Melanosis of the urinary bladder.

Melanosis bladder refers to the urothelium of the bladder appearing black and velvety, with microscopic evaluation describing melanin deposition. Risk factors, pathogenesis and clinical implications are unknown because only sporadic cases are reported in the literature, both with and without the presence of urinary tract symptoms or malignancy. We report a case of melanosis bladder in a male patient with voiding urinary symptoms and an untreated hypospadias.

An epigenome-wide association study meta-analysis of educational attainment.

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications-in our case, CpG methylation-and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

Exploring residual risk for diabetes and microvascular disease in the Diabetes Prevention Program Outcomes Study (DPPOS).

AIM: Approximately half of the participants in the Diabetes Prevention Outcomes Study (DPPOS) had diabetes after 15 years of follow-up, whereas nearly all the others remained with pre-diabetes. We examined whether formerly unexplored factors in the DPPOS coexisted with known risk factors that posed additional risk for, or protection from, diabetes as well as microvascular disease. METHODS: Cox proportional hazard models were used to examine predictors of diabetes. Sequential modelling procedures considered known and formerly unexplored factors. We also constructed models to determine whether the same unexplored factors that associated with progression to diabetes also predicted the prevalence of microvascular disease. Hazard ratios (HR) are per standard deviation change in the variable. RESULTS: In models adjusted for demographics and known diabetes risk factors, two formerly unknown factors were associated with risk for both diabetes and microvascular disease: number of medications taken (HR = 1.07, 95% confidence intervals (95% CI) 1.03 to 1.12 for diabetes; odds ratio (OR) = 1.10, 95% CI 1.04 to 1.16 for microvascular disease) and variability in HbA1c (HR = 1.02, 95% CI 1.01 to 1.03 for diabetes; OR = 1.06, 95% CI 1.04 to 1.09 for microvascular disease per sd). Total comorbidities increased risk for diabetes (HR = 1.10, 95% CI 1.04 to 1.16), whereas higher systolic (OR = 1.22, 95% CI 1.13 to 1.31) and diastolic (OR = 1.14, 95% CI 1.05 to 1.22) blood pressure, as well as the use of anti-hypertensives (OR = 1.41, 95% CI 1.23 to 1.62), increased risk of microvascular disease. CONCLUSIONS: Several formerly unexplored factors in the DPPOS predicted additional risk for diabetes and/or microvascular disease - particularly hypertension and the use of anti-hypertensive medications - helping to explain some of the residual disease risk in participants of the DPPOS.

Changes in Weight and Glucose Can Protect Against Progression in Early Diabetes Independent of Improvements in ß-Cell Function.

CONTEXT: Evidence-based strategies to prevent progression of dysglycemia in newly diagnosed type 2 diabetes are needed. OBJECTIVE: To undertake a secondary analysis of the Early Diabetes Intervention Program (EDIP) in order to understand the features that were protective against worsening glycemia. DESIGN: EDIP was a randomized, placebo-controlled trial. SETTING: Two university diabetes centers. PATIENTS: A total of 219 overweight individuals with fasting glucose < 7.8 mmol/L and 2-hour oral glucose tolerance test (OGTT) glucose > 11.1 mmol/L. INTERVENTIONS: Acarbose versus placebo, on a background of dietary recommendations, with quarterly visits to assess glycemia and intervention adherence for up to 5 years. MAIN OUTCOME MEASURES: Progression of fasting glucose ≥ 7.8 mmol/L on two consecutive quarterly visits. Cox proportional hazards modeling and ANOVA were performed to evaluate determinants of progression. RESULTS: Progression-free status was associated with reductions in weight, fasting glucose, 2-hour OGTT glucose, and increases in the high-density lipoprotein/triglyceride ratio. The reduction in fasting glucose was the only effect that remained significantly associated with progression-free status in multivariable Cox modeling. The reduction in fasting glucose was in turn primarily associated with reductions in weight and in 2-hour OGTT glucose. Acarbose treatment did not explain these changes. CONCLUSIONS: In early diabetes, reductions in glucose, driven by reductions in weight, can delay progressive metabolic worsening. These observations underscore the importance of lifestyle management including weight loss as a tool to mitigate worsening of glycemia in newly diagnosed diabetes.

