Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes

Relationship between fatty liver and glucose metabolism: A cross-sectional study in 571 obese children

BACKGROUND AND AIMS: Early onset type 2 diabetes mellitus (T2DM) is associated with obesity, insulin resistance and impaired beta-cell function. Non-alcoholic fatty liver disease (NAFLD) may be an independent risk factor for T2DM. We investigated the relationship between NAFLD and glucose metabolism in a large sample of obese children. METHODS AND RESULTS: A total of 571 obese children (57% males and 43% females) aged 8-18 years were consecutively studied at a tertiary care centre specialised in paediatric obesity. Liver ultrasonography was used to diagnose NAFLD after exclusion of hepatitis B and C and alcohol consumption. Oral-glucose tolerance testing (OGTT) was performed; insulin sensitivity was evaluated by using the insulin sensitivity index (ISI) and beta-cell function by using the ratio between the incremental areas under the curve (AUC) of insulin and glucose (incAUCins/incAUCglu). A total of 41% of the obese children had NAFLD. Impaired glucose tolerance or T2DM was present in 25% of the children with NAFLD versus 8% of those without it (p<0.001). Children with NAFLD had higher body mass index (BMI), fasting glucose, 120-min OGTT glucose, incAUCins/incAUCglu and lower ISI as compared with children without NAFLD (p</=0.002). At bootstrapped multivariable median regression analysis controlling for gender, age, pubertal status and BMI, NAFLD was an independent predictor of 120-min OGTT glucose and ISI, but not of incAUCins/incAUCglu. Similar findings were obtained using continuous liver steatosis as the predictor, instead of dichotomous NAFLD. CONCLUSION: NAFLD was present in 41% of our obese children and was associated with higher insulin resistance, but not with impaired beta-cell function

Biodiversity of grapevines (Vitis vinifera L.) grown in the Province of Verona

PCR-based DNA microsatellite analysis has been applied to define the genetic relationships among 7 most representative grapevine cultivars grown in the province of Verona, 5 ancient grapevine and two varieties grown in different regions of Italy. For each variety three different clones or accessions were investigated to assess genotypical uniformity; in 5 cases we found out intravarietal dissimilarity. SSR data were used to create a distance matrix and then a polylogenetic tree. Results show a polygenetic relationship among some cultivated (Corvina, Rondinella, Molinara, Trebbiano di Soave-Verdicchio) and ancient (Dindarella-Pelara, Oseleta, Rossetta di montagna) varieties all grown in the Valpolicella hills, suggesting the possibility that their evolution occurred in the same area and with few common anchestors. Two situations of synonyms that had already described between Trebbiano di Soave and Verdicchio, and between Dindarella and Pelara, were confirmed by a molecular method as SSR analysis. Amplification of Trebbiano di Soave/Verdicchio locus VVMD36 yielded a fragment of 500 bp, this allele provides a fast and reliable tool to differentiate among Trebbiano grapevines.

Elevated Concentrations of Liver Enzymes and Ferritin Identify a New Phenotype of Insulin Resistance: Effect of Weight Loss After Gastric Banding

BACKGROUND: Several studies have associated elevated liver enzymes (LFTs), obesity, and type 2 diabetes (T2DM), and a link has been established between insulin resistance (IR) and elevated ferritin concentrations. We examined the relationship between LFTs, ferritin, and IR in morbid obese subjects and the effect of weight loss after bariatric surgery. METHODS: We measured liver enzymes, ferritin, insulin resistance, and glucose tolerance (by OGTT) in 159 morbid obese subjects (BMI = 44.4 +/- 0.4 kg/m(2)) at baseline, 6 months and 1 year after laparoscopic-adjustable-gastric banding (LAGB). Subjects were divided in two groups: increased LFTs (ALT > 30; AST/ALT < 1) vs. normal LFTs. RESULTS: A large proportion of morbid obese subjects had increased LFTs (44%) which were associated with increased IR and ferritin, suggesting potential liver disease. A majority of the morbidly obese with increased LFTs, IGT, and T2DM, were male and had almost double ferritin concentrations, strongly correlated with ALT (r = 0.43, p < 0.0001). Both ferritin and ALT correlated with waist circumference and IR. One year after, LAGB glucose tolerance improved, LFTs and IR were reduced; ferritin did not change significantly, but was still correlated with IR. CONCLUSIONS: Ferritin may be an additional useful marker for more severe hepatic IR

Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35

The human adenovirus serotype 35 (HAdV-35, short Ad35) causes kidney and urinary tract infections, and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here, we show that infectious entry of Ad35 into HeLa, human kidney HK-2 cells and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate, and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180 which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against the serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3 or the sodium-proton exchange inhibitor EIPA blocked the endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1 or the Pak1 effector C-terminal binding protein 1 (CtBP1) potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy and live cell imaging showed that Ad35 colocalized with fluid phase markers in large endocytic structures that were positive for CD46, alpha v integrins and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3), and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells

Liver Enzymes Are Associated With Hepatic Insulin Resistance, Insulin Secretion, and Glucagon Concentration in Healthy Men and Women

International audienceOBJECTIVE: The pathophysiological mechanisms to explain the association between risk of type 2 diabetes and elevated concentrations of γ-glutamyltransferase (GGT) and alanineaminotransferase (ALT) remain poorly characterized. We explored the association of liver enzymes with peripheral and hepatic insulin resistance, insulin secretion, insulin clearance, and glucagon concentration. RESEARCH DESIGN AND METHODS: We studied 1,309 nondiabetic individuals from the Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) study; all had a euglycemic-hyperinsulinemic clamp and an oral glucose tolerance test (OGTT) with assessment of insulin secretion and hepatic insulin extraction. The hepatic insulin resistance index was calculated in 393 individuals. RESULTS: In both men and women, plasma concentrations of GGT and ALT were inversely related with insulin sensitivity (M/I) (all P < 0.01). Likewise, the hepatic insulin resistance index was positively correlated with both GGT (r = 0.37, P < 0.0001, men; r = 0.36, P < 0.0001, women) and ALT (r = 0.25, P = 0.0005, men; r = 0.18, P = 0.01, women). These associations persisted in multivariable models. Increased GGT and ALT were significantly associated with higher insulin secretion rates and with both reduced endogenous clearance of insulin and hepatic insulin extraction during the OGTT (P = 0.0005 in men; P = 0.003 in women). Plasma fasting glucagon levels increased over ALT quartiles (men, quartile 4 vs. quartile 1 11.2 ± 5.1 vs. 9.3 ± 3.8 pmol/L, respectively, P = 0.0002; women, 9.0 ± 4.3 vs. 7.6 ± 3.1, P = 0.001). CONCLUSIONS: In healthy individuals, increased GGT and ALT were biomarkers of both systemic and hepatic insulin resistance with concomitant increased insulin secretion and decreased hepatic insulin clearance. The novel finding of a positive correlation between ALT and fasting glucagon level concentrations warrants confirmation in type 2 diabetes

Effects of Probiotic Supplementation on Gastrointestinal, Sensory and Core Symptoms in Autism Spectrum Disorders: A Randomized Controlled Trial

The microbiota-gut-brain axis has been recently recognized as a key modulator of neuropsychiatric health. In this framework, probiotics (recently named “psychobiotics”) may modulate brain activity and function, possibly improving the behavioral profiles of children with Autism Spectrum Disorder (ASD). We evaluated the effects of probiotics on autism in a double-blind randomized, placebo-controlled trial of 85 preschoolers with ASD (mean age, 4.2 years; 84% boys). Participants were randomly assigned to probiotics (De Simone Formulation) (n=42) or placebo (n=43) for six months. Sixty-three (74%) children completed the trial. No differences between groups were detected on the primary outcome measure, the Total Autism Diagnostic Observation Schedule - Calibrated Severity Score (ADOS-CSS). An exploratory secondary analysis on subgroups of children with or without Gastrointestinal Symptoms (GI group, n= 30; NGI group, n=55) revealed in the NGI group treated with probiotics a significant decline in ADOS scores as compared to that in the placebo group, with a mean reduction of 0.81 in Total ADOS CSS and of 1.14 in Social-Affect ADOS CSS over six months. In the GI group treated with probiotics we found greater improvements in some GI symptoms, adaptive functioning, and sensory profiles than in the GI group treated with placebo. These results suggest potentially positive effects of probiotics on core autism symptoms in a subset of ASD children independent of the specific intermediation of the probiotic effect on GI symptoms. Further studies are warranted to replicate and extend these promising findings on a wider population with subsets of ASD patients which share targets of intervention on the microbiota-gut-brain axis. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02708901

