Adaptive evolution of the lactose utilization network in experimentally evolved populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.     

Field Evaluation and Impact on Clinical Management of a Rapid Diagnostic Kit That Detects Dengue NS1, IgM and IgG

Background

Dengue diagnosis is complex and until recently only specialized laboratories were able to definitively confirm dengue infection. Rapid tests are now available commercially making biological diagnosis possible in the field. The aim of this study was to evaluate a combined dengue rapid test for the detection of NS1 and IgM/IgG antibodies. The evaluation was made prospectively in the field conditions and included the study of the impact of its use as a point-of-care test for case management as well as retrospectively against a panel of well-characterized samples in a reference laboratory.

Methodology/Principal Findings

During the prospective study, 157 patients hospitalized for a suspicion of dengue were enrolled. In the hospital laboratories, the overall sensitivity, specificity, PPV and NPV of the NS1/IgM/IgG combination tests were 85.7%, 83.9%, 95.6% and 59.1% respectively, whereas they were 94,4%, 90.0%, 97.5% and 77.1% respectively in the national reference laboratory at Institut Pasteur in Cambodia. These results demonstrate that optimal performances require adequate training and quality assurance. The retrospective study showed that the sensitivity of the combined kit did not vary significantly between the serotypes and was not affected by the immune status or by the interval of time between onset of fever and sample collection. The analysis of the medical records indicates that the physicians did not take into consideration the results obtained with the rapid test including for care management and use of antibiotic therapy.

Conclusions

In the context of our prospective field study, we demonstrated that if the SD Bioline Dengue Duo kit is correctly used, a positive result highly suggests a dengue case but a negative result doesn''t rule out a dengue infection. Nevertheless, Cambodian pediatricians in their daily practice relied on their clinical diagnosis and thus the false negative results obtained did not directly impact on the clinical management.     

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK₂ complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.     

Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.     

Disregarding the edaphic dimension in species distribution models leads to the omission of crucial spatial information under climate change: the case of Quercus pubescens in France

Species distribution modelling is an easy, persuasive and useful tool for anticipating species distribution shifts under global change. Numerous studies have used only climate variables to predict future potential species range shifts and have omitted environmental factors important for determining species distribution. Here, we assessed the importance of the edaphic dimension in the niche‐space definition of Quercus pubescens and in future spatial projections under global change over the metropolitan French forest territory. We fitted two species distribution models (SDM) based on presence/absence data (111 013 plots), one calibrated from climate variables only (mean temperature of January and climatic water balance of July) and the other one from both climate and edaphic (soil pH inferred from plants) variables. Future predictions were conducted under two climate scenarios (PCM B2 and HadCM3 A2) and based on 100 simulations using a cellular automaton that accounted for seed dispersal distance, landscape barriers preventing migration and unsuitable land cover. Adding the edaphic dimension to the climate‐only SDM substantially improved the niche‐space definition of Q. pubescens, highlighting an increase in species tolerance in confronting climate constraints as the soil pH increased. Future predictions over the 21st century showed that disregarding the edaphic dimension in SDM led to an overestimation of the potential distribution area, an underestimation of the spatial fragmentation of this area, and prevented the identification of local refugia, leading to an underestimation of the northward shift capacity of Q. pubescens and its persistence in its current distribution area. Spatial discrepancies between climate‐only and climate‐plus‐edaphic models are strengthened when seed dispersal and forest fragmentation are accounted for in predicting a future species distribution area. These discrepancies highlight some imprecision in spatial predictions of potential distribution area of species under climate change scenarios and possibly wrong conclusions for conservation and management perspectives when climate‐only models are used.     

Heart rate responses and technical-tactical aspects of official 5-a-side youth soccer matches played on clay and artificial turf

Using a randomized crossover design, this study aimed to compare the heart rate (HR) responses and match analysis parameters (i.e., type of action; the number of players involved in an action; the number of passes performed in a collective action; precision of the shots; lost balls; ball interceptions; dribblings; and tackles) of official 5-a-side youth male soccer matches played over 2 pitch surface (i.e., clay vs. artificial turf) conditions. Twenty-two young male soccer players (age 8.3 ± 0.4 years) engaged in 2 consecutive 15-minute periods in each experimental condition. During the match, 53% of HR responses exceeded 85% of the individual HR(peak). No difference emerged between both pitch surfaces and match periods for HR and match analysis indicators. The two 15-minute periods resulted in being appropriate for 8-year-old players, in resembling the intensity of play and the intermittent nature of adult soccer. The similar HR responses and technical-tactical patterns observed on the 2 surfaces indicate that youth match play is not affected by differences in pitch surface. To support the development of individual and team skills of young soccer players, coaches are advised to prioritize game knowledge based on divergent thinking and experience, rather than traditional teaching based on instructions.     

Selection of floral resources to optimise conservation of agriculturally-functional insect groups

Non-crop vegetation in agricultural landscapes can provide a means of conserving insects in farmed landscapes and optimising on-farm ecosystem services as a result. Inclusion of floral resources may be particularly useful in conserving many beneficial insects, where groups including pollinators and pest natural enemies often rely on nectar and pollen during (at least part of) their life-cycle. As not all flowers are equally suited to all beneficial insects, selection of appropriate flowering plants is key to ensuring that conservation targets are met and benefits to ecosystems services realised as a result. This short paper describes an experiment conducted to assess the ‘total temporal attractiveness’ of a range of British wildflowers to selected functional insect groups. The results obtained demonstrate that flowering period alone is a poor indicator of plant suitability to insects, where no relationship existed between this and attraction to insects overall. Data also suggest that, based on attraction over a season, certain flowering plants are more likely to be of general insect conservation value and/or benefit to functional insect groups than others. Attraction to pest insects was also considered, with relatively high catches of thrips and pollen beetles observed in flowering stands of some plants.     

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK₂ in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK₂ is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of P_i limiting, and (iii) the additional presence of maltose stimulates release of P_i, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK₂ in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and P_i release. This concerted action explains why ATPase activity of MalFGK₂ depends on maltose, and why MalE is essential for transport.     

APIS: An auto‐adaptive parentage inference software that tolerates missing parents

In the context of parentage assignment using genomic markers, key issues are genotyping errors and an absence of parent genotypes because of sampling, traceability or genotyping problems. Most likelihood‐based parentage assignment software programs require a priori estimates of genotyping errors and the proportion of missing parents to set up meaningful assignment decision rules. We present here the R package APIS, which can assign offspring to their parents without any prior information other than the offspring and parental genotypes, and a user‐defined, acceptable error rate among assigned offspring. Assignment decision rules use the distributions of average Mendelian transmission probabilities, which enable estimates of the proportion of offspring with missing parental genotypes. APIS has been compared to other software (CERVUS, VITASSIGN), on a real European seabass (Dicentrarchus labrax) single nucleotide polymorphism data set. The type I error rate (false positives) was lower with APIS than with other software, especially when parental genotypes were missing, but the true positive rate was also lower, except when the theoretical exclusion power reached 0.99999. In general, APIS provided assignments that satisfied the user‐set acceptable error rate of 1% or 5%, even when tested on simulated data with high genotyping error rates (1% or 3%) and up to 50% missing sires. Because it uses the observed distribution of Mendelian transmission probabilities, APIS is best suited to assigning parentage when numerous offspring (>200) are genotyped. We have demonstrated that APIS is an easy‐to‐use and reliable software for parentage assignment, even when up to 50% of sires are missing.