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Crotamine (Ctm) is a peptide isolated from Crotalus durissus terrificus venom. This molecule has been demonstrated to diminish body weight gain and enhance browning in adipose tissue, glucose tolerance, and insulin sensitivity; hence, it has been postulated as an anti-obesogenic peptide. However, the mechanism to elicit the anti-obesogenic effects has yet to be elucidated. Thus, we investigated the possible interaction of Ctm with receptors involved in obesity-related metabolic pathways through protein-protein docking and molecular dynamics refinement. To test the anti-obesogenic mechanism of Ctm, we selected and retrieved 18 targets involved in obesity-related drug discovery from Protein Data Bank. Then, we performed protein-protein dockings. The best three Ctm-target models were selected and refined by molecular dynamics simulations. Molecular docking demonstrated that Ctm was able to interact with 13 of the 18 targets tested. Having a better docking score with glucagon-like peptide-1 receptor (GLP-1R) (-1430.2 kcal/mol), DPP-IV (dipeptidyl peptidase-IV) (-1781.7 kcal/mol) and α-glucosidase (-1232.3 kcal/mol). These three models were refined by molecular dynamics. Ctm demonstrated a higher affinity for GLP-1R (ΔG: -41.886 ± 2.289 kcal/mol). However, Ctm interaction was more stable with DPP-IV (RMSD: 0.360 ± 0.015 nm, Radius of gyration: 2.781 ± 0.009 nm). Moreover, the number of interactions and the molecular mechanics energies of Ctm residues suggest that the interaction of Ctm with these receptors is mainly mediated by basic-hydrophobic dyads Y1-K2, W31-R32, and W33-R34. Together, all these results allow elucidating a possible molecular mechanism behind the previously described anti-obesogenic effects., Competing Interests: Declaration of competing interest 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. The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn 2+ ), at their catalytic site. They are classified into three classes (P-I to P-III). BjussuMP-II, a P-I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II's effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1β, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5-50 μg/mL) for 3-24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II's proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1β, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juliana Pavan Zuliani reports financial support, administrative support, article publishing charges, statistical analysis, travel, and writing assistance were provided by conselho nacional de pesquisa - CNPq - Brazil. Juliana P Zuliani reports a relationship with Conselho Nacional de Pesquisa - CNPq - Brazil that includes: funding grants and non-financial support. No has patent No pending to NO. Nothing. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake ( Crotalus adamanteus ), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus . Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly., Competing Interests: Competing interests statement:The authors declare no competing interest.

Phospholipases A 2 (PLA 2 s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA 2 s (svPLA 2 s) are extracellular proteins with a molecular mass of 13-14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA 2 targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA 2 s were inconsistent, while the data on the dimeric svPLA 2 crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA 2 HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA 2 s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 μM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA 2 s. These are the first indications of the cardiac and vascular effects of true vipers' heterodimeric svPLA 2 s.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects., Competing Interests: Declaration of competing interest The authors have no financial or non-financial interests to disclose. The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus , with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

