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Snake venoms contain various molecules known for activating innate immunity and causing local effects associated with increased vascular permeability, such as vascular leakage and edema, common symptoms seen in snakebite envenomings. We have demonstrated that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability. This study aimed to explore the functional role of CRiSP isolated from Mojave rattlesnake ( Crotalus scutulatus scutulatus ) venom (Css-CRiSP) on the activation of inflammatory responses in different models. We measured the release of inflammatory mediators in cultured human dermal blood endothelial cells (HDBEC), lymphatic endothelial cells (HDLEC) and monocyte-derived macrophages (MDM) at 0.5, 1, 3, 6, and 24 h after treatment with Css-CRiSP (1 μM). We also determined the acute inflammatory response in BALB/c mice 30 min after intraperitoneal injection of the toxin (2 μg/mouse). Css-CRiSP induced the production of IL-8 and IL-6, but not TNF-α, in HDBEC and HDLEC in a time-dependent manner. In addition, Css-CRiSP significantly enhanced the production of IL-6, TNF-α, IL-8, and IL-1β in MDM. Moreover, it caused a remarkable increase of chemotactic mediators in the exudates of experimental mice. Our results reveal that Css-CRiSPs can promote a sustained release of inflammatory mediators on cell lines and an acute activation of innate immunity in a murine model. These findings contribute to the growing body of evidence supporting the involvement of svCRiSPs in the augmentation of envenomation effects, specifically, the role of svCRiSPs in inducing vascular dysfunction, initiating early inflammatory responses, and facilitating the activation of leukocytes and releasing mediators. These findings will lead to a better understanding of the pathophysiology of envenoming by Mojave rattlesnakes, allowing the development of more efficient therapeutic strategies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

The peripheral nervous system harbors a remarkable potential to regenerate after acute nerve trauma. Full functional recovery, however, is rare and critically depends on peripheral nerve Schwann cells that orchestrate breakdown and resynthesis of myelin and, at the same time, support axonal regrowth. How Schwann cells meet the high metabolic demand required for nerve repair remains poorly understood. We here report that nerve injury induces adipocyte to glial signaling and identify the adipokine leptin as an upstream regulator of glial metabolic adaptation in regeneration. Signal integration by leptin receptors in Schwann cells ensures efficient peripheral nerve repair by adjusting injury-specific catabolic processes in regenerating nerves, including myelin autophagy and mitochondrial respiration. Our findings propose a model according to which acute nerve injury triggers a therapeutically targetable intercellular crosstalk that modulates glial metabolism to provide sufficient energy for successful nerve repair., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Beta-cardiotoxin (β-CTX) from the king cobra venom (Ophiophagus hannah) was previously proposed as a novel β-adrenergic blocker. However, the involvement of β-adrenergic signaling by this compound has never been elucidated. The objectives of this study were to investigate the underlying mechanisms of β-CTX as a β-blocker and its association with the β-adrenergic pathway. The effects of β-CTX on isolated cardiac myocyte functions, calcium homeostasis, the phosphorylation level of targeted proteins, and the myofibrillar ATPase activity were studied. Healthy Sprague Dawley rats were used for cardiomyocytes isolation. Like propranolol, β-CTX attenuated the cardiomyocyte inotropy and calcium transient alterations as induced by isoproterenol stimulation. In contrast, these effects were not observed in forskolin-treated cells. Interestingly, cardiomyocytes treated with β-CTX showed no changes in phosphorylation level at any PKA-targeted sites in the myofilaments as demonstrated in Western blot analysis. The skinned fibers study revealed no change in myofilament kinetics by β-CTX. However, this protein exhibited the direct inhibition of myofibrillar ATPase activity with calcium de-sensitization of the enzyme. In summary, the negative inotropic mechanism of β-CTX was discovered. β-CTX exhibits an atypical β-blocker mechanism. These properties of β-CTX may benefit in developing a novel agent aid to treat hypertrophic cardiomyopathy., (© 2021. The Author(s).)

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

What factors influence the evolution of a heavily selected functional trait in a diverse clade? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity in the wider context of phylogenetics, natural history, and biology. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the rattlesnake phylogenetic tree to reconstruct the evolution of coagulotoxicity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships in rattlesnakes, suggesting the importance of diet and/or other environmental variables in driving venom evolution. Moreover, the striking inter- and intraspecific variability in venom activity on human blood highlights the considerable variability faced by physicians treating envenomation. This study is the most comprehensive effort to date to describe and characterize the evolutionary and biological aspects of coagulotoxins in rattlesnake venom. Further research at finer taxonomic levels is recommended to elucidate patterns of variation within species and lineages., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca 2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC 50  = 11.44 μM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Maternal unbalanced nutritional habits during embryonic development and perinatal stages perturb hypothalamic neuronal programming of the offspring, thus increasing obesity-associated diabetes risk. However, the underlying molecular mechanisms remain largely unknown. In this study we sought to determine the translatomic signatures associated with pro-opiomelanocortin (POMC) neuron malprogramming in maternal obesogenic conditions., Methods: We used the RiboTag mouse model to specifically profile the translatome of POMC neurons during neonatal (P0) and perinatal (P21) life and its neuroanatomical, functional, and physiological consequences., Results: Maternal high-fat diet (HFD) exposure did not interfere with offspring's hypothalamic POMC neuron specification, but significantly impaired their spatial distribution and axonal extension to target areas. Importantly, we established POMC neuron-specific translatome signatures accounting for aberrant neuronal development and axonal growth. These anatomical and molecular alterations caused metabolic dysfunction in early life and adulthood., Conclusions: Our study provides fundamental insights on the molecular mechanisms underlying POMC neuron malprogramming in obesogenic contexts., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

An acute inflammatory response, cellular infiltrates, anemia, hemorrhage and endogenous fibrinolysis activation were previously described in C57BL/6 mice injected with M. tener tener venom (Mtt). As the endothelium and innate immunity may participate in these disturbances and due to our poor understanding of the alterations produced by these venoms when the neurotoxic component is not predominant, we evaluated the effects in an in vitro model. At 24 h, the release of pro-inflammatory mediators was detected in peritoneal macrophages. At different times, the release of pro-inflammatory (TNF-α, IL-6, NO and E-Selectin), pro-coagulant (vWF and TF) and pro-fibrinolytic (uPA) mediators were seen in liver sinusoidal endothelial cells (LSECs). These results suggest that Mtt venom activates macrophages and endothelium, thus inducing the release of mediators, such as TNF-α, that orchestrate the acute inflammatory response and the later infiltration of mononuclear cells into liver in C57BL/6 mice. In addition, endothelium activation promotes TF expression, which may in turn modulate the inflammatory and hemostatic response. These findings suggest crosstalk between inflammation and hemostasis in the alterations observed in Micrurus envenomation, where the neurotoxic manifestations do not predominate., (Copyright © 2019. Published by Elsevier Ltd.)

To enhance our ability to monitor poliovirus circulation and certify eradication, we evaluated the performance of the bag-mediated filtration system (BMFS) against the two-phase separation (TPS) method for concentrating wastewater samples for poliovirus detection. Sequential samples were collected at two sites in Mexico; one L was collected by grab and ~ 5 L were collected and filtered in situ with the BMFS. In the laboratory, 500 mL collected by grab were concentrated using TPS and the sample contained in the filter of the BMFS was eluted without secondary concentration. Concentrates were tested for the presence of poliovirus and non-poliovirus enterovirus (NPEV) using Global Poliovirus Laboratory Network standard procedures. Between February 16, 2016, and April 18, 2017, 125 pairs of samples were obtained. Collectors spent an average (± standard deviation) of 4.3 ± 2.2 min collecting the TPS sample versus 73.5 ± 30.5 min collecting and filtering the BMFS sample. Laboratory processing required an estimated 5 h for concentration by TPS and 3.5 h for elution. Sabin 1 poliovirus was detected in 37 [30%] samples with the TPS versus 24 [19%] samples with the BMFS (McNemar's mid p value = 0.004). Sabin 3 poliovirus was detected in 59 [47%] versus 49 (39%) samples (p = 0.043), and NPEV was detected in 67 [54%] versus 40 [32%] samples (p < 0.001). The BMFS method without secondary concentration did not perform as well as the TPS method for detecting Sabin poliovirus and NPEV. Further studies are needed to guide the selection of cost-effective environmental surveillance methods for the polio endgame.

The expense of production and distribution of snakebite antivenom, as well as its relatively infrequent use, has caused antivenom to be increasingly difficult to obtain and ultimately producing an alarming global shortage. Unused, expired antivenom may represent a significant, untapped resource to ameliorate this crisis. This study examines the efficacy of expired antivenom over time using in vitro, whole blood clotting, and platelet function statistics. Representatives from three years for four different global brands of polyvalent antivenom were chosen and tested against their corresponding venoms as well as other venoms that could display cross-reactivity. These antivenoms include Wyeth Polyvalent (U.S.; exp. 1997, 2001, 2003), Antivipmyn ® (Mexico; exp. 2005, 2013, 2017), Biotecfars Polyvalent (Venezuela; exp. 2010, 2014, 2016), and SAIMR (South Africa; exp. 1997, 2005, 2017). Venoms of species tested were Crotalus atrox against Wyeth; C. atrox and Crotalus vegrandis against Antivipmyn ® ; C. atrox, C. vegrandis and Bothrops colombiensis against Biotecfar; and Bitis gabonica and Echis carinatus against South African Institute for Medical Research (SAIMR). Parameters recorded were activated clotting time (ACT), clotting rate (CR), and platelet function (PF). Preliminary results are encouraging as the antivenoms maintained significant efficacy even 20 y after their expiration date. We anticipate these results will motivate further studies and provide hope in the cases of snakebite emergencies when preferable treatments are unavailable., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Envenomation by the Venezuelan bushmaster snake ( Lachesis muta muta ) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys , which is a Ca 2+ -dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 μg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.

