Molecular evolution and the role of oxidative stress in the expansion and functional diversification of cytosolic glutathione transferases

<p>Abstract</p> <p>Background</p> <p>Cytosolic glutathione transferases (cGST) are a large group of ubiquitous enzymes involved in detoxification and are well known for their undesired side effects during chemotherapy. In this work we have performed thorough phylogenetic analyses to understand the various aspects of the evolution and functional diversification of cGSTs. Furthermore, we assessed plausible correlations between gene duplication and substrate specificity of gene paralogs in humans and selected species, notably in mammalian enzymes and their natural substrates.</p> <p>Results</p> <p>We present a molecular phylogeny of cytosolic GSTs that shows that several classes of cGSTs are more ubiquitous and thus have an older ancestry than previously thought. Furthermore, we found that positive selection is implicated in the diversification of cGSTs. The number of duplicate genes per class is generally higher for groups of enzymes that metabolize products of oxidative damage.</p> <p>Conclusions</p> <p>1) Protection against oxidative stress seems to be the major driver of positive selection in mammalian cGSTs, explaining the overall expansion pattern of this subfamily;</p> <p>2) Given the functional redundancy of GSTs that metabolize xenobiotic chemicals, we would expect the loss of gene duplicates, but by contrast we observed a gene expansion of this family, which likely has been favored by: i) the diversification of endogenous substrates; ii) differential tissue expression; and iii) increased specificity for a particular molecule;</p> <p>3) The increased availability of sequence data from diversified taxa is likely to continue to improve our understanding of the early origin of the different cGST classes.</p

High-throughput generation of product profiles for arabinoxylan-active enzymes from metagenomes

Metagenomics is an exciting alternative to seek carbohydrate-active enzymes from a range of sources. Typically, metagenomics reveals dozens of putative catalysts that require functional characterization for further application in industrial processes. High-throughput screening methods compatible with adequate natural substrates are crucial for an accurate functional elucidation of substrate preferences. Based on DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) analysis of enzymatic-reaction products, we generated product profiles to consequently infer substrate cleavage positions, resulting in the generation of enzymatic-degradation maps. Product profiles were produced in high throughput for arabinoxylan (AX)-active enzymes belonging to the glycoside hydrolase families GH43 (subfamilies 2 [MG432], 7 [MG437], and 28 [MG4328]) and GH8 (MG8) starting from 12 (arabino)xylo-oligosaccharides. These enzymes were discovered through functional metagenomic studies of feces from the North American beaver (Castor canadensis). This work shows how enzyme loading alters the product profiles of all enzymes studied and gives insight into AX degradation patterns, revealing sequential substrate preferences of AX-active enzymes. IMPORTANCE: Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylooligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degradation requires a set of enzymes with particular characteristics. Therefore, enzyme discovery and the study of substrate preferences are of utmost importance. Beavers, as consumers of woody biomass, are a promising source of a repertoire of enzymes able to deconstruct hemicelluloses into soluble oligosaccharides. High-throughput analysis of the oligosaccharide profiles produced by these enzymes will assist in the selection of the most appropriate enzymes for the biorefinery

The adaptive evolution of the mammalian mitochondrial genome

<p>Abstract</p> <p>Background</p> <p>The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures.</p> <p>Results</p> <p>Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome <it>b </it>in species with more-specialized metabolic requirements (such as adaptation to low energy diet <it>or </it>large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas). Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome <it>c </it>oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F₀, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas.</p> <p>Conclusion</p> <p>Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation.</p

Spatio-temporal expression patterns of anandamide-binding receptors in rat implantation sites: evidence for a role of the endocannabinoid system during the period of placental development

