Major Improvements in Robustness and Efficiency during the Screening of Novel Enzyme Effectors by the 3-Point Kinetics Assay

The throughput level currently reached by automatic liquid handling and assay monitoring techniques is expected to facilitate the discovery of new modulators of enzyme activity. Judicious and dependable ways to interpret vast amounts of information are, however, required to effectively answer this challenge. Here, the 3-point method of kinetic analysis is proposed as a means to significantly increase the hit success rates and decrease the number of falsely identified compounds (false positives). In this post-Michaelis–Menten approach, each screened reaction is probed in three different occasions, none of which necessarily coincide with the initial period of constant velocity. Enzymology principles rather than subjective criteria are applied to identify unwanted outliers such as assay artifacts, and then to accurately distinguish true enzyme modulation effects from false positives. The exclusion and selection criteria are defined based on the 3-point reaction coordinates, whose relative positions along the time-courses may change from well to well or from plate to plate, if necessary. The robustness and efficiency of the new method is illustrated during a small drug repurposing screening of potential modulators of the deubiquinating activity of ataxin-3, a protein implicated in Machado–Joseph disease. Apparently, intractable Z factors are drastically enhanced after (1) eliminating spurious results, (2) improving the normalization method, and (3) increasing the assay resilience to systematic and random variability. Numerical simulations further demonstrate that the 3-point analysis is highly sensitive to specific, catalytic, and slow-onset modulation effects that are particularly difficult to detect by typical endpoint assays.

Epitope Selection for Fighting Visceral Leishmaniosis: Not All Peptides Function the Same Way

Visceral leishmaniosis (VL) caused by Leishmania infantum is a disease with an increasing prevalence worldwide. Treatments are expensive, toxic, and ineffective. Therefore, vaccination seems to be a promising approach to control VL. Peptide-based vaccination is a useful method due to its stability, absence of local side effects, and ease of scaling up. In this context, bioinformatics seems to facilitate the use of peptides, as this analysis can predict high binding affinity epitopes to MHC class I and II molecules of different species. We have recently reported the use of HisAK70 DNA immunization in mice to induce a resistant phenotype against L. major, L. infantum, and L. amazonensis infections. In the present study, we used bioinformatics tools to select promising multiepitope peptides (HisDTC and AK) from the polyprotein encoded in the HisAK70 DNA to evaluate their immunogenicity in the murine model of VL by L. infantum. Our results revealed that both multiepitope peptides were able to induce the control of VL in mice. Furthermore, HisDTC was able to induce a better cell-mediated immune response in terms of reduced parasite burden, protective cytokine profile, leishmanicidal enzyme modulation, and specific IgG2a isotype production in immunized mice, before and after infectious challenge. Overall, this study indicates that the HisDTC chimera may be considered a satisfactory tool to control VL because it is able to activate a potent CD4+ and CD8+ T-cell protective immune responses.

ENZIMAS MICROBIANA: POTENCIAL BIOTECNOLÓGICO E EFEITOS MODULATÓRIOS EM RUMINANTES

The objective of this review was to survey the main microbial species producing enzymes involved in the degradation of compounds such as cellulose, hemicellulose and lignin. Most of the microorganisms isolated from soil, waste and compost material are capable of producing a broad spectrum of degrading enzymes from the plant cell wall. This enzymatic activity is essential for the fermentation of carbohydrates in the ruminal environment, since ruminants are not able to degrade these polymers, thus establishing a symbiosis relationship between these animals and the autochthonous microbiota of the gastrointestinal tract. However, microbial enzymes have not only been receiving attention in animal nutrition, more and more these have been explored by the industry as biotechnological possibilities, such as the production of ethanol. It is observed a microbial diversity in the rumen, and variations in the production of enzymes. Further studies are needed to elucidate the mechanisms of enzyme modulation in the rumen of production animals, in order to obtain an increase in zootechnical indexes and a higher digestibility of the fiber