The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples

Intervenção em saúde aos portadores de hipertensão arterial e diabetes mellitus pela ESF São Pedro 1 em Governador Valadares - MG

A cidade de Governador Valadares possui 41 equipes de Estratégia Saúde da Família (ESF), dentre elas a ESF São Pedro 1, com alta prevalência de hipertensos e diabéticos na área de abrangência, necessitando de acompanhamento multiprofissional contínuo. A Hipertensão Arterial Sistêmica (HAS) e o Diabetes Mellitus (DM) são duas condições de saúde de alta morbimortalidade e muito prevalentes em saúde pública, especialmente na atenção primária à saúde (APS), sendo desafio constante para os profissionais que trabalham nas unidades básicas de saúde. A prevenção, o diagnóstico, o controle e a reabilitação das condições crônicas fazem parte das funções da APS. Pelo caráter multifatorial no acompanhamento da HAS e DM deve-se buscar um trabalho multidisciplinar e multiprofissional para o bom controle individual e coletivo destas doenças. Este estudo pretende demonstrar a importância da atuação de uma equipe multiprofissional dentro de uma unidade de APS no controle da HAS e DM através da criação de uma agenda específica e grupo operativo para o atendimento destes pacientes

Naloxonazine, an amastigote-specific compound, affects Leishmania parasites through modulation of host-encoded functions

Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy