Cytotoxicity of snake venom Lys49 PLA2-like myotoxin on rat cardiomyocytes ex vivo does not involve a direct action on the contractile apparatus.

Viperid snake venoms contain a unique family of cytotoxic proteins, the Lys49 PLA2 homologs, which are devoid of enzymatic activity but disrupt the integrity of cell membranes. They are known to induce skeletal muscle damage and are therefore named 'myotoxins'. Single intact and skinned (devoid of membranes and cytoplasm but with intact sarcomeric proteins) rat cardiomyocytes were used to analyze the cytotoxic action of a myotoxin, from the venom of Bothrops asper. The toxin induced rapid hypercontraction of intact cardiomyocytes, associated with an increase in the cytosolic concentration of calcium and with cell membrane disruption. Hypercontraction of intact cardiomyocytes was abrogated by the myosin inhibitor para-aminoblebbistatin (AmBleb). No toxin-induced changes of key parameters of force development were observed in skinned cardiomyocytes. Thus, although myosin is a key effector of the observed hypercontraction, a direct effect of the toxin on the sarcomeric proteins -including the actomyosin complex- is not part of the mechanism of cytotoxicity. Owing to the sensitivity of intact cardiomyocytes to the cytotoxic action of myotoxin, this ex vivo model is a valuable tool to explore in further detail the mechanism of action of this group of snake venom toxins.

Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis.

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

Acute kidney injury (AKI) following Eastern Russell's viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

Venom of the Annulated Sea Snake Hydrophis cyanocinctus: A Biochemically Simple but Genetically Complex Weapon.

Given that the venom system in sea snakes has a role in enhancing their secondary adaption to the marine environment, it follows that elucidating the diversity and function of venom toxins will help to understand the adaptive radiation of sea snakes. We performed proteomic and de novo NGS analyses to explore the diversity of venom toxins in the annulated sea snake (Hydrophis cyanocinctus) and estimated the adaptive molecular evolution of the toxin-coding unigenes and the toxicity of the major components. We found three-finger toxins (3-FTxs), phospholipase A2 (PLA2) and cysteine-rich secretory protein (CRISP) in the venom proteome and 59 toxin-coding unigenes belonging to 24 protein families in the venom-gland transcriptome; 3-FTx and PLA2 were the most abundant families. Nearly half of the toxin-coding unigenes had undergone positive selection. The short- (i.p. 0.09 µg/g) and long-chain neurotoxin (i.p. 0.14 µg/g) presented fairly high toxicity, whereas both basic and acidic PLA2s expressed low toxicity. The toxicity of H. cyanocinctus venom was largely determined by the 3-FTxs. Our data show the venom is used by H. cyanocinctus as a biochemically simple but genetically complex weapon and venom evolution in H. cyanocinctus is presumably driven by natural selection to deal with fast-moving prey and enemies in the marine environment.

BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

"Computational and Functional Characterization of a Hemorrhagic Metalloproteinase Purified from Cerastes cerastes Venom".

Structural and functional aspects of snake venoms metalloproteinases (SVMPs) have been extensively studied due to their role in envenomation. However, in the detection of certain coagulation disorders these biomolecules have been used and applied for the production of new thrombolytic drugs. CcMP-II, a SVMP-II metalloproteinase with a hemorrhagic activity, isolated from the venom of Cerastes cerastes, its sequence of 472 amino acids was identified. Predicted 3D structure showed an arrangement of CcMP-II into two distinct domains: i) a metalloproteinase domain where the zinc-binding motif is found (HXXGHNLGIDH) in addition to the sequence Cys-Ile-Met (CIM) at the Met-turn and ii) disintegrin-like domain containing RGD motif. CcMP-II inhibits platelet aggregation induced by collagen in a dose-dependent manner with an IC50 value estimated of 0.11 nM. This proteinase inhibits also aggregation of platelet stimulated by collagen even if the metal chelating agents (EDTA and 1, 10-phenontroline) are present suggesting that anti-aggregating effect is not due to its metalloproteinase domain, but to its disintegrin-like domain. Capillary pathological modifications caused by CcMP-II following intramuscular injection have as well been examined in mice. The key morphological alterations of the capillary vessels were clearly apparent from the ultrastructural study. The CcMP-II can play a key function in the pathogenesis of disorders that occurs following envenomation of Cerastes cerastes. The three-dimensional model of CcMP-II was built to explain structure-function relationships in ADAM/ADAMTs, a family of proteins having significant therapeutic potential. In order to explain structure-function relationships in ADAM / ADAMT, a family of proteins with considerable therapeutic potential, the three-dimensional model of CcMP-II was constructed.

The synthetic varespladib molecule is a multi-functional inhibitor for PLA2 and PLA2-like ophidic toxins.

BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.

Localization of Myotoxin I and Myotoxin II from the venom of Bothrops asper in a murine model.

Phospholipases A2 (PLA2s) and PLA2-like proteins are significant components of snake venoms. Some of these proteins act as potent toxins causing muscle necrosis, which may lead to amputation in severe envenomings. Fundamental aspects of the mechanism of action of these toxins are still not completely known. Myotoxin-I is a catalytically active Asp49 PLA2 from the venom of Bothrops asper, a medically relevant pit viper from Central America. Myotoxin-II is a catalytically inactive Lys49 PLA2-homolog also present in the venom of this snake. For the first time, the in vivo cellular localization of these myotoxins was studied in mouse skeletal muscle using immunofluorescence. Results showed that after 5 min of injection in the gastrocnemius muscle, both toxins initially interacted with the sarcolemma, and some colocalization with nuclei was already evident, especially for Mt-II. After 3 h of injection, a significant colocalization with the nuclei was observed for both toxins. These in vivo results confirm the importance of the initial interaction of these toxins with the sarcolemma and furthermore highlight the internalization and interaction of the toxins with nuclei during their pathophysiological activities, as observed in recent studies using cell culture.

Extensive Variation in the Activities of Pseudocerastes and Eristicophis Viper Venoms Suggests Divergent Envenoming Strategies Are Used for Prey Capture.

Snakes of the genera Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.

De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia-Next-Generation Sequencing, Functional Annotation and Toxinological Correlation.

Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study.

Cleavage of proteoglycans, plasma proteins and the platelet-derived growth factor receptor in the hemorrhagic process induced by snake venom metalloproteinases.

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

Cysteine-Rich Secretory Proteins (CRISPs) From Venomous Snakes: An Overview of the Functional Diversity in A Large and Underappreciated Superfamily.

The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Variations in neurotoxicity and proteome profile of Malayan krait (Bungarus candidus) venoms.

Malayan krait (Bungarus candidus) is a medically important snake species found in Southeast Asia. The neurotoxic effects of envenoming present as flaccid paralysis of skeletal muscles. It is unclear whether geographical variation in venom composition plays a significant role in the degree of clinical neurotoxicity. In this study, the effects of geographical variation on neurotoxicity and venom composition of B. candidus venoms from Indonesia, Malaysia and Thailand were examined. In the chick biventer cervicis nerve-muscle preparation, all venoms abolished indirect twitches and attenuated contractile responses to nicotinic receptor agonists, with venom from Indonesia displaying the most rapid neurotoxicity. A proteomic analysis indicated that three finger toxins (3FTx), phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitors were common toxin groups in the venoms. In addition, venom from Thailand contained L-amino acid oxidase (LAAO), cysteine rich secretory protein (CRISP), thrombin-like enzyme (TLE) and snake venom metalloproteinase (SVMP). Short-chain post-synaptic neurotoxins were not detected in any of the venoms. The largest quantity of long-chain post-synaptic neurotoxins and non-conventional toxins was found in the venom from Thailand. Analysis of PLA2 activity did not show any correlation between the amount of PLA2 and the degree of neurotoxicity of the venoms. Our study shows that variation in venom composition is not limited to the degree of neurotoxicity. This investigation provides additional insights into the geographical differences in venom composition and provides information that could be used to improve the management of Malayan krait envenoming in Southeast Asia.

Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells.

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P-I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 µg/mL) and Moojase (4 µg/mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RT-qPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies.

Complement System Inhibition Modulates the Pro-Inflammatory Effects of a Snake Venom Metalloproteinase.