A high diversity of rattlesnake species can be found in the Baja California peninsula and the island of the Gulf of California, nevertheless, their venom has been poorly evaluated. The aim of this work was to present the first characterization of endemic Crotalus mitchellii, micro endemic C. polisi and C. thalassoporus venoms. All samples provoke human plasma coagulation showing doses in the rank of 2.3-41.0 μg and also produce rapid hydrolysis of the alpha chain of bovine fibrinogen while the beta chain is attacked at larger incubation periods by C. polisi and especially by C. thalassoporus. Phospholipase activity ranging from 23.2 to 173.8 U/mg. The venoms of C. thalassoporus and C. polisi show very high hemorrhagic activity (from 0.03 to 0.31 μg). A total of 130 toxin-related proteins were identified and classified into ten families. Crotalus mitchellii venom was characterized by high abundance of crotoxin-like and other phospholipase proteins (34.5%) and serine proteinases (29.8%). Crotalus polisi showed a similar proportion of metalloproteinases (34%) and serine proteinases (22.8%) components with important contribution of C-type lectins (14.3%) and CRiSP (14.0%) proteins. Venom of C. thalassoporus is dominated by metalloproteases that amount to more than 66% of total toxin proteins. These results provide a foundation for comprehending the biological, ecological and evolutionary significance of venom composition of speckled rattlesnake from the Baja California peninsula., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Luis Beltran reports financial support was provided by National Council for Science and Technology. Alejandro Carbajal-Saucedo reports financial support was provided by National Council for Science and Technology., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Zika virus is the etiologic agent of Zika fever, and has been previously associated with cases of microcephaly, drawing the attention of the health authorities worldwide. However, no vaccine or antiviral are currently available. Phospholipases A 2 (PLA 2 ) isolated from snake venoms have demonstrated antiviral activity against several viruses. Here we demonstrated the anti-ZIKV activity of bothropstoxins-I and II (BthTX-I and II) isolated from Bothrops jararacussu venom. Vero E6 cells were infected with ZIKVPE243 in the presence of compounds for 72 h, when virus titers were evaluated. BthTX-I and II presented strong dose-dependent inhibition of ZIKV, with a SI of 149.1 and 1.44 × 10 5 , respectively. These toxins mainly inhibited the early stages of the replicative cycle, such as during the entry of ZIKV into host cells, as shown by the potent virucidal effect, suggesting the action of these toxins on the virus particles. Moreover, BthTX-I and II presented significant activity towards post-entry stages of the ZIKV replicative cycle. Molecular docking analyses showed that BthTX-I and II potentially interact with DII and DIII domains from ZIKV Envelope protein. Our findings show that these PLA 2 s could be used as useful templates for the development of future antiviral candidate drugs against Zika fever., Competing Interests: Declaration of competing interest 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Snake venom metalloproteinases (SVMPs) are enzymatic proteins present in large amounts in snake venoms presenting proteolytic, hemorrhagic, and coagulant activities. BjussuMP-II, a class P-I SVMP, isolated from the Bothrops jararacussu snake venom does not have relevant hemorrhagic activity but presents fibrinolytic, fibrinogenolytic, antiplatelet, gelatinolytic, and collagenolytic action. This study aimed to verify the action of BjussuMP-II on human neutrophil functionality focusing on the lipid bodies formation and hydrogen peroxide production, the release of dsDNA through colorimetric and microscopic assays, and cytokines by immunoenzymatic assays. Results showed that BjussuMP-II at concentrations of 1.5 up to 50 μg/mL for 24 h is not toxic to human neutrophils using an MTT assay. Under non-cytotoxic concentrations, BjussuMP-II can induce an increase in the formation of lipid bodies, production of hydrogen peroxide and cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8)] liberation and, the release of dsDNA to form NETs. Taken together, the data obtained show for the first time that BjussuMP-II has a pro-inflammatory action and activates human neutrophils that can contribute to local damage observed in snakebite victims., Competing Interests: Declaration of competing interest 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

Snakebite envenoming is a life threatening neglected tropical disease that represents a considerable public health concern in the tropics. Viperid snakes of the genus Bothrops are among those of greatest medical importance in Latin America, and they frequently cause severe systemic haemotoxicity and local tissue destructive effects in human victims. Although snakebite antivenoms can be effective therapeutics, their efficacy is undermined by venom toxin variation among snake species. In this study we investigated the extent of paraspecific venom cross-reactivity exhibited by three distinct anti- Bothrops antivenoms (Soro antibotrópico-crotálico, BothroFav and PoliVal-ICP) against seven different Bothrops pit viper venoms from across Latin America. We applied a range of in vitro assays to assess the immunological binding and recognition of venom toxins by the antivenoms and their inhibitory activities against specific venom functionalities. Our findings demonstrated that, despite some variations, the monovalent antivenom BothroFav and the polyvalent antivenoms Soro antibotrópico-crotálico and PoliVap-ICP exhibited extensive immunological recognition of the distinct toxins found in the different Bothrops venoms, with Soro antibotrópico-crotálico generally outperformed by the other two products. In vitro functional assays revealed outcomes largely consistent with the immunological binding data, with PoliVap-ICP and BothroFav exhibiting the greatest inhibitory potencies against procoagulant and fibrinogen-depleting venom activities, though Soro antibotrópico-crotálico exhibited potent inhibition of venom metalloproteinase activities. Overall, our findings demonstrate broad levels of antivenom paraspecificity, with in vitro immunological binding and functional inhibition often highly comparable between venoms used to manufacture the antivenoms and those from related species, even in the case of the monovalent antivenom BothroFav. Our findings suggest that the current clinical utility of these antivenoms could possibly be expanded to other parts of Latin America that currently suffer from a lack of specific snakebite therapies.

