Effect of mental health staffing inputs on initiation of care among recently separated Veterans.

OBJECTIVE: To estimate a causal relationship between mental health staffing and time to initiation of mental health care for new patients. DATA SOURCES AND STUDY SETTING: As the largest integrated health care delivery system in the United States, the Veterans Health Administration (VHA) provides a unique setting for isolating the effects of staffing on initiation of mental health care where demand is high and out-of-pocket costs are not a relevant confounder. We use data from the Department of Defense and VHA to obtain patient and facility characteristics and health care use. STUDY DESIGN: To isolate exogenous variation in mental health staffing, we used an instrumental variables approach-two-stage residual inclusion with a discrete time hazard model. Our outcome is time to initiation of mental health care after separation from active duty (first appointment) and our exposure is mental health staffing (standardized clinic time per 1000 VHA enrollees per pay period). DATA COLLECTION/EXTRACTION METHODS: Our cohort consists of all Veterans separating from active duty between July 2014 and September 2017, who were enrolled in the VHA, and had at least one diagnosis of post-traumatic stress disorder, major depressive disorder, and/or substance use disorder in the year prior to separation from active duty (N = 54,209). PRINCIPAL FINDINGS: An increase of 1 standard deviation in mental health staffing results in a higher likelihood of initiating mental health care (adjusted hazard ratio: 3.17, 95% confidence interval: 2.62, 3.84, p < 0.001). Models stratified by tertile of mental health staffing exhibit decreasing returns to scale. CONCLUSIONS: Increases in mental health staffing led to faster initiation of care and are especially beneficial in facilities where staffing is lower, although initiation of care appears capacity-limited everywhere.

Effect of mental health staffing inputs on suicide-related events.

OBJECTIVE: To estimate the effects of changes in Veterans Health Administration (VHA) mental health services staffing levels on suicide-related events among a cohort of Veterans. DATA SOURCES: Data were obtained from the VHA Corporate Data Warehouse, the Department of Defense and Veterans Administration Infrastructure for Clinical Intelligence, the VHA survey of enrollees, and customized VHA databases tracking suicide-related events. Geographic variables were obtained from the Area Health Resources Files and the Centers for Medicare and Medicaid Services. STUDY DESIGN: We used an instrumental variables (IV) design with a Heckman correction for non-random partial observability of the use of mental health services. The principal predictor was a measure of provider staffing per 10,000 enrollees. The outcome was the probability of a suicide-related event. DATA COLLECTION/EXTRACTION METHODS: Data were obtained for a cohort of Veterans who recently separated from active service. PRINCIPAL FINDINGS: From 2014 to 2018, the per-pay period probability of a suicide-related event among our cohort was 0.05%. We found that a 1% increase in mental health staffing led to a 1.6 percentage point reduction in suicide-related events. This was driven by the first tertile of staffing, suggesting diminishing returns to scale for mental health staffing. CONCLUSIONS: VHA facilities appear to be staffing-constrained when providing mental health care. Targeted increases in mental health staffing would be likely to reduce suicidality.

Tick-borne Encephalitis Identified in Ireland: Diagnosis and Outcome.

Presentation Tick borne encephalitis (TBE) is not endemic in Ireland and diagnostic tests are seldom requested. We describe the first notified case in Ireland. A 50-year-old female returned from Lithuania and presented with fever and new neurologic signs. Diagnosis TBE was diagnosed by detection of TBE virus specific antibodies in serum and cerebrospinal fluid (CSF). Treatment The patient was managed with observation and supportive care consisting of intravenous fluids and analgesia. Discussion The case highlights the importance of awareness of TBE among physicians and travellers to guide appropriate testing and vaccination. TBE is being recognised in non-endemic countries posing an emerging risk to public health.

A cluster randomized controlled trial for a multi-level, clinic-based smoking cessation program with women in Appalachian communities: study protocol for the "Break Free" program.

BACKGROUND: The cervical cancer burden is high among women living in Appalachia. Cigarette smoking, a cervical cancer risk factor, is also highly prevalent in this population. This project aims to increase smoking cessation among women living in Appalachia by embedding a smoking cessation program within a larger, integrated cervical cancer prevention program. METHODS: The broader program, the Take CARE study, is a multi-site research collaborative designed to address three risk factors for cervical cancer incidence and mortality: tobacco use, human papillomavirus (HPV) infection, and cervical cancer screening. Break Free is a primary care clinic-based implementation program that aims to promote smoking cessation among female smokers in Appalachia by standardizing clinical practice protocols. Break Free includes: (1) implementation of a tobacco user identification system in the Electronic Health Record, (2) clinic staff and provider training on the Ask, Advise and Refer (AAR) model, (3) provider implementation of AAR to identify and treat women who want to quit smoking within the next 6 months, (4) facilitated access to cessation phone counseling plus pharmacotherapy, and (5) the bundling of Break Free tobacco cessation with HPV vaccination and cervical cancer screening interventions in an integrated approach to cervical cancer prevention. The study spans 35 Appalachian health clinics across 10 healthcare systems. We aim to enroll 51 adult female smokers per health system (total N = 510). Baseline and follow-up data will be obtained from participant (provider and patient) surveys. The primary outcome is self-reported 12-month point prevalence abstinence among enrolled patients. All randomized patients are asked to complete follow-up surveys, regardless of whether they participated in tobacco treatment. Data analysis of the primary aims will follow intent-to-treat methodology. Secondary outcomes will assess program implementation and cost effectiveness. DISCUSSION: Addressing high tobacco use rates is critical for reducing cervical cancer morbidity and mortality among women living in Appalachia. This study evaluates the implementation and effectiveness of a smoking cessation program in increasing smoking cessation among female smokers. If results demonstrate effectiveness and sustainability, implementation of this program into other health care clinics could reduce both rates of smoking and cervical cancer. Trial registration NCT04340531 (April 9, 2020).

Dietary intake of sulfur amino acids and risk of kwashiorkor malnutrition in eastern Democratic Republic of the Congo.

BACKGROUND: Kwashiorkor is an often-fatal type of severe acute malnutrition affecting hundreds of thousands of children annually, but whose etiology is still unknown. Evidence suggests inadequate sulfur amino acid (SAA) status may explain many signs of the condition but studies evaluating dietary protein intake in relation to the genesis of kwashiorkor have been conflicting. We know of no studies of kwashiorkor that have measured dietary SAAs. OBJECTIVES: We aimed to determine whether children in a population previously determined to have high prevalence of kwashiorkor [high-prevalence population (HPP)] have lower dietary intakes of SAAs than children in a low-prevalence population (LPP). METHODS: A cross-sectional census survey design of 358 children compared 2 previously identified adjacent populations of children 36-59 mo old in North Kivu Province of the Democratic Republic of the Congo. Data collected included urinary thiocyanate (SCN), cyanogens in cassava-based food products, recent history of illness, and a 24-h quantitative diet recall for the child. RESULTS: The HPP and LPP had kwashiorkor prevalence of 4.5% and 1.7%, respectively. A total of 170 children from 141 households in the LPP and 169 children from 138 households in the HPP completed the study. A higher proportion of HPP children had measurable urinary SCN (44.8% compared with 29.4%, P < 0.01). LPP children were less likely to have been ill recently (26.8% compared with 13.6%, P < 0.01). Median [IQR] intake of SAAs was 32.4 [22.9-49.3] mg/kg for the LPP and 29.6 [18.1-44.3] mg/kg for the HPP (P < 0.05). Methionine was the first limiting amino acid in both populations, with the highest risk of inadequate intake found among HPP children (35.1% compared with 23.6%, P < 0.05). CONCLUSIONS: Children in a population with a higher prevalence of kwashiorkor have lower dietary intake of SAAs than children in a population with a lower prevalence. Trial interventions to reduce incidence of kwashiorkor should consider increasing SAA intake, paying particular attention to methionine.

Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro.

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a "blank slate" culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

Effect on VA Patient Satisfaction of Provider's Use of an Integrated Viewer of Multiple Electronic Health Records.