Assessment of myocardial metabolic flexibility and work efficiency in human type 2 diabetes using 16-[18F]fluoro-4-thiapalmitate, a novel PET fatty acid tracer.

Altered myocardial fuel selection likely underlies cardiac disease risk in diabetes, affecting oxygen demand and myocardial metabolic flexibility. We investigated myocardial fuel selection and metabolic flexibility in human type 2 diabetes mellitus (T2DM), using positron emission tomography to measure rates of myocardial fatty acid oxidation {16-[(18)F]fluoro-4-thia-palmitate (FTP)} and myocardial perfusion and total oxidation ([(11)C]acetate). Participants underwent paired studies under fasting conditions, comparing 3-h insulin + glucose euglycemic clamp conditions (120 mU·m(-2)·min(-1)) to 3-h saline infusion. Lean controls (n = 10) were compared with glycemically controlled volunteers with T2DM (n = 8). Insulin augmented heart rate, blood pressure, and stroke index in both groups (all P < 0.01) and significantly increased myocardial oxygen consumption (P = 0.04) and perfusion (P = 0.01) in both groups. Insulin suppressed available nonesterified fatty acids (P < 0.0001), but fatty acid concentrations were higher in T2DM under both conditions (P < 0.001). Insulin-induced suppression of fatty acid oxidation was seen in both groups (P < 0.0001). However, fatty acid oxidation rates were higher under both conditions in T2DM (P = 0.003). Myocardial work efficiency was lower in T2DM (P = 0.006) and decreased in both groups with the insulin-induced increase in work and shift in fuel utilization (P = 0.01). Augmented fatty acid oxidation is present under baseline and insulin-treated conditions in T2DM, with impaired insulin-induced shifts away from fatty acid oxidation. This is accompanied by reduced work efficiency, possibly due to greater oxygen consumption with fatty acid metabolism. These observations suggest that improved fatty acid suppression, or reductions in myocardial fatty acid uptake and retention, could be therapeutic targets to improve myocardial ischemia tolerance in T2DM.

GWAS for executive function and processing speed suggests involvement of the CADM2 gene.

To identify common variants contributing to normal variation in two specific domains of cognitive functioning, we conducted a genome-wide association study (GWAS) of executive functioning and information processing speed in non-demented older adults from the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) consortium. Neuropsychological testing was available for 5429-32,070 subjects of European ancestry aged 45 years or older, free of dementia and clinical stroke at the time of cognitive testing from 20 cohorts in the discovery phase. We analyzed performance on the Trail Making Test parts A and B, the Letter Digit Substitution Test (LDST), the Digit Symbol Substitution Task (DSST), semantic and phonemic fluency tests, and the Stroop Color and Word Test. Replication was sought in 1311-21860 subjects from 20 independent cohorts. A significant association was observed in the discovery cohorts for the single-nucleotide polymorphism (SNP) rs17518584 (discovery P-value=3.12 × 10(-8)) and in the joint discovery and replication meta-analysis (P-value=3.28 × 10(-9) after adjustment for age, gender and education) in an intron of the gene cell adhesion molecule 2 (CADM2) for performance on the LDST/DSST. Rs17518584 is located about 170 kb upstream of the transcription start site of the major transcript for the CADM2 gene, but is within an intron of a variant transcript that includes an alternative first exon. The variant is associated with expression of CADM2 in the cingulate cortex (P-value=4 × 10(-4)). The protein encoded by CADM2 is involved in glutamate signaling (P-value=7.22 × 10(-15)), gamma-aminobutyric acid (GABA) transport (P-value=1.36 × 10(-11)) and neuron cell-cell adhesion (P-value=1.48 × 10(-13)). Our findings suggest that genetic variation in the CADM2 gene is associated with individual differences in information processing speed.

Steroid Sex Hormones, Sex Hormone-Binding Globulin, and Diabetes Incidence in the Diabetes Prevention Program.

