Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families,be ing dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.Fil: Gay, Claudia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Sanz, Libia. Instituto de Biomedicina de Valencia; EspañaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; Españ

Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae)

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal β-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumour-derived cell lines (LC50 = 0.8 ± 0. 2 µM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 µM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 µM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2-22 µM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 µM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 µM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes

Venom-on-a-chip: a fast and efficient method for comparative venomics

Venom research has attracted an increasing interest in disparate fields, from drug development and pharmacology, to evolutionary biology and ecology, and rational antivenom production. Advances in “-omics” technologies have allowed the characterization of an increasing number of animal venoms, but the methodology currently available is suboptimal for large-scale comparisons of venom profiles. Here, we describe a fast, reproducible and semi-automated protocol for investigating snake venom variability, especially at the intraspecific level, using the Agilent Bioanalyzer on-chip technology. Our protocol generated a phenotype matrix which can be used for robust statistical analysis and correlations of venom variation with ecological correlates, or other extrinsic factors. We also demonstrate the ease and utility of combining on-chip technology with previously fractionated venoms for detection of specific individual toxin proteins. Our study describes a novel strategy for rapid venom discrimination and analysis of compositional variation at multiple taxonomic levels, allowing researchers to tackle evolutionary questions and unveiling the drivers of the incredible biodiversity of venoms

Snake venomics and antivenomics of Middle and South America rattlesnakes.Identification of neurotoxin crotoxin as an adaptive trait during Crotalus Durissus invasion of South America

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Antivenomics of Atropoides mexicanus and Atropoides picadoi snake venoms: Relationship to the neutralization of toxic and enzymatic activities

Viperid snakes of the genus Atropoides are distributed in Mexico and Central America and, owing to their size and venom yield, are capable of provoking severe envenomings in humans. This study evaluated, using an ‘antivenomics’ approach, the ability of a polyspecific (polyvalent) antivenom manufactured in Costa Rica to recognize the proteins of Atropoides mexicanus and A. picadoi venoms, which are not included in the immunization mixture. In addition, the neutralization of lethal, hemorrhagic, myotoxic, coagulant, proteinase and phospholipase A2 (PLA2) activities of these venoms by the antivenom was assessed. The antivenom was highly-effective in immunodepleting many venom components, particularly high molecular mass P-III metalloproteinases (SVMPs), L-amino acid oxidases, and some serine proteinases and P-I SVMPs. In contrast, PLA2s, certain serine proteinases and P-I SVMPs, and a C type lectin-like protein were only partially immunodepleted, and two PLA2 molecules were not depleted at all. The antivenom was able to neutralize all toxic and enzymatic activities tested, although neutralization of lethality by A. nummifer venom was achieved when a challenge dose of 3 LD50s of venom was used, but was iffective when 4 LD50s were used. These results, and previously obtained evidence on the immunoreactivity of this antivenom towards homologous and heterologous venoms, revealed the low immunogenicity of a number of venom components (PLA2s, CRISPs, P-I SVMPs, and some serine proteinases), underscoring the need to search for innovative immunization protocols to improve the immune response to these antigens

DNA catabolites in triathletes: effects of supplementation with an aronia–citrus juice (polyphenols-rich juice)

In this study we analyzed whether our aronia–citrus juice (ACJ, the composition is based on a mixture of 95% citrus juice with 5% of Aronia melanocarpa juice), rich in polyphenols, and physical exercise had an effect on seven catabolites of DNA identified in plasma and on a urine isoprostane (8-iso-PGF2α). Sixteen elite triathletes on a controlled diet for triathlon training (45 days) were used in this clinical trial. Our results show a decrease in the 8-hydroxy-2′-deoxyguanosine concentration due to chronic physical exercise. The ACJ intake and physical exercise maintained the guanosine-3′,5′-cyclic monophosphate plasmatic concentrations and decreased the concentration of 8-hydroxyguanine as well as urinary values of 8-iso-PGF2α. Finally, we observed a significant increase in the 8-nitroguanosine levels in triathletes after ACJ intake, compared to the placebo stage. It is concluded that the combination of the intake of ACJ, rich in polyphenolic compounds, with adequate training was able to influence the plasmatic and urinary values of oxidative stress biomarkers. This suggests a positive effect on the oxidative damage and potential associations with DNA repair mechanisms.LAGF was awarded a pre-doctoral FPI fellowship (BES2012-060185) by the Spanish government. This study was supported by the project AGL2011-23690 (CICYT) (Spanish Ministry of Economy and Competitiveness). This work has been partially funded by the “Fundación Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AbstractAWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins

Omics Meets Biology: Application to the Design and Preclinical Assessment of Antivenoms

artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxinsUniversidad de Costa Rica. CSIC.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Lipidomic approach in young adult triathletes: effect of supplementation with a polyphenols-rich juice on neuroprostane and F2-dihomo-isoprostane markers

The aim of the this study was to determine the effect of a polyphenols-rich juice (aronia-citrus juice, ACJ) on F4-neuroprostanes and F2-dihomo-isoprostanes—markers of oxidative stress associated with the central nervous system (CNS)—in 16 elite triathletes under a controlled diet for triathlon training (145 days). In the triathletes, a decrease of the lipid peroxidation markers after ACJ intake, associated with neuronal membrane degradation (10-epi-10-F4t-neuroprostane and 10-F4t-neuroprostane), was observed when compared with placebo stage values. Regarding the F2-dihomo-isoprostanes, a significant decrease of the neuromotor system damage biomarkers (17-F2t-dihomo-isoprostane) with an increase of training load during the study was observed, although the decrease of the load training at the last stage showed a significant increase of the values of ent-7-(RS)-7-F2t-dihomo-IsoP, suggesting a possible role in adaptation post-training. On the other hand, the changes in the excretion of 17-epi-17-F2t-dihomo-IsoP provided a positive connection between physical exercise and ACJ intake. Thus, the results showed in this clinical study in young triathletes will help to elucidate novel interactions and mechanisms between the excretion of lipid peroxidation metabolites from CNS, supplementation of polyphenols-rich juice in the diet and physical exercise during a training season.This study was supported by the project AGL2011-23690 (CICYT) (Spanish Ministry of Economy and Competitiveness). This work has been partially funded by the “Fundación Séneca de la Región de Murcia” Grupo de Excelencia 19900/GERM/15. LAGF was granted a pre-doctoral FPI fellowship (BES2012-060185) by the Spanish government