Snakebite envenoming represents a worldwide public health issue. Suitable technologies have been investigated for encapsulated recombinant or native proteins capable of inducing an effective and long-lasting adaptive immune response. Nanoparticles are colloidal dispersions that have been used as drug delivery systems for bioactive biological compounds. Venom-loaded nanoparticles modulate the protein release and activate the immune response to produce specific antibodies. In this study, biocompatible cationic nanoparticles with Bothrops jararaca venom were prepared to be used as a novel immunoadjuvant that shows a similar or improved immune response in antibody production when compared to a conventional immunoadjuvant (aluminum hydroxide). We prepared stable, small-sized and spherical particles with high Bothrops jararaca venom protein association efficiency. The high protein loading efficiency, electrophoresis, and zeta potential results demonstrated that Bothrops jararaca venom is adsorbed on the particle surface, which remained as a stable colloidal dispersion over 6 weeks. The slow protein release occurred and followed parabolic diffusion release kinetics. The in vivo studies demonstrated that venom-loaded nanoparticles were able to produce an immune response similar to that of aluminum hydroxide. The cationic nanoparticles (CNp) as carriers of bioactive molecules, were successfully developed and demonstrated to be a promising immunoadjuvant.

In Colombia, South America, there is a subspecies of the South American rattlesnake Crotalus durissus , C. d. cumanensis , a snake of the Viperidae family, whose presence has been reduced due to the destruction of its habitat. It is an enigmatic snake from the group of pit vipers, venomous, with large articulated front fangs, special designs on its body, and a characteristic rattle on its tail. Unlike in Brazil, the occurrence of human envenomation by C. durisus in Colombia is very rare and contributes to less than 1% of envenomation caused by snakes. Its venom is a complex cocktail of proteins with different biological effects, which evolved with the purpose of paralyzing the prey, killing it, and starting its digestive process, as well as having defense functions. When its venom is injected into humans as the result of a bite, the victim presents with both local tissue damage and with systemic involvement, including a diverse degree of neurotoxic, myotoxic, nephrotoxic, and coagulopathic effects, among others. Its biological effects are being studied for use in human health, including the possible development of analgesic, muscle relaxant, anti-inflammatory, immunosuppressive, anti-infection, and antineoplastic drugs. Several groups of researchers in Brazil are very active in their contributions in this regard. In this work, a review is made of the most relevant biological and medical aspects related to the South American rattlesnake and of what may be of importance for a better understanding of the snake C. d. cumanensis , present in Colombia and Venezuela.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus , which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Snakes of the genus Bothrops are responsible the most snakebites in the Brazil, causing a diverse and complex pathophysiological condition. Bothrops erythromelas is the main specie of medical relevance found in the Caatinga from the Brazilian Northeast region. The pathophysiological effects involving B. erythromelas snakebite as well as the organism reaction in response to this envenomation are not so explored. Thus, edema was induced in mice paws using 2.5 μg or 5.0 μg of B. erythromelas venom, and the percentage of edema was measured. Plasma was collected 30  minutes after the envenomation-induced in mice and analyzed by mass spectrometry. It was identified a total of 112 common plasma proteins differentially abundant among experimental groups, which are involved with the complement system and coagulation cascades, oxidative stress, neutrophil degranulation, platelets degranulation and inflammatory response. Apolipoprotein A1 (Apoa), serum amyloid protein A-4 (Saa4), adiponectin (Adipoq) showed up-regulated in mice plasma after injection of venom, while fibulin (Fbln1), factor XII (F12) and vitamin K-dependent protein Z (Proz) showed down-regulated. The results indicate a protein pattern of thrombo-inflammation to the B. erythromelas snakebite, evidencing potential biomarkers for monitoring this snakebite, new therapeutic targets and its correlations with the degree of envenomation once showed modulations in the abundance among the different groups according to the amount of venom injected into the mice., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox , Bothrops erythromelas , and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii . Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms.

