Clinical Trial Protocol for PRIMARY2: A Multicentre, Phase 3, Randomised Controlled Trial Investigating the Additive Diagnostic Value of [68Ga]Ga-PSMA-11 Positron Emission Tomography/Computed Tomography in Men with Negative or Equivocal Multiparametric Magnetic Resonance Imaging for the Diagnosis of Clinically Significant Prostate Cancer.

BACKGROUND: Multiparametric magnetic resonance imaging (mpMRI) has an established role for the diagnosis of clinically significant prostate cancer (sPCa). The PRIMARY trial demonstrated that [68Ga]Ga-PSMA-11 positron emission tomography/computed tomography (PET/CT) was associated with a significant improvement in sensitivity and negative predictive value for sPCa detection. OBJECTIVE: To demonstrate that addition of prostate-specific membrane antigen (PSMA) radioligand PET/CT will enable some men to avoid transperineal prostate biopsy without missing sPCa, and will facilitate biopsy targeting of PSMA-avid sites. DESIGN, SETTING, AND PARTICIPANTS: This multicentre, two-arm, phase 3, randomised controlled trial will recruit 660 participants scheduled to undergo biopsy. Eligible participants will have clinical suspicion of sPCa with a Prostate Imaging-Reporting and Data System (PI-RADS) score of 2 and red flags, or a PI-RADS score of 3 on mpMRI (PI-RADS v2). Participants will be randomised at a 1:1 ratio in permuted blocks stratified by centre. The trial is registered on ClinicalTrials.gov as NCT05154162. INTERVENTION: In the experimental arm, participants will undergo pelvic PSMA PET/CT. Local and central reviewers will interpret scans independently using the PRIMARY score. Participants with a positive result will undergo targeted transperineal prostate biopsies, whereas those with a negative result will undergo prostate-specific antigen monitoring alone. In the control arm, all participants undergo template transperineal prostate biopsies. Participants will be followed for subsequent clinical care for up to 2 yr after randomisation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: sPCa is defined as Gleason score 3 + 4 (≥10%) = 7 disease (grade group 2) or higher on transperineal prostate biopsy. Avoidance of transperineal prostate biopsy will be measured at 6 mo from randomisation. The primary endpoints will be analysed on an intention-to-treat basis. CONCLUSIONS: Patient enrolment began in March 2022, with recruitment expected to take 36 mo. PATIENT SUMMARY: For patients with suspected prostate cancer who have nonsuspicious or unclear MRI (magnetic resonance imaging) scan findings, a different type of scan (called PSMA PET/CT; prostate-specific membrane antigen positron emission tomography/computed tomography) may identify men who could avoid an invasive prostate biopsy. This type of scan could also help urologists in better targeting of samples from suspicious lesions during prostate biopsies.

Reviewing the risk of breast cancer recurrence following breast reconstruction.

BACKGROUND: It is widely accepted that both autologous and alloplastic reconstruction are safe. A recent publication reported a significant association between textured implants and metastatic recurrence of breast cancer. This study aims to assess if the published results are reproducible in our cohort and to review the safety of breast reconstruction. METHODS: This is a retrospective cohort study of adult patients undergoing mastectomy and either alloplastic or autologous breast reconstruction at a single quaternary hospital. Outcomes include disease free survival (DFS), local and recurrence free survival (LRRFS) and BIA-ALCL. For time to event endpoints, unadjusted and multivariate adjusted hazard ratios (HRs) were estimated using Cox regression, and penalized Cox regression respectively. RESULTS: Four hundred and twenty-six patients of whom 187 underwent autologous reconstruction and 239 underwent alloplastic. There were 43 cancer recurrences (24 alloplastic and 19 autologous) and 14 local regional recurrences (8 alloplastic and 4 autologous). There were 26 deaths and no instances of BIA-ALCL. Median follow-up time was 4.7 years. No evidence of association was found between breast reconstruction method and DFS (HR 0.87 CI: 0.47-1.58). It is uncertain whether implant texture grade was associated with increased breast cancer recurrence (HR 2.17 CI: 0.65-7.52). CONCLUSION: Both autologous and alloplastic breast reconstruction have been carried out in our cohort and reconstructive modality was not associated with either reduced DFS or LRRFS. The results in this cohort show there is uncertainty between the use of textured breast implants and either local or distant breast cancer recurrence.

