Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

Base Sequence Effects in Double Helical DNA. I. Potential Energy Estimates of Local Base Morphology

Abstract

A series of potential energy calculations have been carried out to estimate base sequence dependent structural differences in B-DNA. Attention has been focused on the simplest dimeric fragments that can be used to build long chains, computing the energy as a function of the orientation and displacement of the 16 possible base pair combinations within the double helix. Calculations have been performed, for simplicity, on free base pairs rather than complete nucleotide units. Conformational preferences and relative flexibilities are reported for various combinations of the roll, tilt, twist, lateral displacement, and propeller twist of individual residues. The predictions are compared with relevant experimental measures of conformation and flexibility, where available. The energy surfaces are found to fit into two distinct categories, some dimer duplexes preferring to bend in a symmetric fashion and others in a skewed manner. The effects of common chemical substitutions (uracil for thymine, 5-methyl cytosine for cytosine, and hypoxanthine for guanine) on the preferred arrangements of neighboring residues are also examined, and the interactions of the sugar-phosphate backbone are included in selected cases. As a first approximation, long range interactions between more distant neighbors, which may affect the local chain configuration, are ignored. A rotational isomeric state scheme is developed to describe the average configurations of individual dimers and is used to develop a static picture of overall double helical structure. The ability of the energetic scheme to account for documented examples of intrinsic B-DNA curvature is presented, and some new predictions of sequence directed chain bending are offered.     

Molecular Dynamics Simulations of Rhodopsin Point Mutants at the Cytoplasmic Side of Helices 3 and 6

Abstract

The present work reports on a structural analysis carried out through different computer simulations of a set of rhodopsin mutants with differential functional features in regard to the wild type. Most of these mutants, whose experimental features had previously been reported [Ramon et al. J Biol Chem 282, 14272–14282 (2007)], were designed to perturb a network of electrostatic interactions located at the cytoplasmic sides of transmembrane helices 3 and 6. Geometric and energetic features derived from the detailed analysis of a series of molecular dynamics simulations of the different rhodopsin mutants, involving positions 134(3.49), 247(6.30), and 251(6.34), suggest that the protein structure is sensitive to these mutations through the local changes induced that extend further to the secondary structure of neighboring helices and, ultimately, to the packing of the helical bundle. Overall, the results obtained highlight the complexity of the analyzed network of electrostatic interactions where the effect of each mutation on protein structure can produce rather specific features.     

Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group,and pairwise genome comparisons of the species of the group by means of ANI calculations

Strain BCT-7112^T was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN^®, an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112^T as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112^T (=CECT 876^T; =NCIMB 14858^T) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis.     

Growth differentiation factor‐15 (GDF‐15) suppresses in vitro angiogenesis through a novel interaction with connective tissue growth factor (CCN2)

Growth differentiation factor‐15 (GDF‐15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF‐15 and CCN2 using yeast two‐hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF‐15 and His‐tagged CCN2 produced in PC‐3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF‐15 blocks CCN2‐mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF‐15 inhibits CCN2‐mediated angiogenesis, activation of α_Vβ₃ integrins and focal adhesion kinase (FAK) was examined. CCN2‐mediated FAK activation was inhibited by GDF‐15 and was accompanied by a decrease in α_Vβ₃ integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF‐15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF‐15 may provide insight into the functional role of GDF‐15 in disease states. J. Cell. Biochem. 114: 1424–1433, 2013. © 2012 Wiley Periodicals, Inc.     

New Insights into the Circadian Rhythm of Acute Myocardial Infarction in Subgroups

The aim of this study was to determine the existence of the circadian rhythm (CR) in the onset of acute myocardial infarction (AMI) in different patient subgroups. Information was collected about 41,244 infarctions from the database of the ARIAM (Analysis of Delay in AMI) Spanish multicenter study. CR in AMI were explored in subgroups of cases categorized by age, gender, previous ischemic heart disease (PIHD), outcome in coronary care unit, infarction electrocardiograph (ECG) characteristics (Q wave or non‐Q wave), and location of AMI. Cases were classified according to these variables in the different subgroups. To verify the presence of CR, a simple test of equality of time series based on the multiple‐sinusoid (24, 12, and 8 h periods) cosinor analysis was developed. For the groups as a whole, the time of pain onset as an indicator of the AMI occurrence showed a CR (p<0.0001), with a morning peak at 10:10 h. All the analyzed subgroups also showed CR. Comparison between subgroups showed significant differences in the PIHD (p<0.01) and infarction ECG characteristics (p<0.01) groups. The CR of the subgroup with Q‐wave infarction differed from that of non‐Q wave subgroup (p<0.01) when the patients had PIHD (23% in Q wave infarction vs. 39.2% in non‐Q wave). AMI onset followed a CR pattern, which is also observed in all analyzed subgroups. Differences in the CR according to the Q/non‐Q wave infarction characteristics could be determined by PIHD. The cosinor model fit with three components (24, 12, and 8 h periods) showed a higher sensitivity than the single 24 h period analysis.     

Fermentation of glycerol and production of valuable chemical and biofuel molecules

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.