Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.     

Matapax: an online high-throughput genome-wide association study pipeline

High-throughput sequencing and genotyping methods are dramatically increasing the number of observable genetic intraspecies differences that can be exploited as genetic markers. In addition, automated phenotyping platforms and "omics" profiling technologies further enlarge the set of quantifiable macroscopic and molecular traits at an ever-increasing pace. Combined, both lines of technological advances create unparalleled opportunities to identify candidate gene regions and, ideally, even single genes responsible for observed variations in a particular trait via association studies. However, as of yet, this new potential is not sufficiently matched by enabling software solutions to easily exploit this wealth of genotype/phenotype information. We have developed Matapax, a Web-based platform to address this need. Initially, we built the infrastructure to support association studies in Arabidopsis (Arabidopsis thaliana) based on several genotyping efforts covering up to 1,375 Arabidopsis accessions. Based on the user-supplied trait information, associated single-nucleotide polymorphism markers and single-nucleotide polymorphism-harboring or -neighboring genes are identified using both the GAPIT and EMMA libraries developed for R. Additional interrogation is facilitated by displaying candidate regions and genes in a genome browser and by providing relevant annotation information. In the future, we plan to broaden the scope of organisms to other plant species as more genotype/phenotype information becomes available. Matapax is freely available at http://matapax.mpimp-golm.mpg.de and can be accessed using any internet browser.     

Morphological evolution in land plants: new designs with old genes

The colonization and radiation of multicellular plants on land that started over 470 Ma was one of the defining events in the history of this planet. For the first time, large amounts of primary productivity occurred on the continental surface, paving the way for the evolution of complex terrestrial ecosystems and altering global biogeochemical cycles; increased weathering of continental silicates and organic carbon burial resulted in a 90 per cent reduction in atmospheric carbon dioxide levels. The evolution of plants on land was itself characterized by a series of radical transformations of their body plans that included the formation of three-dimensional tissues, de novo evolution of a multicellular diploid sporophyte generation, evolution of multicellular meristems, and the development of specialized tissues and organ systems such as vasculature, roots, leaves, seeds and flowers. In this review, we discuss the evolution of the genes and developmental mechanisms that drove the explosion of plant morphologies on land. Recent studies indicate that many of the gene families which control development in extant plants were already present in the earliest land plants. This suggests that the evolution of novel morphologies was to a large degree driven by the reassembly and reuse of pre-existing genetic mechanisms.     

Sex differences in risk factors for cardiovascular disease: the PERU MIGRANT study

Introduction

Although men and women have similar risk factors for cardiovascular disease, many social behaviors in developing countries differ by sex. Rural-to-urban migrants have different cardiovascular risk profiles than rural or urban dwellers. The objective of this study was to evaluate the sex differences with specific cardiovascular risk factors in rural-to-urban migrants.

Methods and Results

We used the rural-to-urban migrant group of the PERU MIGRANT cross-sectional study to investigate the sex differences in specific cardiovascular risk factors: obesity, hypertension, metabolic syndrome, as well as exposures of socioeconomic status, acculturation surrogates and behavioral characteristics. Logistic regression analysis was used to characterize strength of association between sex and our outcomes adjusting for potential confounders. The sample of migrants was 589 (mean age 46.5 years) and 52.4% were female. In the adjusted models, women were more likely to be obese (OR=5.97; 95%CI: 3.21–11) and have metabolic syndrome (OR=2.22; 95%CI: 1.39–3.55) than men, explaining the greatest variability for obesity and metabolic syndrome but not for hypertension.

Conclusions

Our results suggest that interventions for CVD in Peru should be sex-specific and address the unique health needs of migrant populations living in urban shantytowns since the risk factors for obesity and metabolic syndrome differ between males and females.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

On the application of active learning and Gaussian processes in postcryopreservation cell membrane integrity experiments

Biological cell cryopreservation permits storage of specimens for future use. Stem cell cryostorage in particular is fast becoming a broadly spread practice due to their potential for use in regenerative medicine. For the optimal cryopreservation process, ultralow temperatures are needed. However, elevated temperatures are often unavoidable in a typical sample handling cycle which in turn negatively affects the postcryopreservation integrity of cells. In this paper, we present an application of active learning using an underlying Gaussian Process (GP) model in an experimental study on postcryopreservation membrane integrity response to a range of elevated temperature conditions. We tailored this technique for the current investigation and developed an algorithm which enabled identification of the sampling locations for the experiments in order to obtain the highest information return about the process from a limited size sample set. We applied this algorithm in the experimental study investigating the effects of severe temperature elevation (ranging from -40 to 20 °C) over a short term event (48 hours) on the postcryopreservation membrane integrity of Mesenchymal Stem Cells (MSCs) derived from human bone marrow. The algorithm showed excellent performance by selecting the locations which maximized the reduction of variance of the process response estimate. An approximating GP model developed from this experimental data shows that the elevated temperatures during cryopreservation have an imminent detrimental effect on cell integrity.     

Fast spatiotemporal smoothing of calcium measurements in dendritic trees

We discuss methods for fast spatiotemporal smoothing of calcium signals in dendritic trees, given single-trial, spatially localized imaging data obtained via multi-photon microscopy. By analyzing the dynamics of calcium binding to probe molecules and the effects of the imaging procedure, we show that calcium concentration can be estimated up to an affine transformation, i.e., an additive and multiplicative constant. To obtain a full spatiotemporal estimate, we model calcium dynamics within the cell using a functional approach. The evolution of calcium concentration is represented through a smaller set of hidden variables that incorporate fast transients due to backpropagating action potentials (bAPs), or other forms of stimulation. Because of the resulting state space structure, inference can be done in linear time using forward-backward maximum-a-posteriori methods. Non-negativity constraints on the calcium concentration can also be incorporated using a log-barrier method that does not affect the computational scaling. Moreover, by exploiting the neuronal tree structure we show that the cost of the algorithm is also linear in the size of the dendritic tree, making the approach applicable to arbitrarily large trees. We apply this algorithm to data obtained from hippocampal CA1 pyramidal cells with experimentally evoked bAPs, some of which were paired with excitatory postsynaptic potentials (EPSPs). The algorithm recovers the timing of the bAPs and provides an estimate of the induced calcium transient throughout the tree. The proposed methods could be used to further understand the interplay between bAPs and EPSPs in synaptic strength modification. More generally, this approach allows us to infer the concentration on intracellular calcium across the dendritic tree from noisy observations at a discrete set of points in space.     

Structural insight into the bacterial mucinase StcE essential to adhesion and immune evasion during enterohemorrhagic E. coli infection

Mucin glycoproteins with large numbers of O-linked glycosylations comprise the mucosal barrier lining the mammalian gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect the underlying epithelium from infection. Enterohemorrhagic Escherichia coli (EHEC), the mediator of severe food- and water-borne disease, can breach this barrier and adhere to intestinal cells. StcE, a ～100 kDa metalloprotease secreted by EHEC, plays a pivotal role in remodeling the mucosal lining during infection. To obtain mechanistic insight into its function, we have determined the structure of StcE. Our data reveal a dynamic, multidomain architecture featuring an unusually large substrate-binding cleft and a prominent polarized surface charge distribution highly suggestive of an electrostatic role in substrate targeting. The observation of key conserved motifs in the active site allows us to propose the structural basis for the specific recognition of α-O-glycan-containing substrates. Complementary biochemical analysis provides further insight into its distinct substrate specificity and binding stoichiometry.