Envenomation by Bothrops snakes causes prominent local effects, including pain, oedema, local bleeding, blistering and necrosis, and systemic manifestations, such as hemorrhage, hypotension, shock and acute renal failure. These snake venoms are able to activate the complement system and induce the generation of anaphylatoxins, whose mechanisms include the direct cleavage of complement components by snake venom metalloproteinases and serine proteinases present in the venoms. A metalloproteinase able to activate the three complement pathways and generate active anaphylatoxins, named C-SVMP, was purified from the venom of Bothrops pirajai. Considering the inflammatory nature of Bothrops venoms and the complement-activation property of C-SVMP, in the present work, we investigated the inflammatory effects of C-SVMP in a human whole blood model. The role of the complement system in the inflammatory process and its modulation by the use of compstatin were also investigated. C-SVMP was able to activate the complement system in the whole blood model, generating C3a/C3a desArg, C5a/C5a desArg and SC5b-9. This protein was able to promote an increase in the expression of CD11b, CD14, C3aR, C5aR1, TLR2, and TLR4 markers in leukocytes. Inhibition of component C3 by compstatin significantly reduced the production of anaphylatoxins and the Terminal Complement Complex (TCC) in blood plasma treated with the toxin, as well as the expression of CD11b, C3aR, and C5aR on leukocytes. C-SVMP was able to induce increased production of the cytokines IL-1ß and IL-6 and the chemokines CXCL8/IL-8, CCL2/MCP-1, and CXCL9/MIG in the human whole blood model. The addition of compstatin to the reactions caused a significant reduction in the production of IL-1ß, CXCL8/IL-8, and CCL2/MCP-1 in cells treated with C-SVMP. We therefore conclude that C-SVMP is able to activate the complement system, which leads to an increase in the inflammatory process. The data obtained with the use of compstatin indicate that complement inhibition may significantly control the inflammatory process initiated by Bothrops snake venom toxins.

First look into the venom of Roatan Island's critically endangered coral snake Micrurus ruatanus: Proteomic characterization, toxicity, immunorecognition and neutralization by an antivenom.

A proteomic and toxicological study of the venom from one specimen of Micrurus ruatanus, a critically endangered coral snake species endemic to Roatan Island, Honduras, was carried out. Immunorecognition and neutralization of venom lethality by an anticoral antivenom was also evaluated. Forty peaks were collected from RP-HPLC fractionation of the venom. After SDS-PAGE analysis, fifty-eight bands were examined by MALDI-TOF/TOF mass spectrometry. Micrurus ruatanus venom displayed a three-finger toxin (3FTx)-rich venom phenotype, as well as a significant amount of phospholipases A2 (PLA2s). Various other proteins were identified, including Kunitz-type inhibitor proteins, L-amino acid oxidases, C-type lectin/lectin-like, metalloproteinases, serine proteinases, vespryn/ohanin, 5'-nucleotidases, glutathione peroxidases, and phosphodiesterases. Micrurus ruatanus venom displayed significant PLA2 activity in vitro and myotoxicity in vivo. The venom showed high lethal potency in mice, being one of the most lethal in Central America. The anticoral antivenom (SAC-ICP) produced by Instituto Clodomiro Picado neutralized the lethal activity of the venom. Major fractions with relevant lethal activity were also identified by a screening analysis. SIGNIFICANCE: The proteomic characterization, toxicity, immunorecognition and neutralization of Micrurus ruatanus venom have been determined for the first time. This coral snake is endemic to Roatan Island and contains a three-finger toxin-rich venom that displayed a potent lethal activity in mice. The anticoral antivenom produced by Instituto Clodomiro Picado neutralized the lethal activity of this venom in vivo, and therefore should be effective in the treatment of envenomings by this snake.

Defining the pathogenic threat of envenoming by South African shield-nosed and coral snakes (genus Aspidelaps), and revealing the likely efficacy of available antivenom.

