Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation.

The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Atividades práticas no ensino médio: uma abordagem experimental para aulas de genética / Practical activities in high school education: an experimental approach to genetics teaching

A experimentação no ensino se faz necessária para aproximar os alunos dos fenômenos ensinados nas aulas teóricas, no entanto, as atividades práticas são pouco utilizadas por professores de ciências e biologia. Além disso, os alunos apresentam dificuldades para compreender os conceitos e o fato de os professores utilizarem apenas aulas expositivas baseadas nos livros e apostilas, que trazem exemplos distantes do cotidiano, agrava o problema. Diante desses argumentos, o objetivo deste trabalho foi, por meio de um estudo exploratório, propor um cruzamento monohíbrido, utilizando como material a mosca Drosophila melanogaster como estratégia didática no complemento dos princípios mendelianos para alunos do 2º ano do ensino médio. A atividade foi desenvolvida em horário extra aula, com 10 alunos voluntários, teve duração de sete semanas, sendo uma aula por semana, e contemplou, além do cruzamento, leitura de textos complementares, exercícios de probabilidade, debate sobre a importância da ética no uso das aplicações da genética e fases do ciclo de vida do inseto. O desempenho dos alunos foi avaliado através de um questionário aplicado antes e após as atividades. Os principais resultados indicam que a atividade prática auxiliou na promoção da integração dos alunos e na evolução do entendimento sobre os conteúdos estudados.