Colonic microbiome is altered in alcoholism

Several studies indicate the importance of colonic microbiota in metabolic and inflammatory disorders and importance of diet on microbiota composition. The effects of alcohol, one of the prominent components of diet, on colonic bacterial composition is largely unknown. Mounting evidence suggests that gut-derived bacterial endotoxins are cofactors for alcohol-induced tissue injury and organ failure like alcoholic liver disease (ALD) that only occur in a subset of alcoholics. We hypothesized that chronic alcohol consumption results in alterations of the gut microbiome in a subgroup of alcoholics, and this may be responsible for the observed inflammatory state and endotoxemia in alcoholics. Thus we interrogated the mucosa-associated colonic microbiome in 48 alcoholics with and without ALD as well as 18 healthy subjects. Colonic biopsy samples from subjects were analyzed for microbiota composition using length heterogeneity PCR fingerprinting and multitag pyrosequencing. A subgroup of alcoholics have an altered colonic microbiome (dysbiosis). The alcoholics with dysbiosis had lower median abundances of Bacteroidetes and higher ones of Proteobacteria. The observed alterations appear to correlate with high levels of serum endotoxin in a subset of the samples. Network topology analysis indicated that alcohol use is correlated with decreased connectivity of the microbial network, and this alteration is seen even after an extended period of sobriety. We show that the colonic mucosa-associated bacterial microbiome is altered in a subset of alcoholics. The altered microbiota composition is persistent and correlates with endotoxemia in a subgroup of alcoholics.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Erbin regulates mitogen-activated protein (MAP) kinase activation and MAP kinase-dependent interactions between Merlin and adherens junction protein complexes in Schwann cells

Biallelic mutations in the neurofibromatosis 2 (NF2) gene are linked to schwannoma and meningioma tumorigenesis. Cells with NF2 mutations exhibit elevated levels of phosphorylated extracellular signal-regulated kinase (ERK) and aberrant cell-cell and cell-matrix contacts. The NF2 gene product, merlin, associates with adherens junction protein complexes, suggesting that part of its function as a tumor suppressor involves regulating cell junctions. Here, we find that a novel PDZ protein, called erbin, binds directly to the merlin-binding partner, EBP0, and regulates adherens junction dissociation through a MAP kinase-dependent mechanism. Reducing erbin expression using a targeted siRNA in primary cultures of Schwann cells results in altered cell-cell interactions, disruption of E-cadherin adherens junctions, increased cell proliferation, and elevated levels of phosphorylated ERK, all phenotypes observed in cells that lack merlin. Reduction of erbin expression also results in the dissociation of merlin from adherens junction proteins and an increase in the levels of phosphorylated merlin. These phenotypes can be rescued if cells with reduced levels of erbin are treated with a pharmacological inhibitor of ERK kinase. Collectively, these data indicate that erbin regulates MAP kinase activation in Schwann cells and suggest that erbin links merlin to both adherens junction protein complexes and the MAP kinase signaling pathway.     

Profile-based direct kernels for remote homology detection and fold recognition

MOTIVATION: Protein remote homology detection is a central problem in computational biology. Supervised learning algorithms based on support vector machines are currently one of the most effective methods for remote homology detection. The performance of these methods depends on how the protein sequences are modeled and on the method used to compute the kernel function between them. RESULTS: We introduce two classes of kernel functions that are constructed by combining sequence profiles with new and existing approaches for determining the similarity between pairs of protein sequences. These kernels are constructed directly from these explicit protein similarity measures and employ effective profile-to-profile scoring schemes for measuring the similarity between pairs of proteins. Experiments with remote homology detection and fold recognition problems show that these kernels are capable of producing results that are substantially better than those produced by all of the existing state-of-the-art SVM-based methods. In addition, the experiments show that these kernels, even when used in the absence of profiles, produce results that are better than those produced by existing non-profile-based schemes. AVAILABILITY: The programs for computing the various kernel functions are available on request from the authors.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Genetic modulation of PPARgamma phosphorylation regulates insulin sensitivity

Obesity-associated diabetes is epidemic in industrialized societies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in adipose tissue and the presumed molecular target for antidiabetic thiazolidinedione drugs that reverse insulin resistance but also promote weight gain. Phosphorylation reduces the activity of PPARgamma in vitro, but physiological relevance has not been demonstrated. We have studied mice homozygous for a mutation (S112A) that prevents PPARgamma phosphorylation. Surprisingly, the weights and adipose mass of PPARgamma-S112A mice are not greater than wild-type. Remarkably, however, genetic prevention of PPARgamma phosphorylation preserves insulin sensitivity in the setting of diet-induced obesity. Underlying this protection are smaller fat cells, elevated serum adiponectin, and reduced free fatty acid levels. Thus, the phosphorylation state of PPARgamma modulates insulin sensitivity. Compounds that prevent PPARgamma phosphorylation or ligands that induce the conformation of nonphosphorylated PPARgamma may selectively enhance insulin sensitivity without increasing body weight.     

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples

Background

DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (Taq^αI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system.

Results

With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region.

Conclusions

With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.