Crotamine is a lysine and cysteine rich 42 amino acids long bio-active polypeptide, isolated from the venom of a South American rattlesnake, that can also be used as cell penetrating peptide. A facile synthetic scheme for coupling cargo molecules like fluorophores (carboxyfluorescein) or MRI probes (Gd-DO3A-based macrocycle) is presented. The toxicity, cellular internalization and steady-state accumulation after long-term incubation for 18 h, as well as magnetic resonance relaxivities and cellular relaxation rates of crotamine based probes were evaluated and compared to its shorter synthetic fragment CyLoP-1. The longitudinal relaxivity (r 1 ) of the conjugates of CyLoP-1 and crotamine is significantly lower in medium than in water indicating to the lower contrast enhancement efficacy of DO3A-based probes in biological samples. Carboxyfluorescein labeled crotamine did not exhibit toxicity up to a concentration of 2.5 µM. CyLoP-1 accumulated about four times better within the cells compared to crotamine. Fluorescence microscopy suggests different predominant uptake mechanisms for crotamine and CyLoP-1 in 3T3 cells. While crotamine is predominantly localized in vesicular structures (most likely endosomes and lysosomes) within the cell, CyLoP-1 is mainly homogeneously distributed in the cytosol. The cellular relaxation rate (R 1, cell ) of the crotamine based probe was not significantly increased whereas the corresponding CyLoP-1-derivative showed a slightly elevated R 1, cell . This study indicates the potential of crotamine and in particular the shorter fragment CyLoP-1 to be useful for an efficient transmembrane delivery of agents directed to intracellular (cytosolic) targets. However, the applicability of the conjugates synthesized here as contrast agents in MR imaging is limited. Further improvement is needed to prepare more efficient probes for MRI applications, i.e., by replacing the DO3A- with a DOTA-based chelate., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bothrops leucurus is considered as a snake of medical interest in the State of Bahia, Brazil. However, so far, there are no studies that provide a refined mapping of the composition of this venom. The aim of this work was to better understand the protein composition of B. leucurus snake venom and to isolate and biologically characterize the most abundant toxin, a basic PLA 2 -like. Shotgun proteomics approach identified 137 protein hits in B. leucurus venom subdivided into 19 protein families. The new basic PLA 2- like toxin identified was denominated Bleu-PLA 2 -like, it and other proteoforms represents about 25% of the total proteins in the venom of B. leucurus and induces myotoxicity, inflammation and muscle damage. Immunoreactivity assays demonstrated that B. leucurus venom is moderately recognized by bothropic and crotalic antivenoms, and on the other hand, Bleu-PLA 2 -like and its proteoforms are poorly recognized. Our findings open doors for future studies in order to assess the systemic effects caused by this snake venom in order to better understand the toxinological implications of this envenomation and, consequently, to assist in the clinical treatment of victims., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Small RNAs (sRNAs) and microRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs that regulate gene expression in eukaryotes. Experiments in mice and humans have revealed that a typical small RNA can affect the expression of a wide range of genes, implying that small RNAs function as global regulators. Here, we used small RNA deep sequencing to investigate how jararhagin, a metalloproteinase toxin produced from the venom of Bothrops jararaca , affected mmu-miRNAs expression in mice 2 hours (Jar 2hrs) and 24 hours (Jar 24hrs) after injection compared to PBS control. The findings revealed that seven mmu-miRNAs were substantially differentially expressed ( p value ( p (Corr) cut-off 0.05, fold change ≥ 2) at 2 hrs after jararhagin exposure and that the majority of them were upregulated when compared to PBS. In contrast to these findings, a comparison of Jar 24hrs vs. PBS 24hrs demonstrated that the majority of identified mmu-miRNAs were downregulated. Furthermore, the studies demonstrated that mmu-miRNAs can target the expression of several genes involved in the MAPK signaling pathway. The steady antithetical regulation of mmu-miRNAs may correlate with the expression of genes that trigger apoptosis via MAPK in the early stages, and this effect intensifies with time. The findings expand our understanding of the effects of jararhagin on local tissue lesions at the molecular level.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA 2 ) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR ® 2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA 2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA 2 from C. tzabcan . The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini , and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Snake venoms are natural sources of bioactive substances with therapeutic potential. In this work, we evaluated the cytotoxicity of the Crotalus durissus collilineatus, negative crotamine variety and the isolated fraction C0K3N3 in BALB C/3T3 and K562 cell lines. The results indicate that the C0K3N3 protein is more cytotoxic against the K562 tumor cell line than in the 3T3 baseline., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

Phospholipases A 2 (PLA 2 s) are found in almost every venomous snake family. In snakebites, some PLA 2 s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA 2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA 2 -like proteins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Anavip (F(ab') 2 AV) is a lyophilized F(ab') 2 immunoglobulin fragment derived from horses immunized with venom from Bothrops asper and Crotalus durissus . It was approved by the FDA in 2015 for treatment of North American rattlesnake envenomation but not for Agkistrodon envenomation. Published data regarding the efficacy and safety of Anavip in treating Agkistrodon envenomations is limited. We present a case of a patient treated with Anavip after confirmed Agkistrodon laticinctus envenomation., Case Details: A 77 year-old man was bitten on his fifth finger by a captive A. laticinctus . He was taken to a local emergency department where he received a 10 vial initial dose of F(ab') 2 AV for pain and swelling and was transferred. At the receiving facility, his pain had improved and his swelling had not progressed. Over the next 30 h, his platelets declined to 132,000/mm 3 and he received an additional 4 vials of F(ab') 2 AV. The remainder of his course was unremarkable with complete recovery by 3 months., Discussion: This case provides an additional published datapoint on the use of this F(ab') 2 AV in the treatment of envenomation by Agkistrodon .

The global exploration of snakebites requires the use of quantitative omics approaches to characterize snake venom as it enters into the systemic circulation. These omics approaches give insights into the venom proteome, but a further exploration is warranted to analyze the venom-reactome for the identification of snake venom biomarkers. The recent discovery of extracellular vesicles (EVs), and their critical cellular functions, has presented them as intriguing sources for biomarker discovery and disease diagnosis. Herein, we purified EV's from the snake venom (svEVs) of Crotalus atrox and C. oreganus helleri , and from plasma of BALB/c mice injected with venom from each snake using EVtrap in conjunction with quantitative mass spectrometry for the proteomic identification and quantification of svEVs and plasma biomarkers. Snake venom EVs from C. atrox and C. o. helleri were highly enriched in 5' nucleosidase, L-amino acid oxidase, and metalloproteinases. In mouse plasma EVs, a bioinformatic analysis for revealed upregulated responses involved with cytochrome P450, lipid metabolism, acute phase inflammation immune, and heat shock responses, while downregulated proteins were associated with mitochondrial electron transport, NADH, TCA, cortical cytoskeleton, reticulum stress, and oxidative reduction. Altogether, this analysis will provide direct evidence for svEVs composition and observation of the physiological changes of an envenomated organism.

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A 2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts., Competing Interests: The authors have declared that no competing interests exist.

Acute kidney injury (AKI) is a frequent and potentially fatal complication of snakebites. In the setting of snakebites, endothelial biomarkers may be used to predict disease severity and can play a major role in AKI pathophysiology. The aim of this study was to investigate the potential role of endothelial biomarkers in predicting AKI in Bothrops envenoming. Therefore, blood and urine samples were collected from 26 patients admitted to the emergency department after Bothrops envenoming at 3 different post-bite points in time: on admission (up to 8 h post-bite), 12-16 h, and 24-28 h post-bite, to investigate the time course of endothelial biomarkers in AKI following Bothrops snakebites. The diagnostic performance of injury biomarkers in Bothrops envenomation was evaluated. AKI was diagnosed using the Kidney Disease Improving Global Outcomes (KDIGO) criteria. There was an association between endothelial injury and increased risk for AKI in bothropic envenoming. Angiopoietin- 1 (Ang-1) and Vascular cell adhesion protein-1 (VCAM-1) were useful biomarkers to predict mild AKI [AUC-ROC: Ang-1 0.82, VCAM-1 0.76] within the interval of 8-16 h post Bothrops snakebites. The use of endothelial biomarkers VCAM-1 e Ang-1 within 12-16 h post-bite may be useful in the early stage of mild AKI related to Bothrops envenoming and might have an effect on the early intervention for renal protection in less severe Bothrops-related AKI., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "β-switch" region through the β chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis., (© 2021 by The American Society of Hematology.)

