Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Cultivo de Células Madre Mesenquimales Sobre Hidrogeles Compuestos de Nanopartículas de Hidroxiapatita y Quitosano Foto-Entrecruzable / Mesenchymal Stem Cell Culture on Composite Hydrogels of Hydroxyapatite Nanoparticles and Photo-Crosslinking Chitosan

RESUMEN El quitosano (QT) es un biopolímero que ha sido ampliamente utilizado en aplicaciones de ingeniería de tejido óseo, demostrando un gran potencial para este propósito. El presente estudio tiene como objetivo desarrollar un sistema de hidrogel entrecruzable in situ, compuesto de quitosano y nano-partículas de hidroxiapatita (HAP), un equivalente al componente mineral del hueso. El quitosano fue modificado, utilizando reacciones de carbodiimida, con ácido lactobiónico y ácido azidobenzoico para hacerlo soluble a pH fisiológico y foto-entrecruzable, respectivamente. El quitosano modificado fue mezclado en diferentes proporciones con HAP, y luego de una corta exposición a luz UV, se formaron hidrogeles. Células madre mesenquimales de médula ósea de rata (MSC) fueron sembradas sobre estos hidrogeles y cultivadas por 4, 10 y 16 días, bajo condiciones osteogénicas y no-osteogénicas. A través de ensayos de proliferación celular, actividad de fosfatasa alcalina, y microscopía confocal, se observó que la mayoría de las formulaciones soportan la adhesión y proliferación celular, mostrando importantes interacciones célula-biomaterial, y una diferenciación osteogénica temprana destacada en las formulaciones 70:30 y 50:50, QT:HAP. Algunas formulaciones del sistema foto-entrecruzable tienen potencial en aplicaciones de ingeniería de tejido óseo, y se propone estudios más detallados de diferenciación celular.

ABSTRACT Chitosan (QT) is a biopolymer that has been used in widely used in bone tissue engineering applications, demonstrating great potential for that purpose. Therefore, the present study aims to develop an in situ crosslinking hydrogel system, composed of chitosan and hydroxyapatite (HAP). Briefly, chitosan was modified, using carbodiimide chemistry, with lactobionic and azidobenzoic acid to make it soluble at physiological pH and photo-crosslinkable, respectively. The modified chitosan was mixed with HAP, in different proportions, and later exposed to UV light, yielding hydrogels. Mesenchymal stem cells, from rat bone marrow, were seeded onto the hydrogels and cultured for 4, 10 and 16 days, under osteogenic and non-osteogenic conditions. Through cell proliferation and alkaline phosphatase activity assays, and confocal microscopy, it was observed that the majority of formulations supported cell adhesion and proliferation, and a significant early osteogenic differentiation in formulations 70:30 and 50:50, QT:HAP. According to these results, the proposed photo-crosslinking system has potential for tissue engineering applications, and further specific studies are proposed for cell differentiation.

Ergosterone-coupled Triazol molecules trigger mitochondrial dysfunction, oxidative stress, and acidocalcisomal Ca2+ release in Leishmania mexicana promastigotes.

The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.