A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.     

Exploring the HME and HAE1 efflux systems in the genus <Emphasis Type="Italic">Burkholderia</Emphasis>

Background  

The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia.     

Viability and Management of an Endangered Capercaillie (Tetrao urogallus) Metapopulation in the Jura Mountains, Western Switzerland

The populations of Capercaillie (Tetrao urogallus), the largest European grouse, have seriously declined during the last century over most of their distribution in western and central Europe. In the Jura mountains, the relict population is now isolated and critically endangered (about 500 breeding adults). We developed a simulation software (TetrasPool) that accounts for age and spatial structure as well as stochastic processes, to perform a viability analysis and explore management scenarios for this population, capitalizing on a 24 years-long series of field data. Simulations predict a marked decline and a significant extinction risk over the next century, largely due to environmental and demographic stochasticity (average values of life-history parameters would otherwise allow stability). Variances among scenarios mainly stem from uncertainties about the shape and intensity of density dependence. Uncertainty analyses suggest to focus conservation efforts on enhancing, not only adult survival (as often advocated for long-lived species), but also recruitment. The juvenile stage matters when local populations undergo extinctions, because it ensures connectivity and recolonization. Besides limiting human perturbations, a silvicultural strategy aimed at opening forest structure should improve the quality and surface of available patches, independent of their size and localization. Such measures are to be taken urgently, if the population is to be saved.     

Distribution of Lilian's Lovebirds in Malawi

Lilian's Lovebird Agapornis lilianae is regarded as a mopane Colophospermum mopane woodland specialist. Its global population is sparse and is spread along the Zambezi valley with little known about its current distribution and status. We explored the current distribution of Lilian's Lovebird in Malawi, and in particular focused on the distribution and habitat associations of the largest resident population in Liwonde National Park (LNP). Local birders and tourist guides provided distribution information from across Malawi. Transect walks were conducted to collect data in LNP. Five new atlas records are reported; three were within 40–56 km of the LNP population and two were over 150 km south and north of LNP, respectively. One of the sites is about 66 km from the Lilian's Lovebird population in Luangwa Valley, Zambia. New national records were evidence of the importance of seasonal movements to the species. Lilian's Lovebirds occurred throughout LNP with the highest abundance in the central section. Seasonal movements to areas outside the park were also recorded. A variety of vegetation types were used by the lovebirds, but the strongest habitat associations were with seasonally wet grasslands and not mopane woodlands as would be expected. Thus, conservation efforts should also include these other habitats.     

Investigating the molecular determinants for substrate channeling in BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls degradation pathway

BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls (PCBs) degradation pathway, cleaves 4-hydroxy-2-oxoacids to pyruvate and an aldehyde. The enzyme complex was shown to exhibit substrate channeling, whereby linear aldehydes of up to 6 carbons long and branched isobutyraldehyde were directly channeled from the aldolase to the dehydrogenase with greater than 80% efficiency. BphI variants G322F, G322L, and G323F were created and were found to block aldehyde channeling. The dehydrogenase cofactor NADH was able to activate the catalytic activity of the aldol cleavage reaction in these variants, suggesting that activation of BphI by BphJ cofactors is not solely due to faster aldehyde release. A G323L variant was able to channel acetaldehyde but not the larger propionaldehyde while the G323A variant was able to channel butyraldehyde but not its isomer isobutyraldehyde, confirming that the restricted channeling of aldehydes in these glycine variants are due to steric blockage of the channel. Substitution of His-20 and Tyr-290 in BphI led to significant reductions in aldehyde channeling efficiencies. A mechanism of substrate channeling involving these two gating residues is proposed.     

TEMPERATURE‐DEPENDENT TURNOVERS IN SEX‐DETERMINATION MECHANISMS: A QUANTITATIVE MODEL

Sex determination is often seen as a dichotomous process: individual sex is assumed to be determined either by genetic (genotypic sex determination, GSD) or by environmental factors (environmental sex determination, ESD), most often temperature (temperature sex determination, TSD). We endorse an alternative view, which sees GSD and TSD as the ends of a continuum. Both effects interact a priori, because temperature can affect gene expression at any step along the sex‐determination cascade. We propose to define sex‐determination systems at the population‐ (rather than individual) level, via the proportion of variance in phenotypic sex stemming from genetic versus environmental factors, and we formalize this concept in a quantitative‐genetics framework. Sex is seen as a threshold trait underlain by a liability factor, and reaction norms allow modeling interactions between genotypic and temperature effects (seen as the necessary consequences of thermodynamic constraints on the underlying physiological processes). As this formalization shows, temperature changes (due to e.g., climatic changes or range expansions) are expected to provoke turnovers in sex‐ determination mechanisms, by inducing large‐scale sex reversal and thereby sex‐ratio selection for alternative sex‐determining genes. The frequency of turnovers and prevalence of homomorphic sex chromosomes in cold‐blooded vertebrates might thus directly relate to the temperature dependence in sex‐determination mechanisms.     

Expression of LSLCL, a new C-type lectin, is closely restricted, in bone marrow, to immature neutrophils.

In vitro, LSLCL is expressed by numerous myeloid, promyelocytic, and T or B lymphoblastoid cell lines. In vivo, LSLCL is strongly expressed in bone marrow and only faintly in lymphoid organs. We show here that, in bone marrow, LSLCL is detected: (i) concentrated in the cytoplasm of immature neutrophils but not in myeloblasts nor in mature neutrophils, (ii) in extracellular bone marrow fluid. Besides, numerous cDNAs, similar to LSLCL (identity of 93-99%), are found in 'expressed sequence tags' databases from various origins, mostly fetal and undifferentiated tumour tissues. Since LSLCL and various closely related cDNAs are expressed at definite stages of cellular maturation processes, we hypothesize that this class of proteins could play an important role in the control of cellular differentiation.