PURPOSE: To examine associations between patient perceptions that their provider was knowledgeable of their medical history and clinicians' early adoption of an application that presents providers with an integrated longitudinal view of a patient's electronic health records (EHR) from multiple healthcare systems. METHOD: This retrospective analysis utilizes provider audit logs from the Veterans Health Administration Joint Legacy Viewer (JLV) and patient responses to the Survey of Patient Healthcare Experiences Patient-Centered Medical Home (SHEP/PCMH) patient satisfaction survey (FY2016) to assess the relationship between the primary care provider being an early adopter of JLV and patient perception of the provider's knowledge of their medical history. Multivariate logistic regression models were used to control for patient age, race, sex education, health status, duration of patient-provider relationship, and provider characteristics. RESULTS: The study used responses from 203,903 patients to the SHEP-PCMH survey in FY2016 who received outpatient primary care services from 11,421 unique providers. Most (91%) clinicians had no JLV utilization in the 6 months prior to the studied patient visit. Controlling for patient demographics, length of the patient-provider relationship, and provider and facility characteristics, being an early adopter of the JLV system was associated with a 14% (adj OR 1.14, p < 0.000) increased odds that patients felt their provider was knowledgeable about their medical history. When evaluating the interaction between duration of patient-provider relationship and being an early adopter of JLV, a greater effect was seen with patient-provider relationships that were greater than 3 years (adj OR 1.23, p < 0.000), compared to those less than 3 years. CONCLUSIONS: Increasing the interoperability of medical information systems has the potential to improve both patient care and patient experience of care. This study demonstrates that early adopters of an integrated view of electronic health records from multiple delivery systems are more likely to have their patients report that their clinician was knowledgeable of their medical history. With provider payments often linked to patient satisfaction performance metrics, investments in interoperability may be worthwhile.

A Unique Case of Concurrent Long Bone Osteomyelitis and Empyema Caused by Fusobacterium nucleatum.

We describe a patient presenting with constitutional symptoms, poor oral hygiene and leg swelling who was diagnosed with Fusobacterium nucleatum osteomyelitis of femur and empyema. Long bone osteomyelitis is rarely caused by this microorganism. This unusual case was successfully managed with drainage and antimicrobial therapy.

Trastuzumab uptake and its relation to efficacy in an animal model of HER2-positive breast cancer brain metastasis.

PURPOSE: The extent to which efficacy of the HER2 antibody Trastuzumab in brain metastases is limited by access of antibody to brain lesions remains a question of significant clinical importance. We investigated the uptake and distribution of trastuzumab in brain and mammary fat pad grafts of HER2-positive breast cancer to evaluate the relationship of these parameters to the anti-tumor activity of trastuzumab and trastuzumab emtansine (T-DM1). METHODS: Mouse transgenic breast tumor cells expressing human HER2 (Fo2-1282 or Fo5) were used to establish intracranial and orthotopic tumors. Tumor uptake and tissue distribution of systemically administered 89Zr-trastuzumab or muMAb 4D5 (murine parent of trastuzumab) were measured by PET and ELISA. Efficacy of muMAb 4D5, the PI3K/mTOR inhibitor GNE-317, and T-DM1 was also assessed. RESULTS: 89Zr-trastuzumab and muMAb 4D5 exhibited robust uptake into Fo2-1282 brain tumors, but not normal brains. Uptake into brain grafts was similar to mammary grafts. Despite this, muMAb 4D5 was less efficacious in brain grafts. Co-administration of muMAb 4D5 and GNE-317, a brain-penetrant PI3K/mTOR inhibitor, provided longer survival in mice with brain lesions than either agent alone. Moreover, T-DM1 increased survival in the Fo5 brain metastasis model. CONCLUSIONS: In models of HER2-positive breast cancer brain metastasis, trastuzumab efficacy does not appear to be limited by access to intracranial tumors. Anti-tumor activity improved with the addition of a brain-penetrant PI3K/mTOR inhibitor, suggesting that combining targeted therapies is a more effective strategy for treating HER2-positive breast cancer brain metastases. Survival was also extended in mice with Fo5 brain lesions treated with T-DM1.

The Effect of Gene Variants on Levonorgestrel Pharmacokinetics When Combined With Antiretroviral Therapy Containing Efavirenz or Nevirapine.

Reduced levonorgestrel concentrations from the levonorgestrel contraceptive implant was previously seen when given concomitantly with efavirenz. We sought to assess whether single nucleotide polymorphisms (SNPs) in genes involved in efavirenz and nevirapine metabolism were linked to these changes in levonorgestrel concentration. SNPs in CYP2B6, CYP2A6, NR1I2, and NR1I3 were analyzed. Associations of participant demographics and genotype with levonorgestrel pharmacokinetics were evaluated in HIV-positive women using the levonorgestrel implant plus efavirenz- or nevirapine-based antiretroviral therapy (ART), in comparison to ART-naïve women using multivariate linear regression. Efavirenz group: CYP2B6 516G>T was associated with lower levonorgestrel log10 Cmax and log10 AUC. CYP2B6 15582C>T was associated with lower log10 AUC. Nevirapine group: CYP2B6 516G>T was associated with higher log10 Cmax and lower log10 Cmin . Pharmacogenetic variations influenced subdermal levonorgestrel pharmacokinetics in HIV-positive women, indicating that the magnitude of the interaction with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is influenced by host genetics.

