Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping

The extraordinary phenotypic diversity of dog breeds has been sculpted by a unique population history accompanied by selection for novel and desirable traits. Here we perform a comprehensive analysis using multiple test statistics to identify regions under selection in 509 dogs from 46 diverse breeds using a newly developed high-density genotyping array consisting of >170,000 evenly spaced SNPs. We first identify 44 genomic regions exhibiting extreme differentiation across multiple breeds. Genetic variation in these regions correlates with variation in several phenotypic traits that vary between breeds, and we identify novel associations with both morphological and behavioral traits. We next scan the genome for signatures of selective sweeps in single breeds, characterized by long regions of reduced heterozygosity and fixation of extended haplotypes. These scans identify hundreds of regions, including 22 blocks of homozygosity longer than one megabase in certain breeds. Candidate selection loci are strongly enriched for developmental genes. We chose one highly differentiated region, associated with body size and ear morphology, and characterized it using high-throughput sequencing to provide a list of variants that may directly affect these traits. This study provides a catalogue of genomic regions showing extreme reduction in genetic variation or population differentiation in dogs, including many linked to phenotypic variation. The many blocks of reduced haplotype diversity observed across the genome in dog breeds are the result of both selection and genetic drift, but extended blocks of homozygosity on a megabase scale appear to be best explained by selection. Further elucidation of the variants under selection will help to uncover the genetic basis of complex traits and disease.     

A universal homogeneous assay for high-throughput determination of binding kinetics

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug–target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR–FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein–protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

Fine epitope specificity of antibodies against interleukin-2 explains their paradoxical immunomodulatory effects

The functional dichotomy of antibodies against interleukin-2 (IL-2) is thought to depend upon recognition of different cytokine epitopes. Beyond functional studies, the only molecular evidence obtained so far located the epitopes recognized by the immunoenhancing antibodies S4B6 and JES6–5H4 within the predicted interface of IL-2 with the α receptor subunit, explaining the preferential stimulation of effector cells displaying only β and γ receptor chains. A consistent functional map of the epitope bound by the immunoregulatory antibody JES6-1A12 has now been delineated by screening the interactions of phage-displayed antigen variants (with single and multiple mutations) and antigen mimotopes. The target determinant resides in a region between the predicted interfaces with α and β/γ receptor subunits, supporting the dual inhibitory role of the antibody on both interactions. Binding by JES6-1A12 would thus convert complexed IL-2 into a very weak agonist, reinforcing the advantage of T regulatory cells (displaying the high affinity αβγ heterotrimeric receptor) to capture the cytokine by competition and expand over effector cells, ultimately resulting in the observed strong tolerogenic effect of this antibody. Detailed knowledge of the epitopes recognized by anti-IL-2 antibodies with either immunoenhancing or immunoregulatory properties completes the molecular scenario underlying their use to boost or inhibit immune responses in multiple experimental systems. The expanded functional mapping platform now available could be exploited to study other interactions involving related molecular pairs with the final goal of optimizing cytokine and anti-cytokine therapies.     

Probability of fixation under weak selection: a branching process unifying approach

We link two-allele population models by Haldane and Fisher with Kimura's diffusion approximations of the Wright-Fisher model, by considering continuous-state branching (CB) processes which are either independent (model I) or conditioned to have constant sum (model II). Recent works by the author allow us to further include logistic density-dependence (model III), which is ubiquitous in ecology. In all models, each allele (mutant or resident) is then characterized by a triple demographic trait: intrinsic growth rate r, reproduction variance sigma and competition sensitivity c. Generally, the fixation probability u of the mutant depends on its initial proportion p, the total initial population size z, and the six demographic traits. Under weak selection, we can linearize u in all models thanks to the same master formula u = p + p(1 - p)[g(r)s(r) + g(sigma)s(sigma) + g(c)s(c)] + o(s(r),s(sigma),s(c), where s(r) = r' - r, s(sigma) = sigma-sigma' and s(c) = c - c' are selection coefficients, and g(r), g(sigma), g(c) are invasibility coefficients (' refers to the mutant traits), which are positive and do not depend on p. In particular, increased reproduction variance is always deleterious. We prove that in all three models g(sigma) = 1/sigma and g(r) = z/sigma for small initial population sizes z. In model II, g(r) = z/sigma for all z, and we display invasion isoclines of the 'mean vs variance' type. A slight departure from the isocline is shown to be more beneficial to alleles with low sigma than with high r. In model III, g(c) increases with z like ln(z)/c, and g(r)(z) converges to a finite limit L > K/sigma, where K = r/c is the carrying capacity. For r > 0 the growth invasibility is above z/sigma when z < K, and below z/sigma when z > K, showing that classical models I and II underestimate the fixation probabilities in growing populations, and overestimate them in declining populations.     

