Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

Unveiling the Venom Composition of the Colombian Coral Snakes Micrurus helleri, M. medemi, and M. sangilensis.

Little is known of the biochemical composition and functional features of the venoms of poorly known Colombian coral snakes. Here, we provide a preliminary characterization of the venom of two Colombian endemic coral snake species, Micrurus medemi and M. sangilensis, as well as Colombian populations of M. helleri. Electrophoresis and RP-HPLC techniques were used to identify venom components, and assays were conducted to detect enzyme activities, including phospholipase A2, hyaluronidase, and protease activities. The median lethal dose was determined using murine models. Cytotoxic activities in primary cultures from hippocampal neurons and cancer cell lines were evaluated. The venom profiles revealed similarities in electrophoretic separation among proteins under 20 kDa. The differences in chromatographic profiles were significant, mainly between the fractions containing medium-/large-sized and hydrophobic proteins; this was corroborated by a proteomic analysis which showed the expected composition of neurotoxins from the PLA2 (~38%) and 3FTx (~17%) families; however, a considerable quantity of metalloproteinases (~12%) was detected. PLA2 activity and protease activity were higher in M. helleri venom according to qualitative and quantitative assays. M. medemi venom had the highest lethality. All venoms decreased cell viability when tested on tumoral cell cultures, and M. helleri venom had the highest activity in neuronal primary culture. These preliminary studies shed light on the venoms of understudied coral snakes and broaden the range of sources that could be used for subsequent investigations of components with applications to specific diseases. Our findings also have implications for the clinical manifestations of snake envenoming and improvements in its medical management.

Phenolic Compounds from Wild Plant and In Vitro Cultures of Ageratina pichichensis and Evaluation of Their Antioxidant Activity.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Chemical characterization of yellow-orange and purple varieties of Opuntia ficus-indica fruits and thermal stability of their betalains.

Betalains are plant pigments with biological properties and can be used instead of synthetic colorants to confer color and functional properties to foods. The objective of this work was to carry out the chemical characterization of two varieties of prickly pear of Opuntia ficus-indica, one of yellow-orange coloration (Mandarina) and the other of purple coloration (Vigor), through measurements of chemical parameters and color in pulp, antioxidant activity, total phenolic compounds, and betalain content. Considering the thermolability of betalains and their potential applications in food, the thermal stability and activation energy of betacyanins from Vigor variety and betaxanthins from the Mandarina variety were also evaluated and compared with those from beetroot, the main source of betalains. Results for chemical characterization agreed with previous prickly pear reports of other regions, while the thermal degradation kinetics of betalains showed a first-order degradation pattern with respect to time and temperature treatment. Betacyanins from Vigor prickly pear showed similar thermal stability to those from beetroot, which was reflected in similar values of activation energy, while betaxanthins from Mandarina prickly pear showed a higher stability, and therefore a higher activation energy, than those from beetroot. Based on the results, the prickly pear varieties used in this study can be considered as a good source of betalains with potential applications in food and, in addition, the methodology for the evaluation of thermostability can be used to compare the stability of betalains from different sources in a temperature range of 50-90°C. PRACTICAL APPLICATION: The varieties of prickly pear used in this study can be considered a good source of red-purple and yellow-orange easily extractable pigments. In addition, we report a methodology that can be used for the evaluation of the thermal stability of these pigments and to compare this stability between different plant sources. Gaining knowledge on betalain thermal stability will make it possible to propose specific applications, for example, in processed foods requiring different pigment stabilities.

Giardia lamblia G6PD::6PGL Fused Protein Inhibitors Decrease Trophozoite Viability: A New Alternative against Giardiasis.

Treatments to combat giardiasis have been reported to have several drawbacks, partly due to the drug resistance and toxicity of current antiparasitic agents. These constraints have prompted many researchers to investigate new drugs that act against protozoan parasites. Enzyme inhibition is an important means of regulating pathogen metabolism and has recently been identified as a significant alternative target in the search for new treatments. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (G6PD::6PGL) is a bifunctional enzyme involved in the pentose phosphate pathway (PPP) in Giardia lamblia (G. lamblia). The G. lamblia enzyme is unusual since, unlike the human enzyme, it is a fused enzyme. Here, we show, through inhibition assays, that an in-house chemical library of 120 compounds and four target compounds, named CNZ-7, CNZ-8, CMC-1, and FLP-2, are potent inhibitors of the G. lamblia G6PD::6PGL fused enzyme. With a constant (k2) of 2.3, 3.2, and 2.8 M−1 s−1, respectively, they provoke alterations in the secondary and tertiary protein structure and global stability. As a novel approach, target compounds show antigiardial activity, with IC50 values of 8.7, 15.2, 15.3, and 24.1 µM in trophozoites from G. lamblia. Moreover, these compounds show selectivity against G. lamblia, since, through counter-screening in Caco-2 and HT29 human cells, they were found to have low toxicity. This finding positions these compounds as a potential and attractive starting point for new antigiardial drugs.

Chemical structure of three basic Asp-49 phospholipases A2 isolated from Crotalus molossus nigrescens venom with cytotoxic activity against cancer cells.

Snake venoms are complex mixtures of molecules with several biological activities. Among these molecules, the enzymes with phospholipase A2 activity have been extensively studied in the venoms from snakes because of their importance in the envenomation process and symptoms. The Mexican rattlesnake Crotalus molossus nigrescens is widely distributed in the Mexican plateau. Unlike other crotalids, its venom components have been poorly studied. Here, we characterized the phospholipase activity of one fraction isolated from the venom of this snake and we determined the cytotoxic and neurotoxic effects on brain tumor cells and neuronal primary cultures, respectively. After reverse phase chromatography, we obtained a fraction which was analyzed by mass spectrometry showing higher activity than that from a PLA2 from bee venom used as control. This fraction was enriched with three basic Asp49 phospholipases with molecular masses of 12.5, 13.9 and 14.2 kDa. Their complete amino acid sequences were determined, and their predicted tertiary structures were generated using the model building softwares I-tasser and Chimera. Viability assays revealed that the fraction showed cytotoxic activity against brain tumor cells (C6, RG2 and Daoy) with IC50 values ranging between 10 and 100 ng/ml, whereas an IC50 > 100 ng/ml was exerted in rat primary astrocytes. These findings might be relevant in oncological medicine due to their potential as anticancer agents and low neurotoxic effects compared to conventional drugs.