CONTEXT: Steroid sex hormones and SHBG may modify metabolism and diabetes risk, with implications for sex-specific diabetes risk and effects of prevention interventions. OBJECTIVE: This study aimed to evaluate the relationships of steroid sex hormones, SHBG and SHBG single-nucleotide polymorphisms (SNPs) with diabetes risk factors and with progression to diabetes in the Diabetes Prevention Program (DPP). DESIGN AND SETTING: This was a secondary analysis of a multicenter randomized clinical trial involving 27 U.S. academic institutions. PARTICIPANTS: The study included 2898 DPP participants: 969 men, 948 premenopausal women not taking exogenous sex hormones, 550 postmenopausal women not taking exogenous sex hormones, and 431 postmenopausal women taking exogenous sex hormones. INTERVENTIONS: Participants were randomized to receive intensive lifestyle intervention, metformin, or placebo. MAIN OUTCOMES: Associations of steroid sex hormones, SHBG, and SHBG SNPs with glycemia and diabetes risk factors, and with incident diabetes over median 3.0 years (maximum, 5.0 y). RESULTS: T and DHT were inversely associated with fasting glucose in men, and estrone sulfate was directly associated with 2-hour post-challenge glucose in men and premenopausal women. SHBG was associated with fasting glucose in premenopausal women not taking exogenous sex hormones, and in postmenopausal women taking exogenous sex hormones, but not in the other groups. Diabetes incidence was directly associated with estrone and estradiol and inversely with T in men; the association with T was lost after adjustment for waist circumference. Sex steroids were not associated with diabetes outcomes in women. SHBG and SHBG SNPs did not predict incident diabetes in the DPP population. CONCLUSIONS: Estrogens and T predicted diabetes risk in men but not in women. SHBG and its polymorphisms did not predict risk in men or women. Diabetes risk is more potently determined by obesity and glycemia than by sex hormones.

Failure of hyperglycemia and hyperinsulinemia to compensate for impaired metabolic response to an oral glucose load.

OBJECTIVE: To evaluate whether the augmented insulin and glucose response to a glucose challenge is sufficient to compensate for defects in glucose utilization in obesity and type 2 diabetes, using a breath test measurement of integrated glucose metabolism. METHODS: Non-obese, obese normoglycemic and obese type 2 diabetic subjects were studied on 2 consecutive days. A 75g oral glucose load spiked with ¹³C-glucose was administered, measuring exhaled breath ¹³CO2 as an integrated measure of glucose metabolism and oxidation. A hyperinsulinemic euglycemic clamp was performed, measuring whole body glucose disposal rate. Body composition was measured by DEXA. Multivariable analyses were performed to evaluate the determinants of the breath ¹³CO2. RESULTS: Breath ¹³CO2 was reduced in obese and type 2 diabetic subjects despite hyperglycemia and hyperinsulinemia. The primary determinants of breath response were lean mass, fat mass, fasting FFA concentrations, and OGTT glucose excursion. Multiple approaches to analysis showed that hyperglycemia and hyperinsulinemia were not sufficient to compensate for the defect in glucose metabolism in obesity and diabetes. CONCLUSIONS: Augmented insulin and glucose responses during an OGTT are not sufficient to overcome the underlying defects in glucose metabolism in obesity and diabetes.

Hydrous mantle transition zone indicated by ringwoodite included within diamond.

The ultimate origin of water in the Earth's hydrosphere is in the deep Earth--the mantle. Theory and experiments have shown that although the water storage capacity of olivine-dominated shallow mantle is limited, the Earth's transition zone, at depths between 410 and 660 kilometres, could be a major repository for water, owing to the ability of the higher-pressure polymorphs of olivine--wadsleyite and ringwoodite--to host enough water to comprise up to around 2.5 per cent of their weight. A hydrous transition zone may have a key role in terrestrial magmatism and plate tectonics, yet despite experimental demonstration of the water-bearing capacity of these phases, geophysical probes such as electrical conductivity have provided conflicting results, and the issue of whether the transition zone contains abundant water remains highly controversial. Here we report X-ray diffraction, Raman and infrared spectroscopic data that provide, to our knowledge, the first evidence for the terrestrial occurrence of any higher-pressure polymorph of olivine: we find ringwoodite included in a diamond from Juína, Brazil. The water-rich nature of this inclusion, indicated by infrared absorption, along with the preservation of the ringwoodite, is direct evidence that, at least locally, the transition zone is hydrous, to about 1 weight per cent. The finding also indicates that some kimberlites must have their primary sources in this deep mantle region.