Use of the English Health Literacy Questionnaire (HLQ) with Health Science University Students in Nepal: A Validity Testing Study.

Research evidence shows that health literacy development is a key factor influencing non-communicable diseases care and patient outcomes. Healthcare professionals with strong health literacy skills are essential for providing quality care. We aimed to report the validation testing of the Health Literacy Questionnaire (HLQ) among health professional students in Nepal. A cross-sectional study was conducted with 419 health sciences students using the HLQ in Nepal. Validation testing and reporting were conducted using five sources outlined by 'the 2014 Standards for Educational and Psychological Testing'. The average difficulty was lowest (17.4%) for Scale 4. Social support for health, and highest (51.9%) for Scale 6. Ability to actively engage with healthcare providers. One factor Confirmatory Factor Analysis (CFA) model showed a good fit for Scale 2, Scale 7 and Scale 9 and a reasonable fit for Scale 3 and Scale 4. The restricted nine-factor CFA model showed a satisfactory level of fit. The use of HLQ is seen to be meaningful in Nepal and warrants translation into native Nepali and other dominant local languages with careful consideration of cultural appropriateness using cognitive interviews.

MicroRNA-132 may be associated with blood pressure and liver steatosis-preliminary observations in obese individuals.

Recent findings in experimental models have shown that the microRNA miR-132 (mir-132) is an important regulator of liver homeostasis and lipid metabolism. We aimed to assess miR-132 expression in liver and fat tissues of obese individuals and examine its association with blood pressure (BP) and hepatic steatosis. We examined obese individuals undergoing bariatric surgery for weight loss (n = 19). Clinical and demographic information was obtained. Quantitative PCR was performed to determine tissue expression of miR-132 in liver and subcutaneous and visceral fat biopsies obtained during bariatric surgery. Liver biopsies were read by a single liver pathologist and graded for steatosis, inflammation and fibrosis. Participants (aged 39 ± 8.1 years) had a body mass index (BMI) of 42 ± 4.5 kg/m2 and presented with 2.2 ± 1.2 metabolic abnormalities. Supine BP was 127 ± 16/74 ± 11 mmHg. Hepatic and visceral fat expression of miR-132 were correlated (r = 0.59, P = 0.033). There was no correlation between subcutaneous and visceral expression of miR-132 (r = -0.31, P = 0.20). Hepatic and visceral fat miR-132 expression were associated with BMI (r = 0.62 and r = 0.68, P = 0.049 respectively) and degree of liver steatosis (r = 0.60 and r = 0.55, P < 0.05, respectively). Subcutaneous fat miRNA-132 expression was correlated to office systolic BP (r = 0.46, P < 0.05), several aspects of 24 h BP (24 h systolic BP: r = 0.52; day systolic BP: r = 0.59, P < 0.05 for all), plasma triglycerides (r = 0.51, P < 0.01) and liver enzymes (ALT: r = -0.52; AST: r = -0.48, P < 0.05 for all). We found an association between miR-132 and markers of cardiovascular and metabolic disease. Reduction of miR-132 may be a target for the regulation of liver lipid homeostasis and control of obesity-related blood pressure.

Effect of pavement condition and geometrics at signalised intersections on casualty crashes.