Interventions to Improve the Treatment of Malaria in an Acute Teaching Hospital in Ireland.

Malaria is the most serious parasitic infection. At our institution over a two year period there were treatment errors in 18% (n=3) of cases. The aim of this multidisciplinary study was to ensure appropriate and timely treatment of malaria by implementation of a cluster of interventions: reconfiguration of existing guidelines, provision of prescribing information; delivery of education sessions to front-line staff and enabling rapid access to medication. Staff feedback was assessed through a questionnaire. Perceived benefits gained included awareness of guidelines (91%, n= 39), how to diagnose (81%, n =35), how to treat (86%, n=37), that treatment must be prompt (77%, n=33) and where to find treatment out of hours (84%, n=36). 'Others' perceived benefits (5% n= 2) noted referred to treatment in pregnancy. Going forward, a programme of on-going staff education, repeated audits of guideline compliance and promotion of reporting of medication errors should help ensure that these benefits are sustained.

Prevalence and type of drug-drug interactions involving ART in patients attending a specialist HIV outpatient clinic in Kampala, Uganda.

OBJECTIVES: Scale-up of HIV services in sub-Saharan Africa has rapidly increased, necessitating evaluation of medication safety in these settings. Drug-drug interactions (DDIs) involving antiretrovirals (ARVs) in sub-Saharan Africa are poorly characterized. We evaluated the prevalence and type of ARV DDIs in Ugandan outpatients and identified the patients most at risk. METHODS: A total of 2000 consecutive patients receiving ARVs at the Infectious Diseases Institute, Kampala were studied. The most recent prescription for each patient was screened for clinically significant DDIs using www.hiv-druginteractions.org. Univariable and multivariable logistic regression were used to identify risk factors for DDIs. A screening tool was developed using significant risk factors and tested in a further 500 patients. RESULTS: Clinically significant DDIs were observed in 374 (18.7%) patients, with a total of 514 DDIs observed. Only 0.2% of DDIs involved a contraindicated combination. Comedications commonly associated with DDIs were antibiotics (4.8% of 2000 patients), anthelmintics (2.2%) and antifungals (3.5%). Patient age, gender, CD4 count and weight did not affect risk of DDIs. In multivariable analysis, the patient factors that independently increased risk of DDIs were two or more comedications (P < 0.0001), a PI-containing ARV regimen (P < 0.0001), use of an anti-infective (P < 0.0001) and WHO clinical stage 3-4 (P = 0.04). A scoring system based on having at least two of these risk factors identified between 75% and 90% of DDIs in a validation cohort. CONCLUSIONS: Significant ARV DDIs occur at similar rates in resource-limited settings and developed countries; however, the comedications frequently causing DDIs differ. Development of tools that are relevant to particular settings should be a priority to assist with prevention and management of DDIs.

Use of flow cytometry for characterization and fractionation of cell populations based on their expression of heparan sulfate epitopes.

The ability to characterize alterations in heparan sulfate (HS) structure during development or as a result of loss or mutation of one or more components of the HS biosynthetic pathway is essential for broad understanding of the effects these changes may have on cell/tissue function. The use of anti-HS antibodies provides an opportunity to study HS chain composition in situ, with a multitude of different antibodies having been generated that recognize subtle differences in HS patterning, with the number and positioning of sulfate groups influencing antibody binding affinity. Flow cytometry is a valuable technique to enable the rapid characterization of the changes in HS-specific antibody binding in situ, allowing multiple cell types to be directly compared. Additionally fluorescent-activated cell sorting (FACS) allows fractionation of cells based on their HS-epitope expression.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma.

Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1(R132H) exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1(R132H) exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

Infective endocarditis.

