Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model.

Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.

Optimized method for the extraction of contaminant-free IgY antibodies from egg yolk using PEG 6000.

Hens are oviparous vertebrates and produce IgY antibodies, which is the main type of immunoglobulin in the egg yolk, and high concentrations can be obtained by using a simple method that does not require sophisticated equipment and reagents. The Polyethylene Glycol 6000 method allows the removal of lipids and the precipitation of IgY in two days with an approximated purity of around 80%, however during the original protocol other contaminant proteins can be precipitated. To overcome the issue of contamination with other proteins and extraction time, we optimized the previously method described by Pauly et al. (2011) by adding some changes that improved the aforementioned problems. • Our protocol is customized by the addition of one more filtration step or one more step with PEG 6000 at 3.5% to avoid the contamination with lipids. • Additionally, the changes in the type of agitation, centrifugation and the skip of dialysis make the method more accessible for all the laboratories. • In summary, these modifications serve to enhance the purity, reduce the time for IgY extraction from egg yolk and make it more accessible for every basic research laboratory.

Current Advances in the Development of Diagnostic Tests based on Aptamers in Parasitology: A Systematic Review.

Aptamers are single-stranded DNA or RNA sequences of 20-80 nucleotides that interact with different targets such as: proteins, ions, viruses, or toxins, through non-covalent interactions and their unique three-dimensional conformation. They are obtained in vitro by the systematic evolution of ligands by exponential enrichment (SELEX). Because of their ability of target recognition with high specificity and affinity, aptamers are usually compared to antibodies. However, they present many advantages that make them promising molecules for the development of new methods for the diagnosis and treatment of human diseases. In medical parasitology, aptamers also represent an attractive alternative for the implementation of new parasite detection methods, easy to apply in endemic regions. The aim of this study was to describe the current advances in the development of diagnostic tests based on aptamers in parasitology. For this, articles were selected following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, with specific inclusion and exclusion criteria. The 26 resulting articles deal with the use of aptamers for the detection of six important protozoa that affect human health. This systematic review clearly demonstrates the specificity, sensitivity and selectivity of aptamers and aptasensors, that certainly will soon become standard methods in medical parasitology.