specificity phosphatase 1 (DUSP1) serine and arginine rich splicing factor (SRSF) 3, SRSF9, SRSF5, and acid phosphatase 1 (ACP1). Linkage disequilibrium and haplotype analysis were then performed, followed by statistical analysis (Statistica v.12; Stasoft, Krakow, Poland). RESULTS: We found a correlation between stressful life events at first episode and worse response to lithium (P=.019). In single marker analysis, we observed a significant association between three FKBP5 polymorphisms (rs1360780, rs7748266 and rs9296158), one ACP1 variant (rs300774) and one glucocorticoid-induced transcript 1 gene (GLCC1) variant (rs37972) and the degree of lithium response. Five out of seven FKBP5 polymorphisms showed strong linkage with one haplotype demonstrating an association with lithium efficacy (P=.008). No relationship was found between the other analyzed polymorphisms and lithium response. CONCLUSION: The response to lithium may depend on the variants of genes regulating the HPA axis and stressful life events in bipolar patients.

Brain-derived neurotrophic factor (BDNF) serum level in women with first-episode depression, correlation with clinical and metabolic parameters.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) influences neuron differentiation during development, as well as the synaptic plasticity and neuron survival in adulthood. BDNF has been implicated in the pathogenesis of psychiatric disorders and its serum level is a potential biomarker for depression. The aim of this study was to examine serum levels of BDNF in first-episode depression and its correlation with clinical and metabolic parameters. MATERIALS AND METHODS: The study was performed on a group of 60 women: 30 diagnosed with a first-episode of depression and 30 healthy controls. 17-Item Hamilton Depression Rating Scale (HDRS-17) was used to assess the severity of depression. Patients were randomly chosen for treatment with sertraline or venlafaxine. BDNF serum levels and metabolic parameters: fasting serum glucose, cholesterol, triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C) were measured at baseline and week 8 of treatment. RESULTS: There were no differences between BDNF level in depressed patients compared with the healthy controls. Lack of differences in medication effect of sertraline or venlafaxine on HDRS-17 scores during 8 weeks of treatment was observed. Correlation of BDNF at baseline and fasting serum glucose at baseline and week 8 was detected. CONCLUSIONS: Correlations of BDNF serum levels with metabolic parameters were observed.

Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes.

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

Can Psychological, Social and Demographical Factors Predict Clinical Characteristics Symptomatology of Bipolar Affective Disorder and Schizophrenia?

Schizophrenia (SCH) is a complex, psychiatric disorder affecting 1 % of population. Its clinical phenotype is heterogeneous with delusions, hallucinations, depression, disorganized behaviour and negative symptoms. Bipolar affective disorder (BD) refers to periodic changes in mood and activity from depression to mania. It affects 0.5-1.5 % of population. Two types of disorder (type I and type II) are distinguished by severity of mania episodes. In our analysis, we aimed to check if clinical and demographical characteristics of the sample are predictors of symptom dimensions occurrence in BD and SCH cases. We included total sample of 443 bipolar and 439 schizophrenia patients. Diagnosis was based on DSM-IV criteria using Structured Clinical Interview for DSM-IV. We applied regression models to analyse associations between clinical and demographical traits from OPCRIT and symptom dimensions. We used previously computed dimensions of schizophrenia and bipolar affective disorder as quantitative traits for regression models. Male gender seemed protective factor for depression dimension in schizophrenia and bipolar disorder sample. Presence of definite psychosocial stressor prior disease seemed risk factor for depressive and suicidal domain in BD and SCH. OPCRIT items describing premorbid functioning seemed related with depression, positive and disorganised dimensions in schizophrenia and psychotic in BD. We proved clinical and demographical characteristics of the sample are predictors of symptom dimensions of schizophrenia and bipolar disorder. We also saw relation between clinical dimensions and course of disorder and impairment during disorder.

Clock gene variants differentiate mood disorders.

Genetic variations in clock-related genes were hypothesized to be involved to in the susceptibility of mood disorders MD (both unipolar (UPD) and bipolar (BPD) disorders). In our work we investigated role of gene variants form four core period proteins: CLOCK, ARNTL, TIM and PER3. The total sample comprised from 744 mood disorders inpatients (UPD = 229, BPD = 515) and 635 healthy voluntary controls. The 42 SNPs from four genes of interest were genotyped. We used single polymorphisms, haplotypes, SNPs interactions and prediction analysis using classical statistical and machine learning methods. We observed association between two polymorphisms of CLOCK (rs1801260 and rs11932595) with BPDII and two polymorphisms of TIM (rs2291739, rs11171856) with UPD. We also detected ARNTL haplotype variant (rs1160996C/rs11022779G/rs1122780T) to be associated with increased risk of MD, BPD (both types). We established significant epistatic interaction between PER3 (rs2172563) and ARNTL (rs4146388 and rs7107287) in case of BPD. Additionally relation between PER3 (rs2172563) and CLOCK (rs1268271 and rs3805148) appeared in case of UPD. Classification and Regression Trees (C and RT) showed significant predictive value for 10 polymorphisms in all analyzed genes. However we failed to obtain model with sufficient predictive power. During analyses of sleep disturbances sample, we found carriers of homozygote variants (ARNTL: rs11022778 TT, rs1562438 TT, rs1982350 AA and PER3: rs836755 CC) showing more frequent falling asleep difficulties when compare to other genotypes carriers. Our study suggested a putative role of the CLOCK, TIM, ARNTL and PER3 and polymorphisms in MD susceptibility. In our analyses we showed association of specific gene variants with particular types of MD. We also confirmed necessity of performing separate analyzes for BPD and UPD patients. Comprehensive statistical approach is required even with individual symptoms analyses.

Bioorthogonal deprotection on the dendritic cell surface for chemical control of antigen cross-presentation.

The activation of CD8(+) T-cells requires the uptake of exogenous polypeptide antigens and proteolytic processing of these antigens to octamer or nonamer peptides, which are loaded on MHC-I complexes and presented to the T-cell. By using an azide as a bioorthogonal protecting group rather than as a ligation handle, masked antigens were generated-antigens that are not recognized by their cognate T-cell unless they are deprotected on the cell using a Staudinger reduction.