Development of biosensor-based assays to identify anti-infective oligosaccharides

It is now well accepted that milk oligosaccharides can have a direct inhibitory effect on pathogenic microorganisms by interfering with their adhesion to human cells. Many free oligosaccharides from milk are considered to be soluble receptor analogs of epithelial cell surface carbohydrates and, thus, function as receptor decoys to which pathogens can bind instead of the host. In reality, there are few rapid methods to screen for such oligosaccharides, and much of the research in this area has centered on using human cell line models of infection that are time-consuming. Therefore, a quick and sensitive method is required for detecting the binding of microorganisms to milk oligosaccharides. Our study describes a number of biosensor-based methods to achieve these aims. Our approach involved the exposure of whole bacterial cells to the well-characterized human milk oligosaccharide, 2′-fucosyllactose, immobilized to a pretreated gold chip surface. The technique was validated by screening a range of pathogenic bacteria, including Campylobacter jejuni, to which 2′-fucosyllactose is known to bind. Where binding was detected, its specificity was confirmed by preincubation studies using unlabeled 2′-fucosyllactose. The techniques described represent a quick, cost-effective, and highly reproducible detection method for identifying anti-infective oligosaccharides.     

1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.     

Genome-wide association studies of the PR interval in African Americans

The PR interval on the electrocardiogram reflects atrial and atrioventricular nodal conduction time. The PR interval is heritable, provides important information about arrhythmia risk, and has been suggested to differ among human races. Genome-wide association (GWA) studies have identified common genetic determinants of the PR interval in individuals of European and Asian ancestry, but there is a general paucity of GWA studies in individuals of African ancestry. We performed GWA studies in African American individuals from four cohorts (n = 6,247) to identify genetic variants associated with PR interval duration. Genotyping was performed using the Affymetrix 6.0 microarray. Imputation was performed for 2.8 million single nucleotide polymorphisms (SNPs) using combined YRI and CEU HapMap phase II panels. We observed a strong signal (rs3922844) within the gene encoding the cardiac sodium channel (SCN5A) with genome-wide significant association (p<2.5×10⁻⁸) in two of the four cohorts and in the meta-analysis. The signal explained 2% of PR interval variability in African Americans (beta  = 5.1 msec per minor allele, 95% CI  = 4.1–6.1, p = 3×10⁻²³). This SNP was also associated with PR interval (beta = 2.4 msec per minor allele, 95% CI = 1.8–3.0, p = 3×10⁻¹⁶) in individuals of European ancestry (n = 14,042), but with a smaller effect size (p for heterogeneity <0.001) and variability explained (0.5%). Further meta-analysis of the four cohorts identified genome-wide significant associations with SNPs in SCN10A (rs6798015), MEIS1 (rs10865355), and TBX5 (rs7312625) that were highly correlated with SNPs identified in European and Asian GWA studies. African ancestry was associated with increased PR duration (13.3 msec, p = 0.009) in one but not the other three cohorts. Our findings demonstrate the relevance of common variants to African Americans at four loci previously associated with PR interval in European and Asian samples and identify an association signal at one of these loci that is more strongly associated with PR interval in African Americans than in Europeans.     

Feeding Frequency and Appetite in Lean and Obese Prepubertal Children

To determine the effect of feeding frequency on appetite in normal weight (NW) and obese (OB) prepubertal children, we carried out a prospective, randomized interventional study of 18 NW and 17 OB children ages 6–10. Children received three or five feedings in random order on separate days. Total calories, carbohydrate, protein, and fat composition on each day were equal. Two hours following the last feeding, children were offered ice cream ad lib. The major outcome variable was kilocalories ice cream consumed. A visual analog scale to assess fullness was also administered before consumption of ice cream. We observed that OB children consumed 73.0 ± 37.4 kcal more after five feedings than after three feedings whereas the NW children consumed 47.1 ± 27.8 kcal less. There was significant interaction between meal pattern and weight group indicating that this change in ice cream consumption differed significantly between groups (P = 0.014 by two‐factor analysis). Ice cream intake/kg was less in OB compared to NW subjects (P = 0.012). Fullness ratings before ice cream did not differ by meal pattern or weight group. However, pre‐ice cream fullness predicted ice cream intake in NW but not OB children. In summary, OB and NW children differed in appetite response to meal frequency. Our data suggest that: (i) satiety in OB children is related more to proximity of calories (larger supper) than to antecedent distribution of calories and; (ii) NW children may be more prone to restrict intake based on subjective fullness.     

Genome-wide linkage scan to identify loci associated with type 2 diabetes and blood lipid phenotypes in the Sikh Diabetes Study

In this investigation, we have carried out an autosomal genome-wide linkage analysis to map genes associated with type 2 diabetes (T2D) and five quantitative traits of blood lipids including total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, and triglycerides in a unique family-based cohort from the Sikh Diabetes Study (SDS). A total of 870 individuals (526 male/344 female) from 321 families were successfully genotyped using 398 polymorphic microsatellite markers with an average spacing of 9.26 cM on the autosomes. Results of non-parametric multipoint linkage analysis using S_all statistics (implemented in Merlin) did not reveal any chromosomal region to be significantly associated with T2D in this Sikh cohort. However, linkage analysis for lipid traits using QTL-ALL analysis revealed promising linkage signals with p≤0.005 for total cholesterol, LDL cholesterol, and HDL cholesterol at chromosomes 5p15, 9q21, 10p11, 10q21, and 22q13. The most significant signal (p = 0.0011) occurred at 10q21.2 for HDL cholesterol. We also observed linkage signals for total cholesterol at 22q13.32 (p = 0.0016) and 5p15.33 (p = 0.0031) and for LDL cholesterol at 10p11.23 (p = 0.0045). Interestingly, some of linkage regions identified in this Sikh population coincide with plausible candidate genes reported in recent genome-wide association and meta-analysis studies for lipid traits. Our study provides the first evidence of linkage for loci associated with quantitative lipid traits at four chromosomal regions in this Asian Indian population from Punjab. More detailed examination of these regions with more informative genotyping, sequencing, and functional studies should lead to rapid detection of novel targets of therapeutic importance.     

Improved xenobiotic metabolism and reduced susceptibility to cancer in gluten-sensitive macaques upon introduction of a gluten-free diet

Background

A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta).

Methodology

Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes.

Principal Findings

When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories - cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function - this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes.

Conclusions/Significance

A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research.     

Finite element analysis of defibrillation fields in a human torso model for ventricular defibrillation

In order to optimize defibrillation electrode systems for ventricular defibrillation thresholds (DFTs), a Finite Element Torso model was built from fast CT scans of a patient who had large cardiac dimensions (upper bound of normal) but no heart disease. Clinically used defibrillation electrode configurations, i.e. Superior Vena Cava (SVC) to Right Ventricle (RV) (SVC-RV), left pectoral Can to RV (Can-RV) and Can + SVC-RV, were analyzed. The DFTs were calculated based on 95% ventricular mass having voltage gradient> 5 V/cm and these results were also compared with clinical data. The low voltage gradient regions with voltage gradient < 5 V/cm were identified and the effect of electrode dimension and location on DFTs were also investigated for each system. A good correlation between the model results and the clinical data supports the use of Finite Element Analysis of a human torso model for optimization of defibrillation electrode systems. This correlation also indicates that the critical mass hypothesis is the primary mechanism of defibrillation. Both the FEA results and the clinical data show that Can + SVC-RV system offers the lowest voltage DFTs when compared with SVC-RV and Can-RV systems. Analysis of the effect of RV, SVC and Can electrode dimensions and locations can have an important impact on defibrillation lead designs.