Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.     

Increased Carotid Intima-Media Thickness and Reduced Distensibility in Human Class III Obesity: Independent and Differential Influences of Adiposity and Blood Pressure on the Vasculature

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m² (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m² and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m²). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133⁺/KDR⁺ angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10⁻³kpa⁻¹ vs. 29.91±12.37 10⁻³kpa⁻¹, P<0.05) in individuals with BMI>40.0 kg/m². Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m². In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.     

Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained K_m and k_cat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s^-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (K_i of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn²⁺-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.     

Salt Intake and Health Risk in Climate Change Vulnerable Coastal Bangladesh: What Role Do Beliefs and Practices Play?

BackgroundHigh salt consumption is an important risk factor of elevated blood pressure. In Bangladesh about 20 million people are at high risk of hypertension due to climate change induced saline intrusion in water. The objective of this study is to assess beliefs, perceptions, and practices associated with salt consumption in coastal Bangladesh.MethodsThe study was conducted in Chakaria, Bangladesh between April-June 2011. It was a cross sectional mixed method study. For the qualitative study 6 focus group discussions, 8 key informant interviews, 60 free listing exercises, 20 ranking exercises and 10 observations were conducted. 400 adults were randomly selected for quantitative survey. For analysis we used SPSS for quantitative data, and Anthropac and Nvivo for qualitative data.ResultsSalt was described as an essential component of food with strong cultural and religious roots. People described both health benefits and risks related to salt intake. The overall risk perception regarding excessive salt consumption was low and respondents believed that the cooking process can render the salt harmless. Respondents were aware that salt is added in many foods even if they do not taste salty but did not recognize that salt can occur naturally in both foods and water.ConclusionsIn the study community people had low awareness of the risks associated with excess salt consumption and salt reduction strategies were not high in their agenda. The easy access to and low cost of salt as well as unrecognised presence of salt in drinking water has created an environment conducive to excess salt consumption. It is important to design general messages related to salt reduction and test tailored strategies especially for those at high risk of hypertension.