INTRODUCTION: This study investigated the effects of pavement surface condition and other control factors on casualty crashes at signalized intersections. It involved conducting a before and after study for road surface condition and situational factors. It also included assessing the effects of geometric characteristics on safety performance of signalized intersections post resurfacing to control for the effect of pavement surface condition. Pavement surface condition included roughness, rutting, and skid resistance. The control factors included traffic volume, light and surface moisture condition, and speed limit. The geometric characteristics included approach width, number of lanes, intersection depth, presence of median, presence of shared lane, and presence of bus stop. METHOD: To account for the repeated observations of the effect of light and surface moisture conditions in four occasions (day-dry, day-wet, night-dry and night-wet) Generalized Estimating Equation (GEE) with Negative Binomial (NB) and log link function was applied. For each signalized intersection in the sample, condition data are collected for the year before and after the year of surface treatment. Crash data, however, are collected for a minimum of three and maximum of five years before and after treatment years. RESULTS: The results show that before treatment, light condition, road surface moisture condition, and skid resistance interaction with traffic volume are the significant contributors to crash occurrence. For after treatment; light condition, road surface moisture condition, their interaction product, and roughness interaction with light condition, surface moisture condition, and traffic volume are the significant contributors. The geometric variables that were found to have significant effects on crash frequency post resurfacing were approach width interactions with presence of shared lane, bus stop, or median. CONCLUSIONS: The findings confirm that resurfacing is significant in reducing crash frequency and severity levels. Practical Applications: The study findings would help for better understanding of how geometric characteristics can be improved to reduce crash occurrence.

Beta-blocker use is an independent risk factor for thunderstorm asthma.

OBJECTIVE: To identify risk factors for thunderstorm asthma (TA) in subjects ≥15 years of age from information available in routine clinical records. METHODS: Retrospective and hospital-based case-control study of various clinical factors in all TA cases (n = 53) who presented to a single-site ED in November 2016 (TA16) and in a control group of patients (n = 156) who presented to the same ED with asthma during the pollen season over eight non-TA years. Bivariate analysis and multivariable logistic regression modelling was performed to calculate the odds of TA asthma in the presence of potential risk factors. RESULTS: A logistic regression model revealed that the odds of TA were lower for age (odds ratio [OR] 0.97, 95% confidence interval [CI] 0.95-0.99), higher for Asian country of birth (OR 4.09, 95% CI 1.40-11.95) and higher for oral beta-blocker use (OR 6.43, 95% CI 1.58-26.33) compared to controls. No difference was found between TA16 cases and controls for allergies (to medication, grass pollen, animal), hayfever, smoking, oral non-steroidal anti-inflammatory drugs, or aspirin. Newly diagnosed asthma was higher in TA16 cases versus controls (32.1% vs 12.2%, P = 0.001). CONCLUSIONS: Oral beta-blocker medications, younger age and Asian-born heritage are risk factors for TA. Further study is required to explore the potential association between beta-blockers and TA.

Longitudinal trajectories of mental health in Australian children aged 4-5 to 14-15 years.

Mental health can affect young people's sense of wellbeing and life satisfaction, their ability to participate in employment and education, and their onward opportunities in life. This paper offers a rare opportunity to longitudinally examine mental health in a population-representative study of children aged 4-5 years to 14-15 years. Using data from the Longitudinal Study of Australian Children (LSAC), this study examined maternally-reported child mental health over a 10 year period, in order to understand their initial mental health status early in life and its change over time, as measured by the Strengths and Difficulties Questionnaire. Longitudinal models were fitted from ages 4-5 to 14-15 years. Results showed that child sex, maternal mental health, socio-economic status (family income, maternal education, neighbourhood disadvantage), maternal hostility, and child temperament (persistence, sociability, reactivity) are all independent contributors to child mental health at age 4. These effects largely persist over time, with the effects of maternal mental health increasing slightly over time. Persistence of these effects suggests the need for early intervention and supports. The independent contribution of these factors to child mental health suggests that multi-faceted approaches to child and maternal mental health are needed.

Main nutrient patterns and colorectal cancer risk in the European Prospective Investigation into Cancer and Nutrition study.

BACKGROUND: Much of the current literature on diet-colorectal cancer (CRC) associations focused on studies of single foods/nutrients, whereas less is known about nutrient patterns. We investigated the association between major nutrient patterns and CRC risk in participants of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. METHODS: Among 477 312 participants, intakes of 23 nutrients were estimated from validated dietary questionnaires. Using results from a previous principal component (PC) analysis, four major nutrient patterns were identified. Hazard ratios (HRs) and 95% confidence intervals (CIs) were computed for the association of each of the four patterns and CRC incidence using multivariate Cox proportional hazards models with adjustment for established CRC risk factors. RESULTS: During an average of 11 years of follow-up, 4517 incident cases of CRC were documented. A nutrient pattern characterised by high intakes of vitamins and minerals was inversely associated with CRC (HR per 1 s.d.=0.94, 95% CI: 0.92-0.98) as was a pattern characterised by total protein, riboflavin, phosphorus and calcium (HR (1 s.d.)=0.96, 95% CI: 0.93-0.99). The remaining two patterns were not significantly associated with CRC risk. CONCLUSIONS: Analysing nutrient patterns may improve our understanding of how groups of nutrients relate to CRC.

