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Objective To determine men's satisfaction with and the potential acceptability of 11β-methyl-19-nortestosterone dodecylcarbonate (11β-MNTDC) when used for 28 days as an experimental, once-daily, oral hormonal male contraceptive (HMC). Study design We surveyed participants from a double-blind, randomized, placebo-controlled, phase 1 clinical trial, examining their experience with and willingness to use daily oral 11β-MNTDC for male contraception. Results Of 42 trial participants, 40 (30 11β-MNTDC, 10 placebo) completed baseline and end-of-treatment surveys. Based on a 28-day experience, few cited any baseline concerns about safety and drug adherence. Following treatment, nearly three-quarters (72.5%) of participants reported satisfaction with the study drug and nearly all (92.5%) would recommend the method to others. More than half of participants would be willing to pay for the study drug (62.5%) and indicated that the method exceeded initial expectations (53.9%). Nearly 90% reported that taking the pill was easy to remember and did not interfere with their daily routines. Approximately one-third of participants reported bothersome side effects (37% 11β-MNTDC vs. 20% placebo, p = 0.45). Given the option, 42% of participants would prefer a daily HMC pill over injectable regimens or a daily topical gel. Conclusion A majority of participants in this short-term trial of daily oral 11β-MNTDC reported satisfaction with the regimen, would recommend it to others, and would pay to use the drug as HMC despite some bothersome side effects. Implications Oral 11β-MNTDC would be an acceptable and preferable method among men desiring reversible hormonal male contraception (HMC). These data support further trials of novel oral HMCs such as 11β-MNTDC.

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K+ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

The central circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus drives the 24-hr pattern in cortisol, which functions as the main central synchronizing signal that coordinates peripheral clocks in organs that control whole body metabolism. A superimposed pulsatile pattern of cortisol allows rapid responses that fine tune the body's reaction to changes in the environment. In addition to coordinating metabolic processes to predictable environmental events, cortisol is the main catabolic signal that acts with testosterone, the quintessential male anabolic hormone, to maintain catabolic–anabolic homeostasis in men. Sleep restriction, when sufficiently substantial, increases late afternoon/early evening cortisol levels, but does not alter 24-hr cortisol levels; whereas even maximal acute circadian misalignment only slightly delays the cortisol rhythm. Prolonged circadian misalignment decreases overall cortisol exposure. The implications of these regulatory changes on health and disease require further evaluation.

Background Dimethandrolone (DMA) and 11β-methyl-19-nortestosterone (11β-MNT) are two novel compounds with both androgenic and progestational activity that are under investigation as potential male hormonal contraceptives. Their metabolic effects have never been compared in men. Objective Assess for changes in insulin sensitivity and adiponectin and compare the metabolic effects of these two novel androgens. Materials/methods In two clinical trials of DMA undecanoate (DMAU) and 11β-MNT dodecylcarbonate (11β-MNTDC), oral prodrugs of DMA and 11β-MNT, healthy men received drug, or placebo for 28 days. Insulin and adiponectin assays were performed on stored samples. Mixed model analyses were performed to compare the effects of the two drugs. Student's t test, or the non-parametric Kruskal-Wallis test as appropriate, was used to evaluate for an effect of active drug versus placebo. Results Class effects were seen, with decrease in HDL-C and SHBG, and increase in weight and hematocrit, with no statistically significant differences between the two compounds. No changes in fasting glucose, fasting insulin, or HOMA-IR were seen with either compound. There was a slight decrease in adiponectin with DMAU that was not seen with 11β-MNTDC. An increase in LDL-C was seen with 11β-MNTDC but not with DMAU. Discussion There were no significant changes in insulin resistance after 28 days of oral administration of these novel androgens despite a mild increase in weight. There may be subtle differences in their metabolic impacts that should be explored in future studies. Conclusion Changes in metabolic parameters should be carefully monitored when investigating androgenic compounds.

We identified the prevalence of elevated high-sensitivity C-reactive protein and interleukin-6 in patients with recent cardiovascular (CV) events with or without prediabetes/diabetes, and in a control group of patients with remote CV events. Interleukin-6 was elevated in patients with prediabetes/diabetes and recent CV events (median, 4.84 pg/mL; interquartile range, 3.27-7.45) compared with patients with remote events (2.36 pg/mL; interquartile range, 1.09-4.00). There was a trend for elevated high-sensitivity C-reactive protein in patients with acute events and prediabetes/diabetes (P = 0.147). This supports the notion that patients with prediabetes/diabetes and recent CV events have higher inflammatory burdens than patients without recent CV events or dysglycemia. Résumé: Nous avons défini la prévalence de l’augmentation du taux de protéine C réactive à haute sensibilité et d’interleukine-6 chez des patients ayant récemment subi des événements cardiovasculaires (CV), atteints ou non de prédiabète ou de diabète, et dans un groupe témoin de patients ayant subi des événements CV antérieurement. Le taux d’interleukine-6 était élevé chez les patients atteints de prédiabète ou de diabète ayant récemment subi des événements CV (médiane de 4,84 pg/ml; écart interquartile de 3,27 à 7,45) par rapport aux patients ayant subi des événements antérieurement (2,36 pg/ml; écart interquartile de 1,09 à 4,00). Le taux de protéine C réactive à haute sensibilité avait tendance à être élevé chez les patients atteints de prédiabète ou de diabète ayant subi des événements aigus (p = 0,147). Ces données appuient la notion selon laquelle les patients atteints de prédiabète ou de diabète qui ont récemment subi des événements CV présentent des fardeaux inflammatoires supérieurs à ceux des patients qui n’ont pas récemment subi d’événements CV ou présenté de dysglycémie.

There is a paucity of guidance on HIV pre-exposure prophylaxis (PrEP) implementation in an academic medical center. The objectives of this study were to describe interventions by a multidisciplinary PrEP task force at an academic medical center and compare metrics of PrEP implementation pre- and post-creation of this entity.The interventions of the task force are described within the rubric of the PrEP care continuum. Participants were adults prescribed PrEP for greater than or equal to 30 days at 9 clinical sites across a university health system. Metrics of PrEP implementation were compared over 12-month intervals before and after the creation of the task force.An increased proportion of participants had HIV testing within 7 days of new PrEP prescriptions (92% vs 63%, P.001) and were prescribed PrEP in increments of 90 days or shorter (74% vs 56%, P.001) after the creation of the task force. There were higher rates of testing for bacterial sexually transmitted infections in men who had sex with men and transgender women in the post-intervention compared with pre-intervention period.A multidisciplinary team that focuses on optimizing PrEP delivery along each step of the care continuum may facilitate PrEP scale-up and best practices in an academic setting.

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target., The RNase activity of Inositol-Requiring Enzyme 1 (IRE1) can be allosterically regulated by ATP-competitive inhibitors of the IRE1 kinase domain. Here, the authors identify ATP-competitive IRE1 RNase activators with improved selectivity and cellular activity, and elucidate their mechanism of action.

Context Dimethandrolone undecanoate (DMAU) is being developed as a male contraceptive. Daily oral administration of DMAU, a potent androgen that is not aromatized, markedly suppresses serum testosterone (T) and estradiol (E2) in healthy men. E2 deficiency can increase bone resorption in men. Objective This work aimed to assess changes in bone turnover markers with DMAU administration in a 28-day study. Design A randomized, double-blind, placebo-controlled study was conducted. Setting This study took place at 2 academic medical centers. Participants Healthy men, age 18 to50 years (n = 81), participated. Intervention Men received 0, 100, 200, or 400 mg of oral DMAU for 28 days. Serum C-terminal telopeptide of type I collagen (CTX; bone resorption marker) and procollagen type I amino-terminal propeptide (P1NP; bone formation marker) were measured on days 1 and 28. Main Outcome Measures Changes in bone turnover markers and serum hormones over the treatment period were measured. Results On day 28, median serum T and E2 were markedly suppressed in all treatment groups vs placebo (P < .001 for both). Percentage change (%) in serum P1NP significantly differed across treatment groups (P = .007): Serum P1NP significantly increased in the 200 mg (5%, interquartile range [IQR] –7% to 27%) and 400 mg (22%, IQR –1% to 40%) groups relative to placebo (–8%, IQR –20% to 0%). Change (%) in serum CTX did not differ between groups (P = .09). Conclusions DMAU administration for 28 days to healthy men leads to marked suppression of serum T and E2, yet increases P1NP, a serum marker of bone formation. Longer-term studies of the potent androgen DMAU are warranted to determine its impact on bone health in men.

