Extension temperature of 60°C required for PCR amplification of large DNA fragments (>5 kb) from a low GC bacterium Clostridium acetobutylicum

PCR amplification of DNA fragments has been routinely used in gene cloning and engineering of microbial strains for biotechnological purposes such as production of biofuels and green chemicals. However, it is often a challenge to amplify large DNA fragments (>5 kb) from low GC microorganisms using the standard PCR protocols. In this brief communication, we report a modified PCR method with an extension temperature of 60°C, which efficiently amplified a 5.3 and a 5.5 kb DNA fragment (an extension time of 6 min) from a low GC bacterium Clostridium acetobutylicum (~30% GC). A lower than normal extension temperature (72°C) approach may also facilitate PCR amplification of large DNA fragments (>5 kb) from other low GC microorganisms.     

Genome-wide association studies and susceptibility to infectious diseases

Progress in genomics and the associated technological, statistical and bioinformatics advances have facilitated the successful implementation of genome-wide association studies (GWAS) towards understanding the genetic basis of common diseases. Infectious diseases contribute significantly to the global burden of disease and there is robust epidemiological evidence that host genetic factors are important determinants of the outcome of interactions between host and pathogen. Indeed, infectious diseases have exerted profound selective pressure on human evolution. However, the application of GWAS to infectious diseases has been relatively limited compared with non-communicable diseases. Here we review GWAS findings for important infectious diseases, including malaria, tuberculosis and HIV. We highlight some of the pitfalls recognized more generally for GWAS, as well as issues specific to infection, including the role of the pathogen which also has a genome. We also discuss the challenges encountered when studying African populations which are genetically more ancient and more diverse that other populations and disproportionately bear the main global burden of serious infectious diseases.     

Effects of Bioavailable Heavy Metal Species,Arsenic, and Acid Drainage from Mine Tailings on a Microbial Community Sampled Along a Pollution Gradient in a Freshwater Ecosystem

Sediment and water samples representing a pollution gradient in a long, narrow lake polluted at one end by heavy metals, arsenic, and acid drainage from mine tailings, together with samples from an unpolluted reference lake, were analyzed to determine effects of pollutants on the microbial community of the polluted lake. Ribosomal ribonucleic acid, fatty acid, and phospholipid analyses, along with assays of CO₂ production, denitrification, and enzyme activities, were performed to characterize the microflora; and environmental conditions were defined by various physicochemical analyses, including determination of bioavailable metal species. Mine waste pollution fostered the growth of Holophagal Acidobacteria, green sulphur bacteria, and α-Proteobacteria but inhibited numerous other types of microorganisms, reducing the overall productivity, biomass, and biodiversity of the microflora. The beneficial effects imply toleration of pollutants, suppression of competing or antagonistic species, and utilization of biogenic sulphide; and the toxic effects are attributable to bioavailable metals, arsenic, and sulphuric acid produced by oxidation of sulphides. The bioavailability and toxicity of sediment-bound metals were evidently increased by acidification, elevation of sediment Eh, and inhibition of metal-immobilizing bacteria by pollutants but were decreased by metal-scavenging oxyhydroxides, sulphide, and organic matter. Metal toxicity also depended on specific metal properties (e.g., electronegativity), providing a basis for inferring mechanisms of toxicity and oxidation states of metals and explaining differences in relative toxicity. The pollutants harmed the ecosystem as a whole by inhibiting microorganisms that performed crucial ecological functions, notably oxygen-releasing photosynthesis, decomposition and humification of organic matter, nutrient recycling, and control of metal availability.     

A shotgun lipidomics approach in Sinorhizobium meliloti as a tool in functional genomics

A shotgun lipidomics approach that allowed the analysis of eight lipid classes directly from crude extracts of the soil bacterium Sinorhizobium meliloti is presented. New MS-MS transitions are reported for the analysis of monomethylphosphatidylethanolamines, dimethylphosphatidylethanolamines, and three bacterial non-phosphorus-containing lipid classes [sulfoquinovosyldiacylglycerols, ornithines, and diacylglyceryl-(N,N,N-trimethyl)-homoserines]. Unique MS-MS transitions allowed the analysis of isomeric species from various lipid classes without chromatography. Analyses required small sample amounts and minimal preparation; thus, this methodology has excellent potential to be used as a screening tool for the analysis of large numbers of samples in functional genomics studies. FA distributions within lipid classes of S. meliloti are described for the first time, and the relative positions of fatty acyl substituents (sn-1, sn-2) in phospholipids are presented. FA distributions in diacylglyceryl-(N,N,N-trimethyl)-homoserines were identical to those of phospholipids, indicating a common biosynthetic origin for these lipids. The method was applied to the analysis of mutants deficient in the PhoB regulator protein. Increased lipid cyclopropanation was observed in PhoB-deficient mutants under P_i starvation.     

Magic-factor 1, a partial agonist of Met, induces muscle hypertrophy by protecting myogenic progenitors from apoptosis

Background

Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological activities including cell proliferation, survival, motility and morphogenesis. Its high affinity receptor, the tyrosine kinase Met, is expressed by a wide range of tissues and can be activated by either paracrine or autocrine stimulation. Adult myogenic precursor cells, the so called satellite cells, express both HGF and Met. Following muscle injury, autocrine HGF-Met stimulation plays a key role in promoting activation and early division of satellite cells, but is shut off in a second phase to allow myogenic differentiation. In culture, HGF stimulation promotes proliferation of muscle precursors thereby inhibiting their differentiation.

Methodology/Principal Findings

Magic-Factor 1 (Met-Activating Genetically Improved Chimeric Factor-1 or Magic-F1) is an HGF-derived, engineered protein that contains two Met-binding domains repeated in tandem. It has a reduced affinity for Met and, in contrast to HGF it elicits activation of the AKT but not the ERK signaling pathway. As a result, Magic-F1 is not mitogenic but conserves the ability to promote cell survival. Here we show that Magic-F1 protects myogenic precursors against apoptosis, thus increasing their fusion ability and enhancing muscular differentiation. Electrotransfer of Magic-F1 gene into adult mice promoted muscular hypertrophy and decreased myocyte apoptosis. Magic-F1 transgenic mice displayed constitutive muscular hypertrophy, improved running performance and accelerated muscle regeneration following injury. Crossing of Magic-F1 transgenic mice with α-sarcoglycan knock-out mice –a mouse model of muscular dystrophy– or adenovirus-mediated Magic-F1 gene delivery resulted in amelioration of the dystrophic phenotype as measured by both anatomical/histological analysis and functional tests.

Conclusions/Significance

Because of these features Magic-F1 represents a novel molecular tool to counteract muscle wasting in major muscular diseases such as cachexia or muscular dystrophy.     

Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.