Genetic effects on information processing speed are moderated by age--converging results from three samples.

Information processing is a cognitive trait forming the basis of complex abilities like executive function. The Trail Making Test (TMT) is a well-established test of information processing with moderate to high heritability. Age of the individual also plays an important role. A number of genetic association studies with the TMT have been performed, which, however, did not consider age as a moderating factor. We report the results of genome-wide association studies (GWASs) on age-independent and age-dependent TMT performance in two population-representative community samples (Munich Antidepressant Response Signature, MARS: N1 = 540; Ludwig Maximilians University, LMU: N2 = 350). Age-dependent genome-wide findings were then evaluated in a third sample of healthy elderly subjects (Sydney Memory and Ageing Study, Sydney MAS: N3 = 448). While a meta-analysis on the GWAS findings did not reveal age-independent TMT associations withstanding correction for multiple testing, we found a genome-wide significant age-moderated effect between variants in the DSG1 gene region and TMT-A performance predominantly reflecting visual processing speed (rs2199301, P(meta-analysis) = 1.3 × 10(-7)). The direction of the interaction suggests for the minor allele a beneficial effect in younger adults turning into a detrimental effect in older adults. The detrimental effect of the missense single nucleotide polymorphism rs1426310 within the same DSG1 gene region could be replicated in Sydney MAS participants aged 70-79, but not in those aged 80 years and older, presumably a result of survivor bias. Our findings demonstrate opposing effects of DSG1 variants on information processing speed depending on age, which might be related to the complex processes that DSG1 is involved with, including cell adhesion and apoptosis.

Metabolic syndrome components and their response to lifestyle and metformin interventions are associated with differences in diabetes risk in persons with impaired glucose tolerance.

AIMS: To determine the association of metabolic syndrome (MetS) and its components with diabetes risk in participants with impaired glucose tolerance (IGT), and whether intervention-related changes in MetS lead to differences in diabetes incidence. METHODS: We used the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) revised MetS definition at baseline and intervention-related changes of its components to predict incident diabetes using Cox models in 3234 Diabetes Prevention Program (DPP) participants with IGT over an average follow-up of 3.2 years. RESULTS: In an intention-to-treat analysis, the demographic-adjusted hazard ratios (95% confidence interval) for diabetes in those with MetS (vs. no MetS) at baseline were 1.7 (1.3-2.3), 1.7 (1.2-2.3) and 2.0 (1.3-3.0) for placebo, metformin and lifestyle groups, respectively. Higher levels of fasting plasma glucose and triglycerides at baseline were independently associated with increased risk of diabetes. Greater waist circumference (WC) was associated with higher risk in placebo and lifestyle groups, but not in the metformin group. In a multivariate model, favourable changes in WC (placebo and lifestyle) and high-density lipoprotein cholesterol (placebo and metformin) contributed to reduced diabetes risk. CONCLUSIONS: MetS and some of its components are associated with increased diabetes incidence in persons with IGT in a manner that differed according to DPP intervention. After hyperglycaemia, the most predictive factors for diabetes were baseline hypertriglyceridaemia and both baseline and lifestyle-associated changes in WC. Targeting these cardiometabolic risk factors may help to assess the benefits of interventions that reduce diabetes incidence.

Lifestyle and metformin treatment favorably influence lipoprotein subfraction distribution in the Diabetes Prevention Program.