Validity and calibration of the FFQ used in the Melbourne Collaborative Cohort Study.

OBJECTIVE: To evaluate the reliability and validity of the FFQ administered to participants in the follow-up of the Melbourne Collaborative Cohort Study (MCCS), and to provide calibration coefficients. DESIGN: A random sample stratified by country of birth, age, sex and BMI was selected from MCCS participants. Participants completed two FFQ and three 24 h recalls over 1 year. Reliability was evaluated by intraclass correlation coefficients (ICC). Validity coefficients (VC) were estimated from structural equation models and calibration coefficients obtained from regression calibration models. SETTING: Adults born in Australia, Greece or Italy. SUBJECTS: Nine hundred and sixty-five participants consented to the study; of these, 459 participants were included in the reliability analyses and 615 in the validity and calibration analyses. RESULTS: The FFQ showed good repeatability for twenty-three nutrients with ICC ranging from 0·66 to 0·80 for absolute nutrient intakes for Australian-born and from 0·51 to 0·74 for Greek/Italian-born. For Australian-born, VC ranged from 0·46 (monounsaturated fat) to 0·83 (Ca) for nutrient densities, comparing well with other studies. For Greek/Italian-born, VC were between 0·21 (Na) and 0·64 (riboflavin). Calibration coefficients for nutrient densities ranged from 0·39 (retinol) to 0·74 (Mg) for Australian-born and from 0·18 (Zn) to 0·54 (riboflavin) for Greek/Italian-born. CONCLUSIONS: The FFQ used in the MCCS follow-up study is suitable for estimating energy-adjusted nutrients for Australian-born participants. However, its performance for estimating intakes is poorer for southern European migrants and alternative dietary assessment methods ought to be considered if dietary data are to be measured in similar demographic groups.

Alemtuzumab treatment of multiple sclerosis: long-term safety and efficacy.

OBJECTIVES: Alemtuzumab is a newly licensed treatment of active relapsing-remitting multiple sclerosis (RRMS) in Europe, which in phase II and III studies demonstrated superior efficacy over ß-interferon in reducing disability progression over 2-3 years. In this observational cohort study, we sought to describe our longer-term experience of the efficacy and safety of alemtuzumab in active RRMS. METHODS: Clinical and laboratory data including serial Expanded Disability Status Scale (EDSS) assessments, from all 87 patients treated with alemtuzumab on investigator-led studies in Cambridge, UK, from 1999 to 2012, were collected. The occurrence of adverse events including secondary autoimmunity, malignancy and death, and pregnancy outcomes was recorded. Baseline variables including age, disease duration and relapse rate were compared in univariate and logistic regression analyses between groups with different disability outcomes. RESULTS: Over a median 7-year follow-up (range 33-144 months), most patients (52%) required just two cycles of alemtuzumab. In the remaining patients, relapses triggered re-treatment to a total of three cycles (36%), four cycles (8%) or five cycles (1%). Using a 6-month sustained accumulation of disability definition, 59/87 (67.8%) of patients had an improved or unchanged disability compared with baseline. By an area under the curve analysis, 52/87 (59.8%) patients had an overall improvement or stabilisation of disability. Higher baseline relapse rate was associated with worse long-term disability outcomes, with trends for longer disease duration and older age at first treatment. Secondary autoimmunity was the most frequent adverse event occurring in 41/86 (47.7%) patients, most commonly involving the thyroid gland. CONCLUSIONS: Alemtuzumab is associated with disease stabilisation in the majority of patients with highly active RRMS over an average seven-year follow-up. No new safety concerns arose over this extended follow-up.

Correcting for the bias caused by exposure measurement error in epidemiological studies.

