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.     

Disruption analysis of metallothionein-encoding genes in Candida glabrata.

Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.     

A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo

PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and translocate the catalytic moieties, lethal factor (LF), and edema factor (EF) into the cytosol of mammalian cells. Acidic pH triggers oligomerization and membrane insertion by PA63. A disordered amphipathic loop in domain II of PA (2beta2-2beta3 loop) is involved in membrane insertion by PA63. Because conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2beta2-2beta3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens. It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication. The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization, or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. In addition, PA-I inhibited the channel-forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity. Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo.     

Spectroscopic characterization of heat-induced nonnative beta-lactoglobulin monomers

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.     

Insights into the relation between mrna and protein expression patterns: II. Experimental observations in Escherichia coli

There is a need for improved appreciation of the importance of genome-wide mRNA and protein expression measurements and their role in understanding translation and in relation to genome-wide mathematical frameworks for gene expression regulation. We investigated the use of a high-density microarray technique for mRNA expression analysis and a two-dimensional protein electrophoresis-tandem mass spectrometry method for protein analysis to monitor changes in gene expression. We applied these analytical tools in the context of an environmental perturbation of Escherichia coli cells-the addition of varying amounts of IPTG. We also tested the application of these tools to the study of a genetic perturbation of Escherichia coli cells-the ability of certain strains to hypersecrete the hemolysin protein. We observed a lack of correspondence between mRNA and protein expression profiles. Although our data do not include measurements on all expressed genes (because the ability to measure protein expression profiles is limiting), we observed that the qualitative and quantitative behavior of the measurements of a subset of expressed genes is similar to the behavior of the entire system. The change in observed average mRNA and protein amplification factors for 77 and 52 genes coincided with the observed change in mRNA amplification factor for the entire system. Furthermore, we found that the use of relative changes in expression could be used to elucidate mechanisms of gene expression regulation for the system studied, even when measurements were made on a small subset of the system.