Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21–24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches.

Equilibrative transporters are potential drug targets, however most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64,560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2–2 µM). These nine compounds completely blocked [3H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5–50 µM). Wild-type (WT) parasite IC50 values were up to four-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.

SummaryRheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/ GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1 K120R , we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTPdependent association of Rheb with Tor.

Food allergies are common and estimated to affect 8% of children and 11% of adults in the United States. They pose a significant burden—physical, economic and social—to those affected. There is currently no available cure for food allergies. Emerging evidence suggests that the microbiome contributes to the development and manifestations of atopic disease. According to the hygiene hypothesis, children growing up with older siblings have a lower incidence of allergic disease compared with children from smaller families, due to their early exposure to microbes in the home. Research has also demonstrated that certain environmental exposures, such as a farming environment, during early life are associated with a diverse bacterial experience and reduced risk of allergic sensitization. Dysregulation in the homeostatic interaction between the host and the microbiome or gut dysbiosis appears to precede the development of food allergy, and the timing of such dysbiosis is critical. The microbiome affects food tolerance via the secretion of microbial metabolites (e.g., short chain fatty acids) and the expression of microbial cellular components. Understanding the biology of the microbiome and how it interacts with the host to maintain gut homeostasis is helpful in developing smarter therapeutic approaches. There are ongoing trials evaluating the benefits of probiotics and prebiotics, for the prevention and treatment of atopic diseases to correct the dysbiosis. However, the routine use of probiotics as an intervention for preventing allergic disease is not currently recommended. A new approach in microbial intervention is to attempt a more general modification of the gut microbiome, such as with fecal microbiota transplantation. Developing targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy. Similarly, fecal microbiota transplantation is being explored as a potentially beneficial interventional approach. Overall, targeted bacterial therapies for food allergy may be promising for both the treatment and prevention of food allergy.