<p>Abstract</p> <p>Background</p> <p>Although there is growing evidence that endocannabinoids play a critical role in early pregnancy, there are no studies describing the possible targets for this system after implantation. The endometrial stroma, which undergoes extensive proliferation and differentiation giving rise to the decidua and the trophoblast cells that invade after the initial stages of implantation, are potential targets. Since high anandamide (AEA) levels, the main endocannabinoid, are detrimental to implantation and in order to gain insight into the role of the endocannabinoid system in the development of the fetoplacental unit, the spatio-temporal pattern of expression of the anandamide-binding receptors, CB1, CB2 and the vanilloid receptor (TRPV1), were investigated by quantitative RT-PCR, western blot and immunohistochemistry.</p> <p>Methods</p> <p>Rat uterine maternal tissues from different days of pregnancy were used to investigate the expression of CB1, CB2 and vanilloid receptors by quantitative RT-PCR, western blot and immunohistochemistry.</p> <p>Results</p> <p>The data indicate that all the three receptors were expressed in decidualized cells and placenta. Interestingly, CB1 and CB2 were also expressed in smooth muscle cells of maternal blood vessels and in endovascular trophoblast cells, whereas TRPV1 was mainly expressed in uterine natural killer (uNK) cells and in the longitudinal muscle layer throughout pregnancy. In all tissues, CB2 protein was present at a lower level than CB1.</p> <p>Conclusion</p> <p>These observations support a role for the endocannabinoid system during the period of decidualization and placental development.</p

Towards Stratified Medicine in Plasma Cell Myeloma

Plasma cell myeloma is a clinically heterogeneous malignancy accounting for approximately one to 2% of newly diagnosed cases of cancer worldwide. Treatment options, in addition to long-established cytotoxic drugs, include autologous stem cell transplant, immune modulators, proteasome inhibitors and monoclonal antibodies, plus further targeted therapies currently in clinical trials. Whilst treatment decisions are mostly based on a patient’s age, fitness, including the presence of co-morbidities, and tumour burden, significant scope exists for better risk stratification, sub-classification of disease, and predictors of response to specific therapies. Clinical staging, recurring acquired cytogenetic aberrations, and serum biomarkers such as β-2 microglobulin, and free light chains are in widespread use but often fail to predict the disease progression or inform treatment decision making. Recent scientific advances have provided considerable insight into the biology of myeloma. For example, gene expression profiling is already making a contribution to enhanced understanding of the biology of the disease whilst Next Generation Sequencing has revealed great genomic complexity and heterogeneity. Pathways involved in the oncogenesis, proliferation of the tumour and its resistance to apoptosis are being unravelled. Furthermore, knowledge of the tumour cell surface and its interactions with bystander cells and the bone marrow stroma enhance this understanding and provide novel targets for cell and antibody-based therapies. This review will discuss the development in understanding of the biology of the tumour cell and its environment in the bone marrow, the implementation of new therapeutic options contributing to significantly improved outcomes, and the progression towards more personalised medicine in this disorder

Trial protocol OPPTIMUM : does progesterone prophylaxis for the prevention of preterm labour improve outcome?

Background Preterm birth is a global problem, with a prevalence of 8 to 12% depending on location. Several large trials and systematic reviews have shown progestogens to be effective in preventing or delaying preterm birth in selected high risk women with a singleton pregnancy (including those with a short cervix or previous preterm birth). Although an improvement in short term neonatal outcomes has been shown in some trials these have not consistently been confirmed in meta-analyses. Additionally data on longer term outcomes is limited to a single trial where no difference in outcomes was demonstrated at four years of age of the child, despite those in the “progesterone” group having a lower incidence of preterm birth. Methods/Design The OPPTIMUM study is a double blind randomized placebo controlled trial to determine whether progesterone prophylaxis to prevent preterm birth has long term neonatal or infant benefit. Specifically it will study whether, in women with singleton pregnancy and at high risk of preterm labour, prophylactic vaginal natural progesterone, 200 mg daily from 22 – 34 weeks gestation, compared to placebo, improves obstetric outcome by lengthening pregnancy thus reducing the incidence of preterm delivery (before 34 weeks), improves neonatal outcome by reducing a composite of death and major morbidity, and leads to improved childhood cognitive and neurosensory outcomes at two years of age. Recruitment began in 2009 and is scheduled to close in Spring 2013. As of May 2012, over 800 women had been randomized in 60 sites. Discussion OPPTIMUM will provide further evidence on the effectiveness of vaginal progesterone for prevention of preterm birth and improvement of neonatal outcomes in selected groups of women with singleton pregnancy at high risk of preterm birth. Additionally it will determine whether any reduction in the incidence of preterm birth is accompanied by improved childhood outcome