A novel snake venom cysteine-rich secretory protein (svCRiSP), Hellerin, was purified from C. o. helleri venom using sequential reverse phase and cation-exchange chromatography. Gel electrophoresis, N-terminal sequencing, and LC-MS/MS sequencing identified a single protein with a molecular mass of approximately 24.8 kDa and confirmed its identity as a svCRiSP. Hellerin had cytotoxic effects on human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but not in human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs). Hellerin produced a dramatic increase in both blood vascular permeability in vivo, and in the trans-epithelial permeability of cultured HDLEC and HDBEC cells. This is the first study that describes the effect of a svCRiSP on vascular, blood and lymphatic permeability., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

It has been recently demonstrated that the hemotoxic venom activity of several species of snakes can be inhibited by carbon monoxide (CO) or a metheme forming agent. These and other data suggest that the biometal, heme, may be attached to venom enzymes and may be modulated by CO. A novel fibrinogenolytic metalloproteinase, named CatroxMP-II, was isolated and purified from the venom of a Crotalus atrox viper, and subjected to proteolysis and mass spectroscopy. An ion similar to the predicted singly charged m/z of heme at 617.18 was identified. Lastly, CORM-2 (tricarbonyldichlororuthenium (II) dimer, a CO releasing molecule) inhibited the fibrinogenolytic effects of CatroxMP-II on coagulation kinetics in human plasma. In conclusion, we present the first example of a snake venom metalloproteinase that is heme-bound and CO-inhibited.

