Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

Genetic and chemodiversity in native populations of Schinus terebinthifolia Raddi along the Brazilian Atlantic forest.

Schinus terebinthifolia is a species native to different ecoregions in the Brazilian Atlantic Forest. The plant is listed on the National Relation of Medicinal Plants and recommended as phytomedicine, however while extractive exploitation prevails as the main route of raw material a significant variation of compounds will be detected. To assure the expansion of productive chain it is important to start by studying population diversity and chemical variations. We used SSR markers for studies of genetic structure among populations from dense ombrophilous forest (ES); the deciduous seasonal forest (SM); the savanna (DOU) and the sandbanks (ITA and MSP), and compared the results to their chemical profiles of essential oil. Genetic structure revealed differences among populations and significant fixation rates. Pairwise studies and Bayesian analysis showed similarities between ITA and SM and between DOU and MSP, proving that the patterns of distribution for the species do not follow the isolation by distance or similarity by environmental conditions. The comparison between PCA of genotypes and chemodiversity reinforces the unique profile for each population despite the environmental similarity observed and genetic analysis. The most divergent genotype and chemical group was found at the ombrophilous forest, strong evidence that we should undertake conservation efforts to prevent losses of biodiversity in that area.

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity.

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

Development and characterization of microsatellite markers in Campomanesia adamantium, a native plant of the Cerrado ecoregions of South America.

PREMISE: A novel set of nuclear microsatellite markers was developed and characterized for Campomanesia adamantium (Myrtaceae) and tested for cross-amplification in the related species C. sessiliflora. METHODS AND RESULTS: Forty-one primer pairs were designed for simple sequence repeat loci, of which 36 successfully amplified and were polymorphic. The number of alleles ranged from two to 14, with an average of 8.14 alleles per locus. Additionally, cross-amplification was tested in C. sessiliflora; more than 55.5% of the microsatellite loci amplified, confirming the use of these microsatellite markers in a related species. CONCLUSIONS: We developed a set of microsatellite markers that will be useful for future studies of genetic diversity and population structure of C. adamantium and a closely related species, which will aid in future conservation efforts.