Predicting functional residues in Plasmodium falciparum plasmepsins by combining sequence and structural analysis with molecular dynamics simulations

Plasmepsins are aspartic proteases involved in the initial steps of the hemoglobin degradation pathway, a critical stage in the Plasmodium falciparum life cycle during human infection. Thus, they are attractive targets for novel therapeutic compounds to treat malaria, which remains one of the world's biggest health problems. The three-dimensional structures available for P. falciparum plasmepsins II and IV make structure-based drug design of antimalarial compounds that focus on inhibiting plasmepsins possible. However, the structural flexibility of the plasmepsin active site cavity combined with insufficient knowledge of the functional residues and of those determining the specificity of parasitic enzymes is a drawback when designing specific inhibitors. In this study, we have combined a sequence and structural analysis with molecular dynamics simulations to predict the functional residues in P. falciparum plasmepsins. The careful analysis of X-ray structures and 3D models carried out here suggests that residues Y17, V105, T108, L191, L242, Q275, and T298 are important for plasmepsin function. These seven amino acids are conserved across the malarial strains but not in human aspartic proteases. Residues V105 and T108 are localized in a flap of an interior pocket and they only establish contacts with a specific non-peptide achiral inhibitor. We also observed a rapid conformational change in the L3 region of plasmepsins that closes the active site of the enzyme, which explains earlier experimental findings. These results shed light on the role of V105 and T108 residues in plasmepsin specificities, and they should be useful in structure-based design of novel, selective inhibitors that may serve as antimalarial drugs.     

Molecular modelling of phthalates - PPARs interactions

Di(2-ethylhexyl) phthalate (DEHP) is the most widely plasticizer for polyvinyl chloride (PVC) that is used in plastic tubes, in medical and paramedical devices as well as in food storage packaging. The toxicological profile of DEHP has been evaluated in a number of experimental animal models and has been extensively documented. Its toxicity is in part linked to the activation of the peroxisome proliferator-activated receptor alpha (PPAR(alpha)). As a response, an intensive research for a new, biologically inert plasticizer has been initiated. Among the alternative studied, tri(2-ethylhexyl) trimellitate (TEHTM) or trioctyl trimellitate (TOTM) has attracted increasing interest. However, very little information is available on their biological effects. We proceeded to dock TOTM, DEHP and its metabolites in order to identify compounds that are likely to interact with PPAR(alpha) and PPAR(gamma) binding sites. The results obtained hint that TOTM is not able to bind to PPARs and should therefore be safer than DEHP.     

Phosphorylation relieves autoinhibition of the kinetochore motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.     

Insect herbivores should follow plants escaping their relatives

Numerous observational studies have documented conspecific negative density-dependence that is consistent with the Janzen–Connell Hypothesis (JCH) of diversity maintenance. However, there have been few experimental tests of a central prediction of the JCH: that removing host-specific enemies should lead to greater increases in per capita recruitment in areas of higher host density or lower relative phylogenetic diversity. Using spatially randomized plots of high and low host biomass in a temperate grassland biodiversity experiment, we treated developing seedheads of six prairie perennials to factorial applications of fungicide and insecticide. We measured predispersal seed production, seed viability, and seedling biomass. Results were highly species-specific and idiosyncratic. Effects of insect seed predators and fungal pathogens on predispersal responses varied with neither conspecific biomass nor phylogenetic diversity, suggesting that—at least at the predispersal stage and for the insect and fungal seed predators we were able to exclude—the JCH is not sufficient to contribute to negative conspecific density-dependence for these dominant prairie species.