CONTEXT: Although intensive lifestyle change (ILS) and metformin reduce diabetes incidence in subjects with impaired glucose tolerance (IGT), their effects on lipoprotein subfractions have not been studied. OBJECTIVE: The objective of the study was to characterize the effects of ILS and metformin vs placebo interventions on lipoprotein subfractions in the Diabetes Prevention Program. DESIGN: This was a randomized clinical trial, testing the effects of ILS, metformin, and placebo on diabetes development in subjects with IGT. PARTICIPANTS: Selected individuals with IGT randomized in the Diabetes Prevention Program participated in the study. INTERVENTIONS: Interventions included randomization to metformin 850 mg or placebo twice daily or ILS aimed at a 7% weight loss using a low-fat diet with increased physical activity. MAIN OUTCOME MEASURES: Lipoprotein subfraction size, density, and concentration measured by magnetic resonance and density gradient ultracentrifugation at baseline and 1 year were measured. RESULTS: ILS decreased large and buoyant very low-density lipoprotein, small and dense low-density lipoprotein (LDL), and small high-density lipoprotein (HDL) and raised large HDL. Metformin modestly reduced small and dense LDL and raised small and large HDL. Change in insulin resistance largely accounted for the intervention-associated decreases in large very low-density lipoprotein, whereas changes in body mass index (BMI) and adiponectin were strongly associated with changes in LDL. Baseline and a change in adiponectin were related to change in large HDL, and BMI change associated with small HDL change. The effect of metformin to increase small HDL was independent of adiponectin, BMI, and insulin resistance. CONCLUSION: ILS and metformin treatment have favorable effects on lipoprotein subfractions that are primarily mediated by intervention-related changes in insulin resistance, BMI, and adiponectin. Interventions that slow the development of diabetes may also retard the progression of atherosclerosis.

Effect of acarbose to delay progression of carotid intima-media thickness in early diabetes.

BACKGROUND: The anti-diabetic agent acarbose reduces postprandial glucose excursions. We have evaluated the effect of randomized treatment with acarbose on the progression of carotid intima-media thickness (IMT) in early diabetes. METHODS: The Early Diabetes Intervention Program was a randomized trial of acarbose versus placebo in 219 participants with early diabetes characterized by glucose values over 11.1 mmol/L 2 h after a 75 g oral glucose load and a mean HbA1c of 6.3%. IMT was measured at baseline and yearly. Follow-up was discontinued if participants progressed to the study glucose endpoints; IMT readings were available for a median of 2 years, with 72 subjects followed for 5 years. RESULTS: Progressive increases in IMT were seen in both treatment groups, but progression was reduced in participants randomized to acarbose (p = 0.047). In age, sex and smoking-adjusted analyses, IMT progression was associated with greater fasting and oral glucose tolerance test-excursion glucose, fasting insulin, cholesterol and glycated low-density lipoprotein concentrations. IMT progression was reduced with study-related changes in weight, insulin and non-esterified fatty acids; these features were more strongly associated with reduced IMT progression than acarbose treatment. Despite strong associations of baseline glycemia with IMT progression, study-related changes in glucose were not important determinants of IMT progression. CONCLUSIONS: Acarbose can delay progression of carotid intima-media thickness in early diabetes defined by an oral glucose tolerance test. Glucose, weight, insulin and lipids contributed to risk of progression but reductions in glycemia were not major determinants of reduced rate of IMT progression. Vascular benefits of acarbose may be independent of its glycemic effects.

Long-term effects of the Diabetes Prevention Program interventions on cardiovascular risk factors: a report from the DPP Outcomes Study.

AIMS: Whether long-term cardiovascular risk is reduced by the Diabetes Prevention Program interventions is unknown. The aim of this study was to determine the long-term differences in cardiovascular disease risk factors and the use of lipid and blood pressure medications by the original Diabetes Prevention Program intervention group. METHODS: This long-term follow-up (median 10 years, interquartile range 9.0-10.5) of the three-arm Diabetes Prevention Program randomized controlled clinical trial (metformin, intensive lifestyle and placebo), performed on 2766 (88%) of the Diabetes Prevention Program participants (who originally had impaired glucose tolerance), comprised a mean of 3.2 years of randomized treatment, approximately 1-year transition (during which all participants were offered intensive lifestyle intervention) and 5 years follow-up (Diabetes Prevention Program Outcomes Study). During the study, participants were followed in their original groups with their clinical care being provided by practitioners outside the research setting. The study determined lipoprotein profiles and blood pressure and medication use annually. RESULTS: After 10 years' follow-up from Diabetes Prevention Program baseline, major reductions were seen for systolic (-2 to -3) and diastolic (-6 to -6.5 mmHg) blood pressure, and for LDL cholesterol (-0.51 to -0.6 mmol/l) and triglycerides (-0.23 to -0.25 mmol/l) in all groups, with no between-group differences. HDL cholesterol also rose significantly (0.14 to 0.15 mmol/l) in all groups. Lipid (P = 0.01) and blood pressure (P = 0.09) medication use, however, were lower for the lifestyle group during the Diabetes Prevention Program Outcomes Study. CONCLUSION: Overall, intensive lifestyle intervention achieved, with less medication, a comparable long-term effect on cardiovascular disease risk factors, to that seen in the metformin and placebo groups.