Bothrops venezuelensis snake venoms, from five localities in the North-Central Venezuelan regions, showed biochemical and haemostatic differences. In this study, bioactivities of B. venezuelensis venoms from different regions (Aragua state; Waraira Repano (Capital District); Baruta, La Boyera and Lagunetica (Miranda state)) were compared using both natural and synthetic substrates. The protein contents of these venoms were Lagunetica 89%, La Boyera 79%, Baruta 71%, Waraira Repano 68% and Aragua 64%. Toxic activities effects were: Intraperitoneal LD 50 s: Aragua-14 mg/kg; Waraira Repano-6.4 mg/kg; Baruta: 8.3 mg/kg; La Boyera-4.4 mg/kg; Lagunetica-16.2 mg/kg. The MHD results: Aragua-21.4 μg/mouse; Waraira Repano-2.5 μg/mouse; Baruta-1.2 μg/mouse; La Boyera-1.4 μg/mouse and Lagunetica-12 μg/mouse. The hide powder azure results: Aragua-1.24 U/mg; La Boyera-2.26 U/mg; Baruta-2.83 U/mg; Lagunetica-3.28 U/mg and Waraira Repano-5.77 U/mg. Esterase specific activity on BAEE results: Waraira Repano-666.66 U/mg; La Boyera-805.5 U/mg; Baruta-900.00 U/mg; Lagunetica-922.19 U/mg and Aragua-1960.67 U/mg. Casein zymography showed digestion bands in the molecular weight above 100 and at 66.2 and 21.5 kDa. Analysis of casein degradation by SDS-PAGE showed two different degradation patterns. Fibrinolytic activity (mm 2 /μg) on fibrin plates results: Aragua-6.07; Lagunetica-27.6; Waraira Repano-35.7; La Boyera-44.27 and Baruta-45.63. In the fibrinogenolytic assay, the five venoms completely degraded the α chain after 1 min of incubation. None of the venoms completely degraded the β and γ chains after 24 h incubation. The research indicated that venoms of B. venezuelensis of different geographic areas in Venezuela exhibit variances in composition and component concentrations; except the Aragua venom, all of them had high proteolytic activities., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Crotamine is a cationic, non-enzymatic, protein integrating a minor family of myotoxins, composed of 42 amino acid residues, described in Viperidae and Crotalidae snake's families that has been used in neuroscience research, drug progressing and molecular diversity reports. Crotamine-like protein (CLP) from C.o.helleri venom was isolated in fraction 5 from 7 peaks obtained by sulfopropyl waters protein pak cationic exchange column. In tricine-SDS-PAGE under non-reduced conditions this CLP showed a single band of ~8 kDa molecular weight. CLP induced toxicity of K-562 cells with a CC 50 of 11.09 µM. In mice adrenal gland after 24 h of CLP injection, cortical cells exhibited swollen mitochondria with scarce tubular cristae, some elements of smooth and rough endoplasmic reticula, widened nuclear envelope, slightly osmiophilic lipid droplets, and autophagic vacuoles. In some areas cortical cells plasma membrane and endothelial walls disappeared, which indicated a necrosis process. In other areas, endothelial cell cytoplasm did not present the normal caveolae and pinocytotic vesicles. To our knowledge, this is the first report on mice adrenal gland damages, caused by the injection of CLP from rattlesnakes. Our results propose that adrenal cortex lesions may be significant in the envenoming etiopathogenesis by CLP.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Several in vitro investigations have demonstrated that anticoagulant effects of fibrinogenolytic snake venom metalloproteinases have been abrogated in human plasma by modifying fibrinogen with iron (Fe) and carbon monoxide (CO) to prevent catalysis or by directly inhibiting these enzymes with CO. To translate these findings, we chose to assess the rabbit as a model of envenomation with Crotalus atrox venom. It was determined with thrombelastography that 15 times the concentration of venom noted to compromise coagulation in plasma in vitro was required to cause coagulopathy in vivo, likely secondary to venom binding to blood cells and being cleared from the circulation rapidly. Unlike human plasma, rabbit plasma pre-treated with Fe/CO was not protected from fibrinogenolysis by venom. Consequently, the administration of purified human fibrinogen (with or without Fe/CO) would be required before venom administration to rabbits. Of greater interest, venom exposed to CO had complete loss of fibrinogenolytic effect in rabbit plasma and partial loss of activity in whole blood, indicative of unbinding of CO from venom and binding to haemoglobin. Thus, venom exposed to CO could remain partially or completely inhibited in whole blood long enough for clearance from the circulation, allowing rabbits to be a useful model to test the efficacy of regional CO administration to the bite site. Future investigations are planned to test these novel approaches to attenuate venom-mediated coagulopathy in the rabbit., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper ( Bothrops moojeni ). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X 2 and/or P2X 3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Snake venoms are known to have different venom compositions and toxicity, but differences can also be found within populations of the same species contributing to the complexity of treatment of envenomated victims. One of the first well-documented intraspecies venom variations comes from the Mohave rattlesnake (Crotalus scutulatus scutulatus). Initially, three types of venoms were described; type A venom is the most toxic as a result of ~45% Mojave toxin in the venom composition, type B lacks the Mojave toxin but contains over 50% of snake venom metalloproteases (SVMPs). Also, type A+B venom contains a combination of Mojave toxin and SVMP. The use of an anti-disintegrin antibody in a simple Enzyme-Linked Immunosorbent Assay (ELISA) can be used to identify the difference between the venoms of the type A, B, and A+B Mohave rattlesnakes. This study implements the use of an anti-recombinant disintegrin polyclonal antibody (ARDPA) for the detection of disintegrins and ADAMs (a disintegrin and metalloproteases) in individual crude snake venoms of Mohave rattlesnakes (Crotalus scutulatus scutulatus) of varying geographical locations. After correlation with Western blots, coagulation activity and LD 50 data, it was determined that the antibody allows for a quick and cost-efficient identification of venom types., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2016. Published by Elsevier Ltd.)

Phenotypes frequently vary across and within species. The connection between specific phenotypic effects and function, however, is less understood despite being essential to our understanding of the adaptive process. Snake venoms are ideal for identifying functionally important phenotypic variation because venom variation is common, and venoms can be functionally characterized through simple assays and toxicity measurements. Previous work with the eastern diamondback rattlesnake (Crotalus adamanteus) used multivariate statistical approaches to identify six unique venom phenotypes. We functionally characterized hemolytic, gelatinase, fibrinogenolytic, and coagulant activity for all six phenotypes, as well as one additional venom, to determine if the statistically significant differences in toxin expression levels previously documented corresponded to differences in venom activity. In general, statistical differences in toxin expression predicted the identified functional differences, or lack thereof, in toxic activity, demonstrating that the statistical approach used to characterize C. adamanteus venoms was a fair representation of biologically meaningful differences. Minor differences in activity not accounted for by the statistical model may be the result of amino-acid differences and/or post-translational modifications, but overall we were able to link variation in protein expression levels to variation in function as predicted by multivariate statistical approaches., Competing Interests: statement The authors declare that they have no conflicts of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Background: Bothrops colombiensis is a highly dangerous pit viper and responsible for over 70% of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis, the venom gland transcriptome is currently lacking., Results: We constructed a cDNA library from the venom gland of B. colombiensis, and a set of 729 high quality expressed sequence tags (ESTs) was identified. A total number of 344 ESTs (47.2% of total ESTs) was related to toxins. The most abundant toxin transcripts were metalloproteinases (37.5%), phospholipases A2s (PLA2, 29.7%), and serine proteinases (11.9%). Minor toxin transcripts were linked to waprins (5.5%), C-type lectins (4.1%), ATPases (2.9%), cysteine-rich secretory proteins (CRISP, 2.3%), snake venom vascular endothelium growth factors (svVEGF, 2.3%), L-amino acid oxidases (2%), and other putative toxins (1.7%). While 160 ESTs (22% of total ESTs) coded for translation proteins, regulatory proteins, ribosomal proteins, elongation factors, release factors, metabolic proteins, and immune response proteins. Other proteins detected in the transcriptome (87 ESTs, 11.9% of total ESTs) were undescribed proteins with unknown functions. The remaining 138 (18.9%) cDNAs had no match with known GenBank accessions., Conclusion: This study represents the analysis of transcript expressions and provides a physical resource of unique genes for further study of gene function and the development of novel molecules for medical applications.

