A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

RESUMO:Atualmente, as plantas medicinais movem altos valores financeiros em todo o mundo e representam o tipo de tratamento mais acessível para cerca de 80% da população, principalmente entre os países em desenvolvimento. Entretanto, existe ainda uma falta de conhecimento sobre propriedades químicas, farmacológicas e toxicológicas a fim de assegurar a eficácia e segurança das plantas medicinais. Os critérios de eficácia e segurança de plantas medicinais estão relacionados a qualidade, isto é, as plantas necessitam ser corretamente identificadas, cultivadas e coletadas, devem estar livres de material estranho, partes de outras plantas e contaminações inorgânicas e/ ou microbianas. O objetivo deste estudo foi o de elucidar os diferentes processos e padronizações sobre o controle de drogas vegetais, principalmente no Brasil.Unitermos: Plantas medicinais, fitoquímica, controle de qualidade.ABSTRACT: "Brazil in the context of plants and derivates quality control". Nowadays medicinal plants move high financial values at the world and represent a type of treatment more accessible for about 80% of people, mainly among developing countries. However, there is a lack of knowledge about chemical, pharmacological and toxicological properties towards ensure their efficacy and safety. Efficacy and safety criteria of medicinal plants are related to quality, that is, the plants need to be correctly identified, cultivated and harvested, they must be free of strange material, part of other plants and inorganic and/or microorganisms contamination. The aim of this study was to elucidate different process and standardizations about quality control of vegetal drugs mainly in Brazil. ISSN 0102-695X Keywords INTRODUÇÃOPor longos períodos na história, o uso de plantas medicinais foi presente não só devido ao seu caráter alimentar, mas também às suas propriedades de cura, sejam elas reais ou ritualísticas. Entretanto, até a metade do século XX, o uso de medicamentos de origem sintética tornou-se mais amplo que o uso de plantas medicinais, principalmente na sociedade ocidental, com destaque para os países desenvolvidos visto que os considerados países em desenvolvimento permaneceram fazendo uso das propriedades curativas das plantas, certamente devido às difíceis condições econômicas de aquisição dos medicamentos sintéticos

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65–74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Cytokines expression can be influenced by polymorphisms in their respective coding genes. We associated the CTI/TTD haplotype (Hap-1) and TCI/CCI haplotype (Hap-2) in the IL4 gene formed by the −590, +33 and variable number of tandem repeat polymorphisms with the severity of chronic periodontitis in humans. The functionality of these IL4 haplotypes in the response of immune cells to phorbol 12-myristate 13-acetate (PMA) with Ionomycin and IL-1β (as inflammatory stimuli) was evaluated. Gene expression (quantitative real-time PCR), profile of secreted cytokines (multiplex) and phenotypic polarization of T cells (flow cytometry) were the outcomes assessed. Green fluorescent protein reporter plasmid constructs containing specific IL4 haplotype were transiently transfected into JM cells to assess the influence of the individual haplotypes on promoter activity. In response to inflammatory stimuli the immune cells from Hap-1 haplotype had increased expression of anti-inflammatory IL4; conversely, the Hap-2 haplotype showed higher levels of pro-inflammatory cytokines. The haplotype CTI proved to be the most important for the regulation of IL4 promoter, regardless of the nature of the inflammatory stimulation; whereas the polymorphism in the promoter region had the least functional effect. In conclusion, IL4 haplotypes studied are functional and trigger opposite immune responses: anti-inflammatory (Hap-1) and pro-inflammatory (Hap-2). In addition, we identified the CTI haplotype as the main responsible for the regulation of IL4 transcriptional activity.

The growing list of drug-resistant microorganisms and the persistence of deaths due to diarrhea are compelling reasons to study plants in search of new therapeutic agents. The chemical constitution and popular use of the edible fruits of Campomanesia xanthocarpa O. Berg motivated this study to assess the antimicrobial and antidiarrheal properties of the fruits. An extract in 70% ethanol was prepared, and its antimicrobial activity was tested against several strains of bacteria by the agar diffusion and microdilution methods. Antidiarrheal activity was analyzed by testing intestinal motility in an animal model. Preliminary phytochemical study indicated the presence of flavonoids, saponins, and tannins in the hydroalcoholic extract. Antimicrobial activity was significant, but the minimum inhibitory concentration proved to be higher than the maximum extract concentration tested. The extract did not show significant activity for intestinal motility. Although this fruit extract did not show great results as an antimicrobial or antidiarrheal agent, the study contributes to the search for new plant agents and could be referred to as a research protocol by investigators in this area.