Common variants in genes encoding adiponectin (ADIPOQ) and its receptors (ADIPOR1/2), adiponectin concentrations, and diabetes incidence in the Diabetes Prevention Program.

AIMS: Baseline adiponectin concentrations predict incident Type 2 diabetes mellitus in the Diabetes Prevention Program. We tested the hypothesis that common variants in the genes encoding adiponectin (ADIPOQ) and its receptors (ADIPOR1, ADIPOR2) would associate with circulating adiponectin concentrations and/or with diabetes incidence in the Diabetes Prevention Program population. METHODS: Seventy-seven tagging single-nucleotide polymorphisms (SNPs) in ADIPOQ (24), ADIPOR1 (22) and ADIPOR2 (31) were genotyped. Associations of SNPs with baseline adiponectin concentrations were evaluated using linear modelling. Associations of SNPs with diabetes incidence were evaluated using Cox proportional hazards modelling. RESULTS: Thirteen of 24 ADIPOQ SNPs were significantly associated with baseline adiponectin concentrations. Multivariable analysis including these 13 SNPs revealed strong independent contributions of rs17366568, rs1648707, rs17373414 and rs1403696 with adiponectin concentrations. However, no ADIPOQ SNPs were directly associated with diabetes incidence. Two ADIPOR1 SNPs (rs1342387 and rs12733285) were associated with ∼18% increased diabetes incidence for carriers of the minor allele without differences across treatment groups, and without any relationship with adiponectin concentrations. CONCLUSIONS: ADIPOQ SNPs are significantly associated with adiponectin concentrations in the Diabetes Prevention Program cohort. This observation extends prior observations from unselected populations of European descent into a broader multi-ethnic population, and confirms the relevance of these variants in an obese/dysglycaemic population. Despite the robust relationship between adiponectin concentrations and diabetes risk in this cohort, variants in ADIPOQ that relate to adiponectin concentrations do not relate to diabetes risk in this population. ADIPOR1 variants exerted significant effects on diabetes risk distinct from any effect of adiponectin concentrations.

Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination.

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ~3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

Effects of losartan on whole body, skeletal muscle and vascular insulin responses in obesity/insulin resistance without hypertension.

AIMS: Renin-angiotensin system antagonists have been found to improve glucose metabolism in obese hypertensive and type 2 diabetic subjects. The mechanism of these effects is not well understood. We hypothesized that the angiotensin receptor antagonist losartan would improve insulin-mediated vasodilation, and thereby improve insulin-stimulated glucose uptake in skeletal muscle of insulin-resistant subjects. METHODS: We studied subjects with obesity and insulin resistance but without hypertension, hypercholesterolaemia or dysglycaemia [age 39.0 ± 9.6 yr (mean ± SD), body mass index (BMI) 33.2 ± 5.9 kg/m(2) , BP 115.8 ± 12.2/70.9 ± 7.2 mmHg, LDL 2.1 ± 0.5 mmol/l]. Subjects were randomized to 12 weeks' double-blind treatment with losartan 100 mg once daily (n = 9) or matching placebo (n = 8). Before and after treatment, under hyperinsulinaemic euglycaemic clamp conditions we measured whole-body insulin-stimulated glucose disposal, insulin-mediated vasodilation, and insulin-stimulated leg glucose uptake by the limb balance technique. RESULTS: Whole-body insulin-stimulated glucose disposal was not significantly increased by losartan. Insulin-mediated vasodilation was augmented following both treatments [increase in leg vascular conductance: pretreatment 0.7 ± 0.3 l/min/mmHg (losartan, mean ± SEM) and 0.9 ± 0.3 (placebo), posttreatment 1.0 ± 0.4 (losartan) and 1.3 ± 0.6 (placebo)] but not different between treatment groups (p = 0.53). Insulin's action to augment nitric oxide (NO) production and to augment endothelium-dependent vasodilation was also not improved. Leg glucose uptake was not significantly changed by treatments, and not different between groups (p = 0.11). CONCLUSIONS: These findings argue against the hypothesis that losartan might improve skeletal muscle glucose metabolism by improving insulin-mediated vasodilation in normotensive insulin-resistant obese subjects. The metabolic benefits of angiotensin receptor blockers may require the presence of hypertension in addition to obesity-associated insulin resistance.