In this study, venom samples of rattlesnakes from the United States of America and Venezuela Bolivarian Republic were tested for their gelatinase, fibrinolytic, hemorrhagic and coagulant activities, as well as inhibition of platelet function. Electrophoretic titration curves on polyacrylamide gel were used to compare protein composition. There were different protease activities in the venoms. Hemorrhagic activity showed high geographic variation, but northern specimens had a higher trend in this parameter. Differences in venom protein conformation were palpable between snakes from the North and South America. There was a distinct protein composition among all venoms, several venoms presented hemorrhagic and fibrinolytic activities, and different performance on platelet assays. These results demonstrate the interesting differences between North and South America Crotalus venoms., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead ( Agkistrodon contortrix laticinctus ). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2-3 and 17-19 showed fibrinolytic activity. Fractions 2-6, 8-11, and 16-21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes., Competing Interests: and disclosure The authors declare that they have no conflict of interest. The authors confirm that there are no financial disclosures for this study.

Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

Pancreatic cancer often has a poor prognosis, even when diagnosed early. Pancreatic cancer typically spreads rapidly and is rarely detected in its early stages, which is a major reason it is a leading cause of cancer death. Signs and symptoms may not appear until pancreatic cancer is quite advanced, and complete surgical removal is not possible. Furthermore, pancreatic cancer responds poorly to most chemotherapeutic agents. The importance of integrins in several cell types that affect tumor progression has made them an appealing target for cancer therapy. Some of the proteins found in the snake venom present a great potential as anti-tumor agents. In this study, we summarize the activity of two integrins antagonist, recombinant disintegrins mojastin 1 and viridistatin 2, on human pancreatic carcinoma cell line (BXPC-3). Both recombinant disintegrins inhibited some essential aspects of the metastasis process such as proliferation, adhesion, migration, and survival through apoptosis, making these proteins prominent candidates for the development of drugs for the treatment of pancreatic cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Early detection and control of vaccine-derived poliovirus (VDPV) emergences are essential to secure the gains of polio eradication., Methods: Serial sewage samples were collected in 4 towns of Mexico before, throughout, and after the May 2010 oral poliovirus vaccine (OPV) mass immunization campaign. Isolation and molecular analysis of polioviruses from sewage specimens monitored the duration of vaccine-related strains in the environment and emergence of vaccine-derived polioviruses in a population partially immunized with inactivated poliovirus vaccine (IPV)., Results: Sabin strains were identified up to 5-8 weeks after the campaign in all towns; in Aguascalientes, 1 Sabin 3 was isolated 16 weeks after the campaign, following 7 weeks with no Sabin strains detected. In Tuxtla Gutiérrez, type 2 VDPV was isolated from 4 samples collected before and during the campaign, and type 1 VDPV from 1 sample collected 19 weeks afterward. During 2009-2010, coverage in 4 OPV campaigns conducted averaged only 57% and surveillance for acute flaccid paralysis (AFP) was suboptimal (AFP rate<1 per 100,000 population<15 years of age) in Tuxtla Gutierrez., Conclusions: VDPVs may emerge and spread in settings with inadequate coverage with IPV/OPV vaccination. Environmental surveillance can facilitate early detection in these settings., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Venom fractions isolated from Bothrops venezuelensis were shown to contain a broad spectrum of proteins with varied activities. This study describes venom fractions with coagulant, haemorrhagic, fibrinolytic, proteolytic and antiplatelet activities, and analyses their physico-chemical properties and biological activities via molecular exclusion chromatography, gel electrophoresis and a bioassay battery. The LD50, determined by injecting intraperitoneally serial dilutions of B. venezuelensis venom into mice, was 6.39 mg/kg body weight. Twelve fractions were collected from B. venezuelensis venom using molecular exclusion chromatography. Of these, fractions 1-5 and 7-9 showed haemorrhagic activity, and all fractions except 3 and 6 showed fibrinolytic activity. However, none of the fractions had significant gelatinase activity, and only fractions 4-6 demonstrated activity on hide powder azure. With the exception of fractions 1 and 4, all fractions hydrolysed the insulin B-chain. In addition, all fractions as well as the crude venom showed strong procoagulant activity when tested using a Sonoclot Analyzer. Fractions 1, 3, 5 and 9 inhibited platelet function. In this study we have described the activities of the crude venom and its size-fractions from the scarcely studied B. venezuelensis. Haemorrhagic, fibrinolytic, procoagulant and proteolytic activities, and the inhibition of platelet function were detected. This preliminary study paves