微生物学

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s Glycogen Synthase Kinase-3 (TbGSK-3s). TbGSK-3s represents a potential drug target as it is essential for parasite survival. Small molecules used in our study are indirubin analogues possessing substitutions in different positions in the bis-indole backbone. Presently, the standard laboratory practice for the kinase assays is the incorporation of radiolabeled phosphate from [gamma-³²P]ATP as the efforts for developing non-radioactive assays (ELISA-based assays, fluorescence quenching assays, etc.) exhibit limitations such as lack in sensitivity or limitations for broad applications. This protocol can be a useful starting point for lead discovery, as it surpasses the drawbacks of radioactive kinase assays and it allows for relatively sensitive measurements of kinase inhibition for TbGSK-3s.

Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols for the in vitro screening of a natural product-derived compound required for the determination of its antileishmanial potency. For this purpose, the Total Phenolic Fraction (TPF) derived from extra virgin olive oil is evaluated through the in vitro cell culture method against extracellular promastigote and intracellular amastigote Leishmania spp. forms. The aim of this article is to describe a step-by-step procedure that can be easily applied to accurately estimate the 50% inhibitory concentration (IC₅₀), the 50% cytotoxic concentration (CC₅₀) and the selectivity index (SI) via the resazurin reduction assay. These protocols are based on the ability of resazurin (oxidized blue form) to be irreversibly reduced by enzymes in viable cells and generate a red fluorescent resorufin product and can be easily expanded to the investigation of the antimicrobial activity in other microorganisms.