BACKGROUND: Infective endocarditis continues to pose a therapeutic challenge to treating clinicians. We believe that the successful management of endocarditis mandates a thorough understanding of the risk factors for adverse outcomes and a co-ordinated team approach. METHODS: Between the years 2000 and 2009, 85 patients required surgery for infective endocarditis, with a total of 112 infected valves being treated surgically. Data was analysed to determine factors significantly associated with morbidity and mortality. RESULTS: The mean age was 50.5 years. Nine (10.5%) of these patients had Prosthetic Valve Endocarditis, the remaining 76 (89.5%) had Native Valve Endocarditis. Twenty-nine percent of patients were NYHA 4 pre-operatively, 15% of patients were haemodynamically unstable requiring inotropic support, 34% were persistently febrile despite antibiotic therapy, and 48% had suffered any embolic event, 20% suffered cerebral emboli. The commonest causative organism in our series was Staphylococcus Aureus (54.1%) with 2.3% of cases being due to MRSA. The second commonest organism isolated was Streptococcus spp. at 21.1%. Operative mortality was 12.9%, of which on-table mortality was 2.2%. Mean follow-up was 56 months (range 1-151). Early recurrence rates (<3 months) were 2.3%. Late recurrence was 7.0%. The pre-operative factors associated with increased mortality were age over 65, inotropic requirement, uncontrolled sepsis and cerebral emboli. We summarise our experience and recommendations for a team approach to the management of infective endocarditis.

Mesenchymal stromal cells: inhibiting PDGF receptors or depleting fibronectin induces mesodermal progenitors with endothelial potential.

Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs toward mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle shape with abundant smooth muscle α-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle α-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent upregulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers platelet endothelial cell adhesion molecule 1 and vascular endothelial-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus, MSC potency and vascular induction are regulated by perturbing mesenchymal fate.

Aortic root replacement for ascending aortic disease: a 10 year review.

BACKGROUND: Bentall's procedure and its modifications have been used for over 40 years for the treatment of ascending aortic disease. This study reviewed 10 years of experience with Aortic Root Replacement (ARR) in a major cardiac surgical centre. METHODS: Eighty-nine patients underwent ARR between 1999 and 2009. The records were scrutinised by retrospective chart review. RESULTS: The mean age was 54 years. Seventy-nine percent of patients were male and 21% female. The indications for the procedure were Aortic Root Aneurysm (ARA) (65%), type A dissection (28%), infective endocarditis (4.4%) and prosthetic valve regurgitation (2.2%). Fifty-seven percent of these were performed electively and 43% as an emergency. A bicuspid aortic valve was present in 37%. Arch surgery was required in 15.7%, bypass grafting in 12.3% and mitral valve surgery in 5.6%. The descending aorta was involved in 16.8%. Operative mortality was 3.3% and in-hospital mortality 12.3%. Mean follow-up was 67.05 months (range 2-143). No patients required re-operation. CONCLUSIONS: The factors associated with increased in-hospital mortality were pre-operative haemodynamic instability, concommitant coronary artery disease and acute renal failure. The presence of a bicuspid valve may be associated with lower rates of complications, but no difference in mortality.

Excellent clinical outcomes from a national donation-after-determination-of-cardiac-death lung transplant collaborative.

Donation-after-Determination-of-Cardiac-Death (DDCD) donor lungs can potentially increase the pool of lungs available for Lung Transplantation (LTx). This paper presents the 5-year results for Maastricht category III DDCD LTx undertaken by the multicenter Australian National DDCD LTx Collaborative. The Collaborative was developed to facilitate interaction with the Australian Organ Donation Authority, standardization of definitions, guidelines, education and audit processes. Between 2006 and 2011 there were 174 actual DDCD category III donors (with an additional 37 potentially suitable donors who did not arrest in the mandated 90 min postwithdrawal window), of whom 71 donated lungs for 70 bilateral LTx and two single LTx. In 2010 this equated to an "extra" 28% of donors utilized for LTx. Withdrawal to pulmonary arterial flush was a mean of 35.2 ± 4.0 min (range 18-89). At 24 h, the incidence of grade 3 primary graft dysfunction was 8.5%[median PaO(2)/FiO(2) ratio 315 (range 50-507)]. Overall the incidence of grade 3 chronic rejections was 5%. One- and 5-year actuarial survival was 97% and 90%, versus 90% and 61%, respectively, for 503 contemporaneous brain-dead donor lung transplants. Category III DDCD LTx therefore provides a significant, practical, additional quality source of transplantable lungs.