While envenoming by the southern African shield-nosed or coral snakes (genus Aspidelaps) has caused fatalities, bites are uncommon. Consequently, this venom is not used in the mixture of snake venoms used to immunise horses for the manufacture of regional SAIMR (South African Institute for Medical Research) polyvalent antivenom. Aspidelaps species are even excluded from the manufacturer's list of venomous snakes that can be treated by this highly effective product. This leaves clinicians, albeit rarely, in a therapeutic vacuum when treating envenoming by these snakes. This is a significantly understudied small group of nocturnal snakes and little is known about their venom compositions and toxicities. Using a murine preclinical model, this study determined that the paralysing toxicity of venoms from Aspidelaps scutatus intermedius, A. lubricus cowlesi and A. l. lubricus approached that of venoms from highly neurotoxic African cobras and mambas. This finding was consistent with the cross-genus dominance of venom three-finger toxins, including numerous isoforms which showed extensive interspecific variation. Our comprehensive analysis of venom proteomes showed that the three Aspidelaps species possess highly similar venom proteomic compositions. We also revealed that the SAIMR polyvalent antivenom cross-reacted extensively in vitro with venom proteins of the three Aspidelaps. Importantly, this cross-genus venom-IgG binding translated to preclinical (in a murine model) neutralisation of A. s. intermedius venom-induced lethality by the SAIMR polyvalent antivenom, at doses comparable with those that neutralise venom from the cape cobra (Naja nivea), which the antivenom is directed against. Our results suggest a wider than anticipated clinical utility of the SAIMR polyvalent antivenom, and here we seek to inform southern African clinicians that this readily available antivenom is likely to prove effective for victims of Aspidelaps envenoming. BIOLOGICAL SIGNIFICANCE: Coral and shield-nosed snakes (genus Aspidelaps) comprise two species and several subspecies of potentially medically important venomous snakes distributed in Namibia, Botswana, Zimbabwe, Mozambique and South Africa. Documented human fatalities, although rare, have occurred from both A. lubricus and A. scutatus. However, their venom proteomes and the pathological effects of envenomings by this understudied group of nocturnal snakes remain uncharacterised. Furthermore, no commercial antivenom is made using venom from species of the genus Aspidelaps. To fill this gap, we have conducted a transcriptomics-guided comparative proteomics analysis of the venoms of the intermediate shield-nose snake (A. s. intermedius), southern coral snake (A. l. lubricus), and Cowle's shield snake (A. l. cowlesi); investigated the mechanism of action underpinning lethality by A. s. intermedius in the murine model; and assessed the in vitro immunoreactivity of the SAIMR polyvalent antivenom towards the venom toxins of A. l. lubricus and A. l. cowlesi, and the in vivo capability of this antivenom at neutralising the lethal effect of A. s. intermedius venom. Our data revealed a high degree of conservation of the global composition of the three Aspidelaps venom proteomes, all characterised by the overwhelming predominance of neurotoxic 3FTxs, which induced classical signs of systemic neurotoxicity in mice. The SAIMR polyvalent antivenom extensively binds to Aspidelaps venom toxins and neutralised, with a potency of 0.235 mg venom/mL antivenom, the lethal effect of A. s. intermedius venom. Our data suggest that the SAIMR antivenom could be a useful therapeutic tool for treating human envenomings by Aspidelaps species.

Venom Proteome of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia: Toxicity Correlation, Immunoprofiling and Cross-Neutralization by Sea Snake Antivenom.

The venom proteome of Hydrophis curtus (synonym: Lapemis hardwickii) from Penang, Malaysia was investigated with nano-electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LCMS/MS) of the reverse-phase high-performance liquid chromatography (HPLC) venom fractions. Thirty distinct protein forms were identified as toxins from ten families. The three major protein families were phospholipase A2 (PLA2, 62.0% of total venom proteins), three-finger toxin (3FTX, 26.33%) and cysteine-rich secretory protein (CRiSP, 9.00%). PLA2 comprises diverse homologues (11 forms), predominantly the acidic subtypes (48.26%). 3FTX composed of one short alpha-neurotoxin (SNTX, 22.89%) and four long alpha-neurotoxins (LNTX, 3.44%). Both SNTX and LNTX were lethal in mice (intravenous LD50 = 0.10 and 0.24 µg/g, respectively) but the PLA2 were non-lethal (LD50 >1 µg/g). The more abundant and toxic SNTX appeared to be the main driver of venom lethality (holovenom LD50 = 0.20 µg/g). The heterologous Sea Snake Antivenom (SSAV, Australia) effectively cross-neutralized the venom (normalized potency = 9.35 mg venom neutralized per g antivenom) and the two neurotoxins in vivo, with the LNTX being neutralized more effectively (normalized potency = 3.5 mg toxin/g antivenom) than SNTX (normalized potency = 1.57 mg/g). SSAV immunorecognition was strong toward PLA2 but moderate-to-weak toward the alpha-neurotoxins, indicating that neutralization of the alpha-neurotoxins should be further improved.

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity.

Proteins constitute almost 95% of snake venom's dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5'nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Cell surface nucleolin interacts with and internalizes Bothrops asper Lys49 phospholipase A2 and mediates its toxic activity.

Phospholipases A2 are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA2-like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 °C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 °C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxin.