High-Efficiency Transformation and Expression of Genomic Libraries in Yeast.

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method's efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

Genome-Wide Libraries for Protozoan Pathogen Drug Target Screening Using Yeast Surface Display.

The lack of genetic tools to manipulate protozoan pathogens has limited the use of genome-wide approaches to identify drug or vaccine targets and understand these organisms' biology. We have developed an efficient method to construct genome-wide libraries for yeast surface display (YSD) and developed a YSD fitness screen (YSD-FS) to identify drug targets. We show the efficacy of our method by generating genome-wide libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of ∼105 to 106 clones, representing ∼6- to 30-fold of the parasite's genome. Nanopore sequencing confirmed the libraries' genome coverage with multiple clones for each parasite gene. Western blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS assay, we identified bonafide interactors of metronidazole, a drug used to treat protozoan and bacterial infections. We also found enrichment in nucleotide-binding domain sequences associated with yeast increased fitness to metronidazole, indicating that this drug might target multiple enzymes containing nucleotide-binding domains. The libraries are valuable biological resources for discovering drug or vaccine targets, ligand receptors, protein-protein interactions, and pathogen-host interactions. The library assembly approach can be applied to other organisms or expression systems, and the YSD-FS assay might help identify new drug targets in protozoan pathogens.

In silico, in vitro, and in vivo investigation of antioxidant potential and toxicity of ethyl ferulate.

By submitting this manuscript, each author certifies that they have made a direct and substantial contribution to the work reported in the manuscript. In this manuscript the conception, design, investigation, acquisition of data and analysis, interpretation of data and writing of the article were conducted by author Camila Bomfim de Sá under the guidance of professors Margareth de Fátima Formiga Melo Diniz, Hilzeth de Luna Freire Pessôa and Caliandra Maria Bezerra Luna Lima, who also approved the final version of the manuscript. Professor Damião Pergentino de Sousa and his student Mayara Castro de Morais performed the production, synthesis and chemical characterization of ethyl ferulate (EF). Professor Abrahão Alves de Oliveira Filho assessed the in silico tests. PhD student Andressa Brito Lira participated in the critical review of the text for important intellectual content and assisted in the in vitro antioxidant activity and cytotoxicity tests. Kardilandia Mendes de Oliveira participated in acute oral toxicity tests evaluating the biochemical parameters. Students, Tafaela Dias and Cinthia Rodrigues Melo also assisted in the acute oral toxicity testing and preparing of slides for histopathological analysis. Pathologist Alexandre Rolim da Paz analyzed the histopathology results. EF, a phenolic compound of the large class of phenylpropanoids, is derived from ferulic acid and is produced both naturally and synthetically. Its principal pharmacological activities are: anti-inflammatory and antioxidant activity. This study aimed to investigate the in silico, in vitro and in vivo toxicity and antioxidant activity of EF. The in silico prediction showed more than 20 biological activities as well as good absorption at the biological membranes and no theoretical toxicity. However, EF presented high environmental toxicity. EF presented low hemolytic potential and exerted protective activity for the erythrocyte membrane for only blood type O. EF presented antioxidant activity against H2O2 at all concentrations and all blood types, but no effect against phenylhydrazine, being unable to prevent its oxidative effects. In the acute nonclinical toxicological trial, the treated animals presented behavioral changes (e.g., sedation). Feed intake was higher for the 2000 mg/kg group, but with no significant difference in weight change. The biochemical parameters presented no differences between treated and control animals, and the organs remained intact with no change. Thus, EF presents a low toxic profile and this study provides important information about the toxicity of this compound, suggesting future safe use.