Clinical update on non-alcoholic fatty liver disease and steatohepatitis

clusion of less common causes of fatty liver such as Hepatitis B or C infection, drugs and autoimmune disorders, fatty liver is classified as non-alcoholic (NAFLD) or alcoholic fatty liver disease (AFLD) on the basis of daily ethanol intake. The agreed threshold of ethanol intake used to separate NAFLD from AFLD is 20 g/day, equivalent to 12 drinks per week. 2 The term NAFLD covers a wide spectrum of liver injury, ranging from simple steatosis, i.e. fatty infiltration of hepatocytes > 5%, to non-alcoholic steatohepatitis (NASH). At liver biopsy NASH is characterized by necro-inflammation and/or fibrosis, and is histologically indistinguishable from alcoholic steatohepatitis. The histological classification of NAFLD and NASH is being continuously improved and a refined scoring system has been recently proposed by the National Institutes of Health. 3 NAFLD is presently regarded as the most common liver disease in Western countries and virtually impacts all fields of clinical medicine, being considered a risk factor for advanced liver disease, type 2 diabetes and cardiovascular disease. Although NAFLD had long been recognized as a frequent clinical entity, only recently its importance as a potential cause of progressive and severe liver disease has been fully acknowledged. NAFLD prevalence in the general population ranges from 16 to 25% in adults 4 and from 10 to 19% in children. 5 It increases with age, shows different gender specificity according to geographic areas (generally more prevalent in women in US and viceversa in men in Europe and Japan) and varies with ethnicity, with the highest prevalence among the Hispanic population and the lowest among the AfroAmerican one. 6 Prevalence rates of NAFLD are strikingly increased in certain subpopulations, such as patients with obesity, type 2 diabetes mellitus and dyslipidemia, and the whole spectrum of NAFLD is commonly associated with the metabolic syndrome. The true prevalence of NASH remains largely unknown. According to data derived from autopsy or liver biopsy studies, about 10-15% of NAFLD patients meet the current diagnostic criteria for NASH, pointing to a prevalence of 2-3% in the general population. The incidence and natural course of NAFLD are also difficult to assess because most of the available studies are retrospective and/or performed on clinical series. The few prospective studies are too short to exclude late complications. While simple steatosis has a benign prognosis, NASH may progress to cirrhosis in 30% of cases and is responsible for liver-related death in 3-8%. In clinical series, up to 70% of patients with cryptogenic cirrhosis have clinical features suggestive of NASH.

Angiopoietin-like protein 4 overexpression in visceral adipose tissue from obese subjects with impaired glucose metabolism and relationship with lipoprotein lipase

Angiopoietin-like protein 4 (ANGPTL4) regulates lipid partitioning by inhibiting circulating and tissue lipoprotein lipase (LPL); ANGPTL4 loss-of-function variants improve insulin sensitivity and reduce type 2 diabetes (T2D) risk with mechanisms partially unknown. This study was designed to explore metabolic implications of differential ANGPTL4 and LPL expression in human adipose tissue (AT). We recruited eighty-eight obese individuals, with and without abnormal glucose metabolism (AGM), undergoing bariatric surgery; visceral AT (VAT) fragments were obtained intra-operatively and analyzed by immunohistochemistry and mRNA by rt-PCR. Data on hepatic ANGPTL4 mRNA were available for 40 participants. VAT ANGPTL4 expression was higher in AGM individuals than in those with normal glucose tolerance (NGT) and associated with VAT inflammation, insulin resistance, and presence of adipocyte size heterogeneity. Increased ANGPTL4 was associated with AGM with OR = 5.1 (95% C.I.: 1.2–23; p = 0.02) and AUROC = 0.76 (95% C.I.: 1.2–23; p &lt; 0.001). High LPL was associated with the detection of homogeneous adipocyte size, reduced microvessel density, and higher HIF-1α levels and inversely correlated to blood transaminases. In conclusion, in obese individuals, VAT ANGPTL4 levels are increased in the presence of local inflammation and AGM. Conversely, higher LPL expression describes a condition of increased lipid storage in adipocytes, which may serve as a protective mechanism against ectopic fat accumulation and related metabolic disease in obesity

The antidepressant fluoxetine acts on energy balance and leptin sensitivity via BDNF

Leptin and Brain Derived Neurotrophic Factor (BDNF) pathways are critical players in body weight homeostasis. Noninvasive treatments like environmental stimulation are able to increase response to leptin and induce BDNF expression in the brain. Emerging evidences point to the antidepressant selective serotonin reuptake inhibitor Fluoxetine (FLX) as a drug with effects similar to environmental stimulation. FLX is known to impact on body weight, with mechanisms yet to be elucidated. We herein asked whether FLX affects energy balance, the leptin system and BDNF function. Adult lean male mice chronically treated with FLX showed reduced weight gain, higher energy expenditure, increased sensitivity to acute leptin, increased hypothalamic BDNF expression, associated to changes in white adipose tissue expression typical of "brownization". In the Ntrk2tm1Ddg/J model, carrying a mutation in the BDNF receptor Tyrosine kinase B (TrkB), these effects are partially or totally reversed. Wild type obese mice treated with FLX showed reduced weight gain, increased energy output, and differently from untreated obese mice, a preserved acute response to leptin in terms of activation of the intracellular leptin transducer STAT3. In conclusion, FLX impacts on energy balance and induces leptin sensitivity and an intact TrkB function is required for these effects to take place