the way for the identification of specific molecules in B. venezuelensis venom that could have therapeutic potential for cancer and aberrant haemostasis treatment., Competing Interests: Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Angiogenesis plays a crucial role in the growth and spread of cancer. New vascularization nourishes cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby tissue, spread to other parts of the body, and form new colonies of cancer cells. Tumor angiogenesis consists of endothelial cell proliferation, migration, and tube formation into the tumor mass. The study of natural and synthetic angiogenesis inhibitors is a promising area for therapeutics since tumors cannot grow or spread without the formation of new blood vessels. Anti-angiogenic activities have been identified in peptides known as disintegrins. Disintegrins are a family of small proteins (45-84 amino acids in length), many which are found in snake venom that function as potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. This study reports two recombinant disintegrins (r-mojastin 1 and r-viridistatin 2) inhibiting, with similar effectiveness, distinct steps in angiogenesis such as proliferation, adhesion to fibronectin, migration, and tube formation in vitro and in vivo. Both recombinant disintegrins bind to α(v)β₃ and α(v)β₅ receptors that are upregulated in tumor endothelial cells, having a higher binding activity to α(v)β₃ integrin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report a genus-wide comparison of venom proteome variation across New World pit vipers in the genus Agkistrodon. Despite the wide variety of habitats occupied by this genus and that all its taxa feed on diverse species of vertebrates and invertebrate prey, the venom proteomes of copperheads, cottonmouths, and cantils are remarkably similar, both in the type and relative abundance of their different toxin families. The venoms from all the eleven species and subspecies sampled showed relatively similar proteolytic and PLA2 activities. In contrast, quantitative differences were observed in hemorrhagic and myotoxic activities in mice. The highest myotoxic activity was observed with the venoms of A. b. bilineatus, followed by A. p. piscivorus, whereas the venoms of A. c. contortrix and A. p. leucostoma induced the lowest myotoxic activity. The venoms of Agkistrodon bilineatus subspecies showed the highest hemorrhagic activity and A. c. contortrix the lowest. Compositional and toxicological analyses agree with clinical observations of envenomations by Agkistrodon in the USA and Central America. A comparative analysis of Agkistrodon shows that venom divergence tracks phylogeny of this genus to a greater extent than in Sistrurus rattlesnakes, suggesting that the distinct natural histories of Agkistrodon and Sistrurus clades may have played a key role in molding the patterns of evolution of their venom protein genes., Biological Significance: A deep understanding of the structural and functional profiles of venoms and of the principles governing the evolution of venomous systems is a goal of venomics. Isolated proteomics analyses have been conducted on venoms from many species of vipers and pit vipers. However, making sense of these large inventories of data requires the integration of this information across multiple species to identify evolutionary and ecological trends. Our genus-wide venomics study provides a comprehensive overview of the toxic arsenal across Agkistrodon and a ground for understanding the natural histories of, and clinical observations of envenomations by, species of this genus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze-thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

Unlabelled: Researchers trying to improve the safety and efficacy of fibrinolytic therapy have been isolating fibrinolytic enzymes from snake venoms. Two fibrinolytic enzymes, colombienases 1 and 2, with significant activity have been isolated from the venom of Bothrops colombiensis., Methods: The colombienases were characterized for various biological activities which include hemorrhagic, fibrinogenolytic, proteolytic, hemolytic, edematogenic and cytotoxic., Results: Colombienases directly acted on fibrin by degrading fibrinogen Aα and Bβ chains without activating the fibrinolytic system (plasminogen/plasmin), additionally colombienase-2 degraded fibrinogen γ chains as well as the fibronectin molecule. Laminin and type IV collagen were colombienases resistant. Gelatin-zymogram activity was present in B. colombiensis venom (BcV) bands between 64 and 148 kDa. All activities were abolished by metalloproteinases inhibitors. Both enzymes lacked hemorrhagic, hemolytic, cytotoxic, plasminogen activator and coagulant activities., Conclusions: Both colombienases had direct fibrino(geno)lytic activity without other toxic side effects including in vivo hemorrhaging, which could be promising in terms of therapeutic potential as thrombolytic agents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A 5' truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5'-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, Crotalus atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC(50) of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC(50)s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders., (Published by Elsevier Ltd.)