An important goal of many epidemiological studies is to estimate the magnitude of association between an exposure and an outcome. Exposure measurement error causes bias in such estimates of association and can be substantial. In this article, we describe the problem of exposure measurement error and its effects. We show how a simple hand calculation, in conjunction with validation study data and a calibration equation, can be used to correct estimates for the bias caused by exposure measurement error. Correcting estimates of association for measurement error helps researchers appropriately assess effect size.

Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation.

The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28(-)CD57(+)), highly proliferative (Ki67(+)), oligoclonal, memory-like CD4 and CD8 T cells (CCR7(-)CD45RA(-) or CCR7(-)CD45RA(+)) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopenia-associated autoimmunity in humans.

Long term lymphocyte reconstitution after alemtuzumab treatment of multiple sclerosis.

BACKGROUND: Alemtuzumab is a lymphocyte depleting monoclonal antibody that has demonstrated superior efficacy over interferon ß-1a for relapsing-remitting multiple sclerosis (MS), and is currently under investigation in phase 3 trials. One unresolved issue is the duration and significance of the lymphopenia induced. The long term effects on lymphocyte reconstitution of a single course, and the consequences that this has on disability, morbidity, mortality and autoimmunity, were examined. METHODS: The lymphocyte reconstitution (n=36; 384 person years) and crude safety data (n=37; 447 person years) are reported for the first patients with progressive MS to receive alemtuzumab (1991-1997). Reconstitution time was expressed as a geometric mean or, when a non-negligible number of individuals failed to recover, as a median using survival analysis. RESULTS: Geometric mean recovery time (GMRT) of total lymphocyte counts to the lower limit of the normal range (LLN; ≥1.0×10(9) cells/l) was 12.7 months (95% CI 8.8 to 18.2 months). For B cells, GMRT to LLN (≥0.1×10(9)/l) was 7.1 months (95% CI 5.3 to 9.5); median recovery times for CD8 (LLN ≥0.2×10(9) cells/l) and CD4 lymphocytes (LLN ≥0.4×10(9) cells/l) were 20 months and 35 months, respectively. However, CD8 and CD4 counts recovered to baseline levels in only 30% and 21% of patients, respectively. No infective safety concerns arose during 447 person years of follow-up. CONCLUSIONS: Lymphocyte counts recovered to LLN after a single course of alemtuzumab in approximately 8 months (B cells) and 3 years (T cell subsets), but usually did not recover to baseline values. However, this long lasting lymphopenia in patients with a previously normal immune system was not associated with an increased risk of serious opportunistic infection.

Identifying dietary patterns using a normal mixture model: application to the EPIC study.

BACKGROUND: Finite mixture models posit the existence of a latent categorical variable and can be used for probabilistic classification. The authors illustrate the use of mixture models for dietary pattern analysis. An advantage of this approach is taking classification uncertainty into account. METHODS: Participants were a random sample of women from the European Prospective Investigation into Cancer. Food consumption was measured using dietary questionnaires. Mixture models identified latent classes in food consumption data, which were interpreted as dietary patterns. RESULTS: Among various assumptions examined, models allowing the variance of foods to vary within and between classes fit better than alternatives assuming constant variance (the K-means method of cluster analysis also makes the latter assumption). An eight-class model was best fitting and five patterns validated well in a second random sample. Patterns with lower classification uncertainty tended to be better validated. One pattern showed low consumption of foods despite being associated with moderate body mass index. CONCLUSION: Mixture modelling for dietary pattern analysis has advantages over both factor and cluster analysis. In contrast to these other methods, it is easy to estimate pattern prevalence, to describe patterns and to use patterns to predict disease taking classification uncertainty into account. Owing to substantial error in food consumptions, any analysis will usually find some patterns that cannot be well validated. While knowledge of classification uncertainty may aid pattern evaluation, any method will better identify patterns from food consumptions measured with less error. Mixture models may be useful to identify individuals who under-report food consumption.

Region-specific nutrient intake patterns exhibit a geographical gradient within and between European countries.

