Risk Alleles in/near ADCY5, ADRA2A,CDKAL1, CDKN2A/B,GRB10, and TCF7L2 Elevate Plasma Glucose Levels at Birth and in Early Childhood: Results from the FAMILY Study

BackgroundMetabolic abnormalities that lead to type 2 diabetes mellitus begin in early childhood.ObjectivesWe investigate whether common genetic variants identified in adults have an effect on glucose in early life.Methods610 newborns, 463 mothers, and 366 fathers were included in the present study. Plasma glucose and anthropometric characteristics were collected at birth, 3, and 5 years. After quality assessment, 37 SNPs, which have demonstrated an association with fasting plasma glucose at the genome-wide threshold in adults, were studied. Quantitative trait disequilibrium tests and mixed-effects regressions were conducted to estimate an effect of the SNPs on glucose.ResultsRisk alleles for 6 loci increased glucose levels from birth to 5 years of age (ADCY5, ADRA2A, CDKAL1, CDKN2A/B, GRB10, and TCF7L2, 4.85x10^-3 ≤ P ≤ 4.60x10^-2). Together, these 6 SNPs increase glucose by 0.05 mmol/L for each risk allele in a genotype score (P = 6.33x10^-5). None of the associations described in the present study have been reported previously in early childhood.ConclusionOur data support the notion that a subset of loci contributing to plasma glucose variation in adults has an effect at birth and in early life.     

Microbial ecology of chlorinated solvent biodegradation

This study focused on the microbial ecology of tetrachloroethene (PCE) degradation to trichloroethene, cis‐1,2‐dichloroethene and vinyl chloride to evaluate the relationship between the microbial community and the potential accumulation or degradation of these toxic metabolites. Multiple soil microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymidine analogue, bromodeoxyuridine (BrdU), was added to the microcosms and incorporated into the DNA of actively replicating cells. We compared the total and active bacterial communities during the 50‐day incubations by using phylogenic microarrays and 454 pyrosequencing to identify microorganisms and functional genes associated with PCE degradation to ethene. By use of this integrative approach, both the key community members and the ecological functions concomitant with complete PCE degradation could be determined, including the presence and activity of microbial community members responsible for producing hydrogen and acetate, which are critical for Dehalococcoides‐mediated PCE degradation. In addition, by correlation of chemical data and phylogenic microarray data, we identified several bacteria that could potentially oxidize hydrogen. These results demonstrate that PCE degradation is dependent on some microbial community members for production of appropriate metabolites, while other members of the community compete for hydrogen in soil at low redox potentials.     

Statin therapy, alone or with rapamycin, does not reverse monocrotaline pulmonary arterial hypertension: the rapamcyin-atorvastatin-simvastatin study

Pulmonary arterial hypertension (PAH) is characterized by excessive pulmonary artery smooth muscle cell proliferation and impaired apoptosis leading to obstruction of resistance pulmonary arteries. We hypothesized that antiproliferative (rapamycin) and proapoptotic (statins) agents, already used clinically for other indications, would decrease experimental PAH, facilitating translation to human therapies. Prior studies in the rat monocrotaline-PAH model have indicated that simvastatin regresses and rapamycin prevents, but cannot reverse, PAH. Two PAH regression strategies (rapamycin monotherapy vs. rapamycin + atorvastatin) and one prevention strategy (simvastatin) were tested in a rat monocrotaline-PAH model. Adult male Sprague-Dawley rats were randomized to saline (n = 6) or monocrotaline (60 mg/kg ip, n = 36) treatment groups. Monocrotaline rats were randomized to gavage with vehicle, rapamycin (2.5 mgxkg(-1)xday(-1)), or rapamycin + atorvastatin (10 mgxkg(-1)xday(-1)) treatment groups, beginning 12 days post-monocrotaline. Echocardiographic and hemodynamic end points were assessed 2 wk later. Additional monocrotaline-PAH rats (n = 20) were randomized to vehicle or simvastatin (2 mgxkg(-1)xday(-1)) treatment groups and followed echocardiographically for 4 wk. Monocrotaline-PAH increased lung p70 S6 kinase phosphorylation, and this was reversed by rapamycin, confirming the biological activity of rapamycin. Despite the use of high doses, neither rapamcyin nor rapamycin + atorvastatin improved survival nor reduced PAH, vascular remodeling, and right ventricular hypertrophy. Although prophylactic simvastatin slowed PAH progression, by 4 wk PAH severity and mortality were not different from placebo. Apart from the new finding of p70 S6 kinase phosphorylation in monocrotaline-PAH, this is a negative therapeutic trial (none of these promising therapies improved monocrotaline-PAH). These negative results should be considered as human trials with these agents are underway (simvastatin) or proposed (rapamycin).     

Patterns and implications of gene gain and loss in the evolution of Prochlorococcus

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.     

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

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Temperature-dependent modulation of CaV3 T-type calcium channels by protein kinases C and A in mammalian cells

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.     

Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.