Colombienases are acidic, low molecular weight metalloproteinases (Mr of 23,074.31 Da colombienase-1 and 23,078.80 Da colombienase-2; pI of 6.0 and 6.2, respectively) isolated from Bothrops colombiensis snake venom. The chromatographic profile in RP-HPLC and its partial sequence confirmed its high homogeneity. Both colombienases present fibrino(geno)lytic activity, but did not show any hemorrhagic, amidolytic, plasminogen activator or coagulant activities, and no effect on platelet aggregation induced by collagen or ADP. Both enzymes were strongly active on fibrinogen Aα chains followed by the Bβ chains, and colombienases-2, at high doses, also degraded the γ chains. This activity was stable at temperatures ranging between 4 and 37 °C, with a maximum activity at 25 °C, and at pHs between 7 and 9. The homology demonstrated by the comparison of sequences, with zinc-dependent metalloproteinases, as well as the metal chelant effects on, confirmed that the colombienases were metalloproteinases, particularly to α-fibrinogenases belonging to the P-I class of SVPMs (20-30 kDa), which contain only the single-domain proteins. The biological characteristics of the colombienases confer a therapeutic potential, since they contain a high fibrino(geno)lytic activity, devoid of hemorrhagic activity. These metalloproteinases might be explored as thrombolytic agents given that they dissolve fibrin clots or prevent their formation.

Snake venom disintegrins inhibit platelet aggregation and have anti-cancer activities. In this study, we report the cloning, expression, and functional activities of a recombinant disintegrin, r-viridistatin 2 (GenBank ID: JQ071899), from the Prairie rattlesnake. r-Viridistatin 2 was tested for anti-invasive and anti-adhesive activities against six different cancer cell lines (human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorectal adenocarcinoma (CaCo-2), human breast adenocarcinoma (MDA-MB-231) and murine skin melanoma (B16F10)). r-Viridistatin 2 shares 96% and 64% amino acid identity with two other Prairie rattlesnake medium-sized disintegrins, viridin and viridistatin, respectively. r-Viridistatin 2 was able to inhibit adhesion of T24, SK-MEL-28, HT-1080, CaCo-2 and MDA-MB-231 to various extracellular matrix proteins with different affinities. r-Viridistatin 2 decreased the ability of T24 and SK-MEL-28 cells to migrate by 62 and 96% respectively, after 24 h of incubation and the invasion of T24, SK-MEL-28, HT-1080 and MDA-MB-231 cells were inhibited by 80, 85, 65 and 64% respectively, through a reconstituted basement membrane using a modified Boyden chamber. Finally, r-viridistatin 2 effectively inhibited lung colonization of murine melanoma cells in BALB/c mice by 71%, suggesting that r-viridistatin 2 could be a potent anti-cancer agent in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Twenty-one Mojave rattlesnakes, Crotalus scutulatus scutulatus (C. s. scutulatus), were collected from Arizona and New Mexico U.S.A. Venom proteome of each specimen was analyzed using reverse-phase HPLC and SDS-PAGE. The toxicity of venoms was analyzed using lethal dose 50 (LD(50)). Health severity outcomes between two Arizona counties U.S.A., Pima and Cochise, were determined by retrospective chart review of the Arizona Poison and Drug Information Center (APDIC) database between the years of 2002 and 2009. Six phenotypes (A-F) were identified based on three venom protein families; Mojave toxin, snake venom metalloproteinases PI and PIII (SVMP), and myotoxin-A. Venom changed geographically from SVMP-rich to Mojave toxin-rich phenotypes as you move from south central to southeastern Arizona. Phenotypes containing myotoxin-A were only found in the transitional zone between the SVMP and Mojave toxin phenotypes. Venom samples containing the largest amounts of SVMP or Mojave toxin had the highest and lowest LD(50s), respectively. There was a significant difference when comparing the presence of neurotoxic effects between Pima and Cochise counties (p=0.001). No significant difference was found when comparing severity (p=0.32), number of antivenom vials administered (p=0.17), days spent in a health care facility (p=0.23) or envenomation per 100,000 population (p=0.06). Although not part of the original data to be collected, death and intubations, were also noted. There is a 10× increased risk of death and a 50× increased risk of intubations if envenomated in Cochise County., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A mouse model was established to reproduce the haemorrhagic syndrome which occurs in humans after accidental contact with the hairs of the caterpillar Lonomia achelous (LA) and measures the haemostatic and inflammatory alterations that occur as a result of this contact. Mice were injected intradermally with different doses (0.4, 0.8 and 1.6 mg/animal) of L. achelous haemolymph (LAH). Haematological (haemoglobin, haematocrit, platelet count, differential leukocyte count), haemostatic (fibrinogen, plasminogen, factor XIII [FXIII], fibrinolytic activity) and inflammatory parameters (tumour necrosis factor alpha [TNF-α], nitric oxide [NO]) were measured at different times up to 48 h. C57BL/6 mice responded to LAH injection, in terms of these parameters, in a manner similar to that seen in humans, whereas the BALB/c mice were unresponsive. In C57BL/6 mice injected with LAH, time course measurements showed: a) a reduction in the haemoglobin, haematocrit, fibrinogen, FXIII and plasminogen levels, b) no effect on the platelet count and c) immediate leukocytosis and an increase in the fibrinolytic activity in plasma. An inflammatory response (TNF-α) was observed within 1 h post-injection, followed by a more persistent increase in serum NO. These findings suggest that C57BL/6 mice represent a useful model of the haemorrhagic syndrome observed in humans who have suffered contact with the caterpillar, permitting a deeper understanding of the role of the inflammatory response in the haematological and haemostatic manifestations of this syndrome., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.

Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH < 6.5), MitTx massively potentiates (>100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception., (© 2011 Macmillan Publishers Limited. All rights reserved)

Platelets play a central role in hemostasis during vascular injury. Patients affected with the hemorrhagic syndrome caused by contact with Lonomia achelous caterpillars (Lac) Lepidoptera distributed in various South American countries, show digestive, pulmonary and intraperitoneal bleeding in combination with hematomas and echymosis. In the present study, we have evaluated the effects of Lonomin V (serine protease isolated from Lac hemolymph) on some functional properties of platelets, evaluating its importance in primary hemostasis. Platelet adhesion to fibrinogen was reduced by 19, 20, 36, and 37% after pre-treated with 0.2, 2, 20 and 40 nM of Lonomin V, respectively. Pre-incubation of the platelets with 408 nM of Lonomin V, for 4 min at 37 °C, resulted in complete inhibition of the collagen-induced platelet aggregation, in contrast to 56% inhibition of the ADP - induced platelet aggregation. Lonomin V also inhibited anti-α(IIb)β(3) integrin binding to platelets by 56, 57, 52 and 54% at concentrations of 0.2, 2, 20 and 40 nM respectively. Additionally, Lonomin V inhibited anti-P-selectin binding to platelets by 28, 37, 33 and 33% at the same concentrations. The platelets tested with Lonomin V did not modify their viability. In summary, Lonomin V inhibited platelet aggregation, probably caused by the degradation of collagen. The anti-platelet activity of Lonomin V has been shown to be unique and a potentially useful tool for investigating cell-matrix and cell-cell interactions and for the development of antithrombotic agents in terms of their anti-adhesive activities., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A₂s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD₅₀s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD₅₀ of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD₅₀s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia., Methods: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood., Results: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate., Conclusions: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD₅₀) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

Porthidium lansbergii hutmanni is a small pit viper found on Margarita Island, Venezuela. Local tissue damage is one of the most obvious characteristics of P. l. hutmanni envenomation, which can lead to diverse pathological effects, such as hemorrhage, edema, blistering, necrosis, lymphatic vessel damage and degradation of extracellular matrix. Metalloproteinases are one of the major components in venoms responsible for these effects. To date, very little is known or has been reported on P. l. hutmanni venom. Crude P. l. hutmanni venom had a LD(50) of 2.5 mg/kg and was considered very hemorrhagic (minimal hemorrhagic dose [MHD]: 0.98 μg) when compared to other hemorrhagic (Bothrops) venoms in Venezuela. Crude P. l. hutmanni venom also inhibited ADP-induced platelet aggregation. A metalloproteinase, Porthidin-1, from this venom was isolated by three chromatography steps (Sephadex G100, Superose 12 HR10/30 and Bioscale Q2). Porthidin-1 falls in the SVMP P-I class having a molecular weight of 23 kDa, verified by both SDS-PAGE and mass spectrometry. High-resolution mass spectrometry and a database search identified a peptide from Porthidin-1 (YNGDLDK) belonging to the SVMP family of proteins. Porthidin-1 contained hemorrhagic, fibrino(geno)lytic, caseinolytic and gelatinolytic activities, and these activities were capable of being neutralized by metalloproteinase inhibitors but not serine proteinase inhibitors. The peptide YNGDLDK shared similarities with five venom proteins with a BLAST e-value of <1. This work details the biochemical and pathophysiological effects that can result from envenomations, and highlights the importance and significance for characterizing unknown or poorly documented venoms from different geographical regions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)