Sarcoidosis is a complex disease with heterogeneous clinical presentations that can affect virtually any organ. Although the lung is typically the most common organ involved, combined pulmonary and cardiac sarcoidosis (CS) account for most of the morbidity and mortality associated with this disease. Pulmonary sarcoidosis can be asymptomatic or result in impairment in quality of life and end-stage, severe, and/or life-threatening disease. The latter outcome is seen almost exclusively in those with fibrotic pulmonary sarcoidosis, which accounts for 10% to 20% of pulmonary sarcoidosis patients. CS is problematic to diagnose and may cause significant morbidity and death from heart failure or ventricular arrhythmias. The diagnosis of CS usually requires surrogate cardiac imaging biomarkers, as endomyocardial biopsy has relatively low yield, even with directed electrophysiological mapping. Treatment of CS is often multifactorial, involving a combination of antigranulomatous therapy and pharmacotherapy for cardiac arrhythmias and/or heart failure in addition to device placement and cardiac transplantation.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6–9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10–12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery. Structural and physiological studies show that the inner membrane protein PbgA is a crucial sensor of lipopolysaccharide (LPS) and regulates the activity of the LPS biosynthesis enzyme LpxC.

Background: Interleukin-2 (IL-2), one of the proinflammatory cytokines, is used in the treatment of certain malignancies. In some studies, transient increases in cortisol and ACTH secretion occurred. Thus, this agent may be used as an experimental probe of adrenal cortisol secretion. Objective: This study quantifies the effects of low and moderate doses of IL-2 on cortisol secretion and assesses the modulation by age, dose and body composition. Site: Mayo Clinical Translational Research Unit. Subjects: Study comprised 35 healthy men, 17 young and 18 older. Methods: Randomized prospective double-blind saline-controlled study of IL-2 administration in two doses with concurrent 10-min blood sampling for 24 h. Outcome measures: Deconvolution analysis and approximate entropy of cortisol secretion. Results: Low-dose IL-2 administration increased nocturnal pulsatile cortisol secretion from 1460 ± 160 to 2120 ± 220 nmol/L/8 h in young subjects and from 1680 ± 105 to 1960 ± 125 nmol/L/8 h (treatment P P = 0.02). Comparable results were obtained for total cortisol secretion (P treatment P = 0.005). The higher IL-2 dose caused a large increase in young (P P = 0.90) subjects. This dose also increased approximate entropy from 0.877 ± 0.041 to 1.024 ± 0.049 (P = 0.008), pointing to reduced secretory orderliness. Incremental cortisol (nocturnal) secretion correlated negatively with visceral fat mass (R = −0.41, P = 0.019). Conclusion: In healthy men, IL-2 injection drives pulsatile cortisol secretion in a dose-dependent way in young, but not older, individuals and erodes cortisol secretory orderliness at a higher dose in young subjects. Cortisol responses are diminished with increasing abdominal visceral fat mass.

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme-inhibitor dynamics.

The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the ‘venom-ome’ and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 ‘venom-ome-specific toxins’ (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery., Analysis of a near-chromosomal genome assembly and transcriptome profiling of the Indian cobra identifies genes expressed in the venom glands. These data should help develop a new antivenom.

