Analysis of Allele-Specific Expression in Mouse Liver by RNA-Seq: A Comparison With Cis-eQTL Identified Using Genetic Linkage

We report an analysis of allele-specific expression (ASE) and parent-of-origin expression in adult mouse liver using next generation sequencing (RNA-Seq) of reciprocal crosses of heterozygous F₁ mice from the parental strains C57BL/6J and DBA/2J. We found a 60% overlap between genes exhibiting ASE and putative cis-acting expression quantitative trait loci (cis-eQTL) identified in an intercross between the same strains. We discuss the various biological and technical factors that contribute to the differences. We also identify genes exhibiting parental imprinting and complex expression patterns. Our study demonstrates the importance of biological replicates to limit the number of false positives with RNA-Seq data.     

The Synechocystis PCC6803 MerA-Like Enzyme Operates in the Reduction of Both Mercury and Uranium under the Control of the Glutaredoxin 1 Enzyme

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX₂C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.     

Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments

Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over‐represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over‐represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate‐reducing bacteria and sulphur‐oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico‐chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.     

Search for new tools to combat Gram-negative resistant bacteria among amine derivatives of 5-arylidenehydantoin

A series of amine-alkyl derivatives of 5-arylidenehydantoin 3–21 was evaluated for their ability to improve antibiotic effectiveness in two strains of Gram-negative Enterobacter aerogenes: the reference strain (ATCC-13048) and the chloramphenicol-resistant derivative over-producing the AcrAB-TolC efflux pump (CM-64). Three antibiotics, chloramphenicol, nalidixic acid and sparfloxacin were used as markers of efflux pump activity. New compounds (5–16) were obtained within 3–4 step synthesis using Knoevenagel condensation, Mitsunobu reaction and microwave aided N-alkylation. Molecular modeling based structure–activity relationship (SAR) studies were performed. The most active compounds: (Z)-5-(4-(diethylamino)benzylidene)-3-(2-hydroxy-3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (14) and (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(isopropylamino)propyl)imidazolidine-2,4-dione (15) induced fourfold decrease of minimal inhibition concentration (MIC) of all tested antibiotics in the strain CM-64 overexpressing the AcrAB-TolC pump.     

Genome-wide interval mapping using SNPs identifies new QTL for growth,body composition and several physiological variables in an F2 intercross between fat and lean chicken lines

Background

For decades, genetic improvement based on measuring growth and body composition traits has been successfully applied in the production of meat-type chickens. However, this conventional approach is hindered by antagonistic genetic correlations between some traits and the high cost of measuring body composition traits. Marker-assisted selection should overcome these problems by selecting loci that have effects on either one trait only or on more than one trait but with a favorable genetic correlation. In the present study, identification of such loci was done by genotyping an F₂ intercross between fat and lean lines divergently selected for abdominal fatness genotyped with a medium-density genetic map (120 microsatellites and 1302 single nucleotide polymorphisms). Genome scan linkage analyses were performed for growth (body weight at 1, 3, 5, and 7 weeks, and shank length and diameter at 9 weeks), body composition at 9 weeks (abdominal fat weight and percentage, breast muscle weight and percentage, and thigh weight and percentage), and for several physiological measurements at 7 weeks in the fasting state, i.e. body temperature and plasma levels of IGF-I, NEFA and glucose. Interval mapping analyses were performed with the QTLMap software, including single-trait analyses with single and multiple QTL on the same chromosome.

Results

Sixty-seven QTL were detected, most of which had never been described before. Of these 67 QTL, 47 were detected by single-QTL analyses and 20 by multiple-QTL analyses, which underlines the importance of using different statistical models. Close analysis of the genes located in the defined intervals identified several relevant functional candidates, such as ACACA for abdominal fatness, GHSR and GAS1 for breast muscle weight, DCRX and ASPSCR1 for plasma glucose content, and ChEBP for shank diameter.

Conclusions

The medium-density genetic map enabled us to genotype new regions of the chicken genome (including micro-chromosomes) that influenced the traits investigated. With this marker density, confidence intervals were sufficiently small (14 cM on average) to search for candidate genes. Altogether, this new information provides a valuable starting point for the identification of causative genes responsible for important QTL controlling growth, body composition and metabolic traits in the broiler chicken.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.