A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture.

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.

Association of the SLC30A8 missense polymorphism R325W with proinsulin levels at baseline and after lifestyle, metformin or troglitazone intervention in the Diabetes Prevention Program.

AIMS/HYPOTHESIS: Individuals with impaired glucose tolerance have increased proinsulin levels, despite normal glucose or C-peptide levels. In the Diabetes Prevention Program (DPP), increased proinsulin levels predicted type 2 diabetes and proinsulin levels were significantly reduced following treatment with metformin, lifestyle modification or troglitazone compared with placebo. Genetic and physiological studies suggest a role for the zinc transporter gene SLC30A8 in diabetes risk, possibly through effects on insulin-processing in beta cells. We hypothesised that the risk allele at the type 2 diabetes-associated missense polymorphism rs13266634 (R325W) in SLC30A8 would predict proinsulin levels in individuals at risk of type 2 diabetes and may modulate response to preventive interventions. METHODS: We genotyped rs13266634 in 3,007 DPP participants and examined its association with fasting proinsulin and fasting insulin at baseline and at 1 year post-intervention. RESULTS: We found that increasing dosage of the C risk allele at SLC30A8 rs13266634 was significantly associated with higher proinsulin levels at baseline (p = 0.002) after adjustment for baseline insulin. This supports the hypothesis that risk alleles at SLC30A8 mark individuals with insulin-processing defects. At the 1 year analysis, proinsulin levels decreased significantly in all groups receiving active intervention and were no longer associated with SLC30A8 genotype (p = 0.86) after adjustment for insulin at baseline and 1 year. We found no genotype × treatment interactions at 1 year. CONCLUSIONS/INTERPRETATION: In prediabetic individuals, genotype at SLC30A8 predicts baseline proinsulin levels independently of insulin levels, but does not predict proinsulin levels after amelioration of insulin sensitivity at 1 year.

A cross-sectional evaluation of seasonality as a determinant of endothelial function.

Endothelium-dependent vasodilation is impaired in obese versus lean humans. We set out to evaluate whether agonist-mediated endothelium-dependent vasodilation varies by season in a cross-sectional dataset of lean and obese humans, and whether this effect differed by obesity status. All vascular studies performed in our laboratory over a 12 year period from 1997 to 2009 were evaluated. Endothelium-dependent vasodilation was measured invasively using thermodilution in the leg as the response to intra-arterially infused methacholine chloride. Resting blood pressure was measured concurrently by cuff and intra-arterially. The association of endothelium-dependent vasodilation and blood pressure measurements with season was evaluated, comparing responses in lean and obese subjects. Endothelium-dependent vasodilation differed between lean and obese subjects, and varied across seasons (p=0.02), but without an interaction between season effect and obesity status. The proportion of obese versus lean subjects differed significantly across seasons in our dataset, and after adjusting for this factor the apparent seasonal variation in endothelium-dependent vasodilation was lost (p=0.12), and was not present in either lean or obese subjects separately. Systolic arterial blood pressure varied across seasons, and this effect remained significant after adjusting obesity status (p=0.019 and p=0.043 for blood pressures measured intra-arterially and by cuff respectively). In our cross-sectional dataset, seasonal variations in blood pressure were seen but we did not observe an association between season and endothelium-dependent vasodilation in lean or obese subjects.