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)–dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)–dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB–dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB–dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe −/− mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P P RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet–induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B , E-selectin , and monocyte attachment. Conclusions: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Background Small observational studies have suggested that statin users have a lower risk of dying with COVID‐19. We tested this hypothesis in a large, population‐based cohort of adults in 2 of Canada’s most populous provinces: Ontario and Alberta. Methods and Results We examined reverse transcriptase–polymerase chain reaction swab positivity rates for SARS‐CoV‐2 in adults using statins compared with nonusers. In patients with SARS‐CoV‐2 infection, we compared 30‐day risk of all‐cause emergency department visit, hospitalization, intensive care unit admission, or death in statin users versus nonusers, adjusting for baseline differences in demographics, clinical comorbidities, and prior health care use, as well as propensity for statin use. Between January and June 2020, 2.4% of 226 142 tested individuals aged 18 to 65 years, 2.7% of 88 387 people aged 66 to 75 years, and 4.1% of 154 950 people older than 75 years had a positive reverse transcriptase–polymerase chain reaction swab for SARS‐CoV‐2. Compared with 353 878 nonusers, the 115 871 statin users were more likely to test positive for SARS‐CoV‐2 (3.6% versus 2.8%, P Conclusions Compared with statin nonusers, patients taking statins exhibit the same risk of testing positive for SARS‐CoV‐2 and those younger than 75 years exhibit similar outcomes within 30 days of a positive test. Patients older than 75 years with a positive SARS‐CoV‐2 test and who were taking statins had more emergency department visits and hospitalizations, but exhibited lower 30‐day all‐cause mortality risk.

This chapter looks at urinary tract infections (UTIs), which account for a significant burden of morbidity, cost, and healthcare resource utilization in both the inpatient and outpatient setting. UTIs occur in patients of all ages with a predilection for females throughout life and males at each end of the age spectrum. The term “urinary tract infection” encompasses a group of conditions that includes cystitis, pyelonephritis, and asymptomatic bacteriuria. Appropriate management of a patient with a UTI requires consideration of multiple factors, including age, sex, underlying comorbidities, pregnancy, history of prior UTIs, site of infection, anatomic aberrations, and the pathogen involved. Determination of the location of the infection as upper versus lower tract is essential to selection of optimal therapy. Lower urinary tract infection is infection involving the bladder, or cystitis, and is characterized by dysuria, pyuria, urinary frequency, or urinary urgency. Upper urinary tract infection, or pyelonephritis, is infection involving the bladder and kidney that classically presents with fever and flank pain, with or without the symptoms of lower tract infection.

A promising development in hormonal male contraception (HMC) is a class of bifunctional prodrugs that combine both androgenic and progestogenic activities into a single molecule. Examples of these prodrugs currently being studied are dimethandrolone undecanoate (DMAU) and 11β-methyl-19-nortestosterone-17β-dodecylcarbonate (11β-MNTDC) (1, 2). The inactive prodrugs are cleaved to release active drug over a 24-hour timeframe, providing once-a-day dosing. As potent androgens, these steroids suppress gonadotropin secretion, leading to markedly decreased serum testosterone production and circulating levels. Low testosterone levels might lead to unpleasant symptoms of hypogonadism if DMAU and 11β-MNTDC are not providing sufficient and effective androgenicity. Therefore, we examined the impact of the novel progestogenic androgens on serum testosterone levels and acceptability of varying dosages of these oral prodrugs in a secondary analysis of two Phase 1 placebo-controlled trials. Healthy male participants were randomized to take two or four oral pills of active drug or placebo per day. As DMAU and 11β-MNTDC share similar mechanisms of action and tolerability, we examined the association of dosage as well as testosterone concentrations on combined drug acceptability versus placebo. Survey respondents across the two trials (39 DMAU, 30 11β-MNTDC, 28 combined placebo group) shared similar baseline demographics. After seven days of usage, testosterone levels for those using either prodrug dropped to levels below 100 ng/dL while testosterone levels for those using the placebo (400-600 ng/dL) remained within the reference. Recipients of either DMAU or 11β-MNTDC reported greater willingness to use the active prodrug in the future (75%), compared to placebo recipients (46.4%, p=0. 007). Throughout the 28-day oral pill usage, while average testosterone levels during the period of suppression (day 7 to 28) were very low, they were significantly higher in the 200 mg group than in the 400 mg group (92.7 ng/dL vs. 49.6 ng/dL, p-value (1) Thirumalai et al. J Clin Endocrinol Metab. 2019; 104: 423-32. (2) Yuen et al. J Clin Endocrinol Metab. 2020;105. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.