Next-generation mapping of Arabidopsis genes

Next‐generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time‐consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F₂ population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F₂ population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user‐friendly, web‐based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM . We used NGM to identify three genes involved in cell‐wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F₂ lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.     

A novel series of potent and selective EP(4) receptor ligands: facile modulation of agonism and antagonism

A novel series of EP(4) ligands, based on a benzyl indoline scaffold, has been discovered. It was found that agonism and antagonism in this series can be easily modulated by minor modifications on the benzyl group. The pharmacokinetic, metabolic and pharmacological profiles of these compounds was explored. It was found that these compounds show good pharmacokinetics in rat and are efficacious in pre-clinical models of pain and inflammation.     

Leishmania chagasi: an ecto-3'-nucleotidase activity modulated by inorganic phosphate and its possible involvement in parasite-macrophage interaction

In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3′AMP 10 times more than 5′AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3′nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3′nucleotidase activity was not observed on ecto-5′nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3′nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage–parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3′nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite–macrophage interaction.     

On the electrophysiological response of bone cells using a Stokesian fluid stimulus probe for delivery of quantifiable localized picoNewton level forces

A Stokesian fluid stimulus probe (SFSP), capable of delivering quantifiable pN level hydrodynamic forces, is developed to distinguish the electrophysiological response of the cell process and cell body of osteocyte-like MLO-Y4 cells without touching the cell or its substrate. The hydrodynamic disturbance is a short lived (100 ms), constant strength pressure pulse that propagates nearly instantaneously through the medium creating a nearly spherical expanding fluid bolus surrounding a 0.8 μm micropipette tip. Laboratory model experiments show that the growth of the bolus and the pressure field can be closely modeled by quasi-steady Stokes flow through a circular orifice provided the tip Reynolds number, Re(t)<0.03. By measuring the deflection of the dendritic processes between discrete attachment sites, and applying a detailed ultrastructural model for the central actin filament bundle within the process, one is able to calculate the forces produced by the probe using elastic beam theory. One finds that forces between 1 and 2.3 pN are sufficient to initiate electrical signaling when applied to the cell process, but not the much softer cell body. Even more significantly, cellular excitation by the process only occurs when the probe is directed at discrete focal attachment sites along the cell process. This suggests that electrical signaling is initiated at discrete focal attachments along the cell process and that these sites are likely integrin-mediated complexes associated with stretch-activated ion channels though their molecular structure is unknown.     

Positive and negative effects of a dominant competitor on the settlement, growth, and survival of competing species in an epibenthic community

Because dominant competitors can monopolize resources, any positive effects they have on other species can have large community impacts. The solitary tunicate Ascidia ceratodes is a dominant competitor in the fouling community in Bodega Harbor, CA. This tunicate preempts primary substratum from competitors, but its thick tunic also allows other species to grow on its surface. The net effect of Ascidia on the community as a whole therefore depends on the balance between competitive and facilitative effects. In this study we evaluate the facilitative effects of Ascidia on different life stages of common competing species. We quantified larval settlement onto Ascidia compared to unoccupied space; we quantified the growth rate on Ascidia of small colonies of two common species; and we measured whether established colonies could escape overgrowth by Ascidia by growing onto its tunic. We found evidence for high rates of settlement on Ascidia, with some species showing higher and others lower settlement relative to that observed on free space. The growth rate of settlers was generally lower on Ascidia compared to primary substratum. We also found that colonial species established on primary space commonly escape overgrowth from Ascidia by growing onto Ascidia, as this occurs in about half of all encounters. This study indicates that the total effect of Ascidia on the community will depend on species-specific and life stage-specific effects, with likely long-term consequences for the diversity and composition of the community.