Until recently, the study of nutrient patterns was hampered at an international level by a lack of standardization of both dietary methods and nutrient databases. We aimed to describe the diversity of nutrient patterns in the European Prospective Investigation into Cancer and Nutrition (EPIC) study at population level as a starting point for future nutrient pattern analyses and their associations with chronic diseases in multi-center studies. In this cross-sectional study, 36,034 persons aged 35-74 y were administered a single, standardized 24-h dietary recall. Intake of 25 nutrients (excluding intake from dietary supplements) was estimated using a standardized nutrient database. We used a graphic presentation of mean nutrient intakes by region and sex relative to the overall EPIC means to contrast patterns within and between 10 European countries. In Mediterranean regions, including Greece, Italy, and the southern centers of Spain, the nutrient pattern was dominated by relatively high intakes of vitamin E and monounsaturated fatty acids (MUFA), whereas intakes of retinol and vitamin D were relatively low. In contrast, in Nordic countries, including Norway, Sweden, and Denmark, reported intake of these same nutrients resulted in almost the opposite pattern. Population groups in Germany, The Netherlands, and the UK shared a fatty acid pattern of relatively high intakes of PUFA and SFA and relatively low intakes of MUFA, in combination with a relatively high intake of sugar. We confirmed large variability in nutrient intakes across the EPIC study populations and identified 3 main region-specific patterns with a geographical gradient within and between European countries.

Within- and between-cohort variation in measured macronutrient intakes, taking account of measurement errors, in the European Prospective Investigation into Cancer and Nutrition study.

Multicenter epidemiologic studies provide a unique opportunity to evaluate the association between exposure and disease at the individual and the aggregate levels. The two components can eventually be pooled to corroborate each other, using weights proportional to the intraclass correlation coefficient (ICC), which expresses the amount of between-cohort variability in the exposure variable compared with the total. The greater the ICC, the more the overall estimate will reflect the between-cohort component. Dietary measurements are affected by measurement errors, particularly within a cohort. In 1992-2000, the variability of macronutrient intake distribution before and after calibration for measurement error in the European Prospective Investigation into Cancer and Nutrition was evaluated. A two-level, random-effects model was used. Evaluation of macronutrient densities revealed that energy has a considerable effect on the calibration model, leading to ICC values larger than those for the absolute intakes. Given the shrinkage of the within-center variability, a sizable increase in the ICC was observed for protein in men and women (0.48 and 0.54, respectively) and carbohydrates in men (0.41). Results suggest that the effect of calibration on macronutrient intake variability is greater for the within-cohort component, thus increasing the relative importance of the between-cohort component. After calibration, the two components had a similar weight. This observation has important implications for the analysis of multicenter studies because the between-cohort component provides a large part of the overall heterogeneity.

Seasonal misclassification error and magnitude of true between-person variation in dietary nutrient intake: a random coefficients analysis and implications for the Japan Public Health Center (JPHC) Cohort Study.

OBJECTIVE: We examine (1) the extent to which seasonal diet assessments correctly classify individuals with respect to their usual nutrient intake, and (2) whether the magnitude of true variation in intake between individuals is seasonal. These effects could lead, respectively, to bias in estimates of relative risk for associations between usual nutrient exposure and disease, and to an increase in required sample size. SUBJECTS AND SETTING: One hundred and twenty-seven families in four regions of the Japan Public Health Center (JPHC) Cohort Study. DESIGN: On average, 48 weighed daily food records were collected per family over six seasons of 1994 and 1995. RESULTS: A random slopes regression model was used to predict the correlation between seasonal and annual average intakes, and to estimate true between-person variation in intakes by season. Mean vitamin C intake was greatest in summer and autumn, and seasonal variation was attributable to the consumption of fruit and vegetables. Predicted correlations between seasonal and annual average vitamin C intake ranged from 0.62 to 0.87, with greatest correlations in summer and autumn. True between-person variation in vitamin C intake was also strongly seasonal, ranging from 45 to 78% of total variance, and was again greatest in summer and autumn. These effects were less seasonal among energy and 13 other nutrients. CONCLUSIONS: It may be possible substantially to reduce both seasonal misclassification of individuals with respect to their usual vitamin C intake, and required sample size, by asking subjects to report high-season intake of fruit and vegetables in the JPHC Study.