Multi-Trait GWAS and new candidate genes annotation for growth curve parameters in Brahman cattle

Understanding the genetic architecture of beef cattle growth cannot be limited simply to the genome-wide association study (GWAS) for body weight at any specific ages, but should be extended to a more general purpose by considering the whole growth trajectory over time using a growth curve approach. For such an approach, the parameters that are used to describe growth curves were treated as phenotypes under a GWAS model. Data from 1,255 Brahman cattle that were weighed at birth, 6, 12, 15, 18, and 24 months of age were analyzed. Parameter estimates, such as mature weight (A) and maturity rate (K) from nonlinear models are utilized as substitutes for the original body weights for the GWAS analysis. We chose the best nonlinear model to describe the weight-age data, and the estimated parameters were used as phenotypes in a multi-trait GWAS. Our aims were to identify and characterize associated SNP markers to indicate SNP-derived candidate genes and annotate their function as related to growth processes in beef cattle. The Brody model presented the best goodness of fit, and the heritability values for the parameter estimates for mature weight (A) and maturity rate (K) were 0.23 and 0.32, respectively, proving that these traits can be a feasible alternative when the objective is to change the shape of growth curves within genetic improvement programs. The genetic correlation between A and K was -0.84, indicating that animals with lower mature body weights reached that weight at younger ages. One hundred and sixty seven (167) and two hundred and sixty two (262) significant SNPs were associated with A and K, respectively. The annotated genes closest to the most significant SNPs for A had direct biological functions related to muscle development (RAB28), myogenic induction (BTG1), fetal growth (IL2), and body weights (APEX2); K genes were functionally associated with body weight, body height, average daily gain (TMEM18), and skeletal muscle development (SMN1). Candidate genes emerging from this GWAS may inform the search for causative mutations that could underpin genomic breeding for improved growth rates

Avian Binocularity and Adaptation to Nocturnal Environments: Genomic Insights froma Highly Derived Visual Phenotype

Typical avian eyes are phenotypically engineered for photopic vision (daylight). In contrast, the highly derived eyes of the barn owl (Tyto alba) are adapted for scotopic vision (dim light). The dramatic modifications distinguishing barn owl eyes from other birds include: 1) shifts in frontal orientation to improve binocularity, 2) rod-dominated retina, and 3) enlarged corneas and lenses. Some of these features parallel mammalian eye patterns, which are hypothesized to have initially evolved in nocturnal environments. Here, we used an integrative approach combining phylogenomics and functional phenotypes of 211 eye-development genes across 48 avian genomes representing most avian orders, including the stem lineage of the scotopic-adapted barn owl. Overall, we identified 25 eye-development genes that coevolved under intensified or relaxed selection in the retina, lens, cornea, and optic nerves of the barn owl. The agtpbp1 gene, which is associated with the survival of photoreceptor populations, was pseudogenized in the barn owl genome. Our results further revealed that barn owl retinal genes responsible for the maintenance, proliferation, and differentiation of photoreceptors experienced an evolutionary relaxation. Signatures of relaxed selection were also observed in the lens and cornea morphology-associated genes, suggesting that adaptive evolution in these structures was essentially structural. Four eye-development genes (ephb1, phactr4, prph2, and rs1) evolved in positive association with the orbit convergence in birds and under relaxed selection in the barn owl lineage, likely contributing to an increased reliance on binocular vision in the barn owl. Moreover, we found evidence of coevolutionary interactions among genes that are expressed in the retina, lens, and optic nerve, suggesting synergetic adaptive events. Our study disentangles the genomic changes governing the binocularity and low-light perception adaptations of barn owls to nocturnal environments while revealing the molecular mechanisms contributing to the shift from the typical avian photopic vision to the more-novel scotopic-adapted eye

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I)