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Harnessing phage display technology for discovery of human IgGs targeting clinically relevant toxins from the venom of the Central American coral snake (Micrurus nigrocinctus)
(2019-02-01) Bermúdez Méndez, Erick; Fernández Ulate, Julián; Lomonte Vigliotti, Bruno; Gutiérrez, José María; Laustsen, Andreas Hougaard
Envenomings caused by snakebites represent a serious public health problem in rural tropical areas of Africa, Asia, Latin America, and Oceania. Micrurus nigrocinctus, commonly known as Central American coral snake, is the most abundant and medically important species from the Elapidae family in Central America. The clinically most relevant toxins of M. nigrocinctus venom belong to the phospholipase A2 (PLA2) and three-finger toxin (3FTx) protein families. These toxins induce both myotoxic effects and potent neurotoxic effects that can lead to respiratory arrest due to neuromuscular paralysis. Currently, the only effective treatment available against bites from coral snakes consists of antivenom derived from serum of immunized horses. Although effective in neutralizing toxicity, this antivenom suffers from the drawbacks of having a heterologous nature and therefore being incompatible with the human immune system, difficult to manufacture due to the scarcity of M. nigrocinctus venom, and having a sub-optimally balanced response skewed towards venom components of high immunogenicity, but limited toxicity.
In this project, we harness phage display technology to discover human single-chain variable fragments (scFvs) against key venom toxins of M. nigrocinctus venom. Following identification of high affinity binders employing the IONTAS scFv library, candidate scFv binders will be converted to the IgG format to increase their half-life, providing prolonged systemic protection against the venom. We expect this study will help to pave the way for the development of novel, low-cost antivenoms with improved efficacy and safety against M. nigrocinctus envenomings.
Chapter 208 - Bothrops asper Hemorrhagic Proteinases
(2012) Gutiérrez, José María; Rucavado Romero, Alexandra; Ovadia, Michael; Neil D. Rawlings; Guy Salvesen
This chapter describes the biochemical, structural, and biological characteristics of the hemorrhagic metalloproteinases present in Bothrops asper venom, mainly BaH1, BaH4, and BaP1. These enzymes belong to the M12 family of zinc-dependent metalloproteinases and are responsible for the local and systemic hemorrhagic effects induced by the venom. BaH1 and BaH4 exhibit high hemorrhagic activity, whereas BaP1 shows a milder effect. These proteins hydrolyze several substrates, including type IV collagen, laminin, and fibronectin—key components of the capillary basement membrane. Their proteolytic action leads to blood vessel degradation, muscle necrosis, inflammation, and blister formation in the skin. Structural studies indicate that BaH1 and BaH4 are 64–69 kDa proteins (class III), while BaP1, at 24 kDa, belongs to class I. These enzymes are inhibited by chelating agents and natural antihemorrhagic proteins found in snake and mammalian sera. Research on these metalloproteinases has enhanced the understanding of the pathogenic mechanisms underlying venom-induced tissue damage and contributed to the development of therapeutic strategies complementary to traditional antivenom treatments.
Metalloproteinase inhibitors in snakebite envenomations
(1999-10-25) Gutiérrez, José María; Rucavado Romero, Alexandra; Ovadia, Michael
Pit viper envenomations are characterized by prominent local tissue damage, such as necrosis, hemorrhage and inflammation. These effects are relatively difficult to neutralize with antivenoms because of their rapid onset and development1. When treatment is delayed, as often occurs in tropical regions of the world, patients are at risk of developing permanent sequelae such as tissue loss or dysfunction. Horse- or sheep-derived antivenoms continue to be the mainstay in the treatment of snakebite envenomations, as they effectively neutralize systemically acting venom toxins, and partially decrease the extent of venom-induced local tissue damage. However, there is a need to develop ancillary treatments to inhibit locally acting toxins that could be used in addition to immunotherapy. Metalloproteinases are widely distributed in crotaline and viperine snake venoms2. They play a significant role in local tissue damage by inducing hemorrhage, oedema, myonecrosis, dermonecrosis and inflammation. Inhibitors of venom metalloproteinases, which could be injected directly at the site of venom injection, could offer a means of addressing this problem.
Interaction of snake venom metalloproteinases with type IV collagen: role of the different domains in target biding
(2020-07-09) Apú Leitón, Navilla; Rodrigues da Silva, Aldo; Kalogeropoulos, Konstantinos; Herrera Arias, Cristina; Camacho, Erika; Rucavado Romero, Alexandra; Gutiérrez, José María; Escalante Muñoz, Teresa
Snake venom metalloproteinases (SVMPs) disrupt capillary vessels leading to local and systemic hemorrhage. It has been proposed that type IV collagen degradation is one of the key steps in SVMPs-induced hemor- rhage, since hydrolysis of this protein results in a significant weakening of the mechanical stability of the capillary wall. However, the sequences in SVMPs (‘exosites’) which enable them to specifically interact with type IV collagen have not been identified, although it is hypothesized that they are located in the disintegrin-like and cysteine-rich domains characteristic of multi-domain SVMPs. In this study, the interactions of several SVMPs with type IV collagen were investigated in vitro by western blot, ELISA and af- finity chromatography. Hemorrhagic PI, PII and PIII SVMPs, in addition to the DC domain, were compared in the different assays in the presence or absence of a metalloproteinase inhibitor. In all the assays, the strongest binding was observed with CsH1, a PIII SVMP, whereas the PI SVMP BaP1 exhibited the lowest binding, and the PII SVMP BlatH1 presented a mod- erate binding. Interestingly, the binding of the isolated DC domain of CsH1 was lower than the native CsH1 suggesting a role of the metalloproteinase domain in the interaction with type IV collagen.
Mathematical modelling of the order-promising process for fruit supply chains considering the perishability and subtypes of products
(2017-05-08) Grillo Espinoza, Hanzel; Alemany Díaz, María del Mar Eva; Ortiz Bas, Ángel; Fuertes Miquel, Vicente Samuel
This paper proposes a mixed integer mathematical programming model to support the complex order promising process in fruit supply chains. Due to natural factors, such as land, weather or harvesting time, these supply chains present units of the same product that differ in certain relevant attributes to customers (subtypes). This becomes a managerial problem when customers require specific subtypes in their orders. Additionally, the deterioration of the original characteristics of subtypes over time generates waste and gives rise to a shelf life-based pricing policy. Therefore, the developed model should ensure that customers are served not only the quantities and dates, but also the required homogeneity and freshness. The model aims to maximise two conflicting objectives: total profit and mean product freshness. The novelty of the model derives from considering both homogeneity in subtypes as a requirement in customer orders and the traceability of product deterioration over time. Different scenarios are defined according to the weight assigned to each objective, shelf-life length and pricing policy in a rolling horizon scheme. The numerical experiments conducted for a real orange and tangerine supply chain, show the model's validity and the conflicting behaviour of the two objectives. The highest profit is made at the expense of the lowest mean freshness delivered, which is reinforced by the narrower the price variation with freshness. Finally, the positive impact of prolonging the product's shelf life on both objectives is shown.