Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. E. Paux and F. Legeai contributed equally to this work.     

Influence of histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the PD1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA1 and psbA2 genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT*) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA3 gene. The O2-evolving activity of His-tagged PSII isolated from WT* was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA1 is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT*. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P680, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to PD1 is not an important determinant of the redox and spectroscopic properties of P680 in T. elongatus.     

Synthesis of biodegradable poly-epsilon-caprolactone microspheres by dispersion ring-opening polymerization in supercritical carbon dioxide

A series of fluorinated diblock and triblock copolymers of poly(epsilon-caprolactone) and poly(heptadecafluorodecylacrylate) were prepared by combining ring-opening polymerization of epsilon-CL and atom transfer radical polymerization of the acrylate. These copolymers with well-controlled molecular weight and composition were characterized by (1)H NMR spectroscopy and used as stabilizers for the dispersion ring-opening polymerization of epsilon-CL in supercritical carbon dioxide. The effect of composition and architecture of the polymeric stabilizers on the stabilization of PCL microspheres was investigated. Finally, purification of PCL was successfully implemented by reactive supercritical fluid extraction of the tin catalyst.     

Evaluation of the Impact of the Cancer Therapy Everolimus on the Central Nervous System in Mice

Cancer and treatments may induce cognitive impairments in cancer patients, and the causal link between chemotherapy and cognitive dysfunctions was recently validated in animal models. New cancer targeted therapies have become widely used, and their impact on brain functions and quality of life needs to be explored. We evaluated the impact of everolimus, an anticancer agent targeting the mTOR pathway, on cognitive functions, cerebral metabolism, and hippocampal cell proliferation/vascular density in mice. Adult mice received everolimus daily for 2 weeks, and behavioral tests were performed from 1 week after the last treatment. Everolimus-treated mice displayed a marked reduction in weight gain from the last day of the treatment period. Ex vivo analysis showed altered cytochrome oxidase activity in selective cerebral regions involved in energy balance, food intake, reward, learning and memory modulation, sleep/wake cycle regulation, and arousal. Like chemotherapy, everolimus did not alter emotional reactivity, learning and memory performances, but in contrast to chemotherapy, did not affect behavioral flexibility or reactivity to novelty. In vivo hippocampal neural cell proliferation and vascular density were also unchanged after everolimus treatments. In conclusion, two weeks daily everolimus treatment at the clinical dose did not evoke alteration of cognitive performances evaluated in hippocampal- and prefrontal cortex-dependent tasks that would persist at one to four weeks after the end of the treatment completion. However, acute everolimus treatment caused selective CO modifications without altering the mTOR effector P70S6 kinase in cerebral regions involved in feeding behavior and/or the sleep/wake cycle, at least in part under control of the solitary nucleus and the parasubthalamic region of the hypothalamus. Thus, this area may represent a key target for everolimus-mediating peripheral modifications, which has been previously associated with symptoms such as weight loss and fatigue.     

Enrichment of N-terminal cysteinyl-peptides by covalent capture

The detection of low abundance proteins in complex biological samples is still a challenge in proteomics. To circumvent this obstacle a number of strategies involving the targeting of subsets of proteins or peptides were developed.The following work describes a new approach to simplify peptide mixtures by enrichment of N-terminal cysteinyl peptides (and to some extent N-terminal threonine peptides). The strategy is based on the use of an isolation method, so-called covalent capture (CC), which relies on the formation of a covalent bond between an N-terminal free cysteine or N-terminal free threonine and an aldehyde fixed on a solid support. The CC is highly selective. It permits extensive washes of the resin for the elimination of non-specific moieties before the release of the captured peptides. The application of the CC to proteomics was evaluated on tryptic peptides of standard proteins and test protein mixtures. The procedure demonstrated a significant reduction in sample complexity, while allowing the identification of N-terminal cysteinyl peptides hidden in the non-fractionated samples.This new strategy provides an efficient tool to existing proteomics approaches to reduce sample complexity and potentially identify less abundance proteins.