Bothrops leucurus is the major causative agent of venomous snakebites in Northeastern Brazil. Severe pain is the most frequent symptom in these envenomings, with an important inflammatory component. This work characterized the pronociceptive effects evoked by B. leucurus venom (BLV) in mice and the role of inflammatory mediators in these responses. The nociceptive behaviors were quantified by the modified formalin test. The mechanical hyperalgesia was assessed by the digital von Frey test. Pharmacological assays were performed with different antagonists and synthesis inhibitors to investigate the involvement of inflammatory mediators in both nociceptive events. BLV (1-15 μg/paw) injection in mice evoked intense and dose-dependent nociceptive behaviors that lasted for up to 1 h. BLV (10 μg/paw) also caused sustained mechanical hyperalgesia. Histamine and serotonin played a role in the nociception, but not in the BLV-induced mechanical hyperalgesia. Nitric oxide contributed to both responses, but only to the late stages of mechanical hyperalgesia. Eicosanoids were also present in both nociceptive responses. Prostanoid synthesis by COX-1 seemed to be more relevant for the nociception, whereas COX-2 had a more prominent role in the mechanical hyperalgesia. Leukotrienes were the most relevant mediators of BLV-induced mechanical hyperalgesia, hence inhibiting lipoxygenase pathway could be an efficient therapeutic strategy for pain management during envenoming. Our behavioral data demonstrates that BLV promotes nociceptive transmission mediated by biogenic amines, nitric oxide and eicosanoids, and nociceptor sensitization through nitric oxide and eicosanoids. Moreover, phospholipases A 2 (PLA 2 ), an important class of toxins present in bothropic venoms, appear to play an important role in the nociceptive and hypernociceptive response induced by BLV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species' geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting.

Introduction: Protobothrops mucrosquamatus bite induces wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and acute renal failure. The severity of the hematological derangements and associated factors for wound necrosis and subsequent surgery and the appropriate management of these conditions have not been well characterized. Although severe renal failure requiring hemodialysis has been reported following P . mucrosquamatus bite, the culprit snake may be erroneously classified., Materials and Methods: A total of 186 patients with P . mucrosquamatus bites were retrospectively evaluated. They were categorized into group 1 (patients receiving debridement or finger/toe amputation) and group 2 (all other patients) to identify the associated factors for surgery. Characteristic data were compared between groups 1 and 2 and between definite and suspected cases., Results: No differences were observed between definite and suspected cases in terms of symptomatology and management. Of the 186 patients, 7 (3.8%) were asymptomatic, 179 (96.2%) experienced tissue swelling and pain, and 107 (57.5%) had local ecchymosis. Coagulopathy, thrombocytopenia, and renal impairment were found in 13 (7%), 19 (10.2%), and 7 (3.8%) patients, respectively. None of the patients required transfusion therapy or hemodialysis. Furthermore, no systemic bleeding or death occurred. Antivenom was administered to all 179 envenomed patients at a median of 1.5 h post-bite. The median total dose of the specific antivenom was 5.5 vials. In multivariate logistic regression analysis, finger as the bite site, bullae and blister formation, and wound infection were significantly associated with wound necrosis; whereas finger as the bite site and bullae and blister formation were related to debridement or finger/toe amputation., Discussion and Conclusions: Protobothrops mucrosquamatus envenomation mainly exerts effects on local tissue. Systemic effects are uncommon and generally nonsevere and transient after the treatment with the specific antivenom. We speculated that severe renal failure requiring hemodialysis is not a typical finding of P . mucrosquamatus envenomation. Patients with finger as the bite site and bullae or blister formation should be carefully examined for wound necrosis, secondary infection, and subsequent surgery. Further evaluations of the efficacy of antivenom against local tissue effects and the effect of selective antibiotics in the management of bite wound infection are urgently required. Although the antivenom manufacturer suggested a skin test prior to use, we believed that it could be omitted because it does not accurately predict the allergic responses.

Snakebite envenomation causes > 81,000 deaths and incapacities in another 400,000 people worldwide every year. Snake venoms are complex natural secretions comprised of hundreds of different molecules with a wide range of biological functions that after injection cause local and systemic manifestations. Although several studies have investigated snake venoms, the majority have focused on the protein portion (toxins), without significant attention paid to the lipid fraction. Therefore, an untargeted lipidomic approach based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) was applied to investigate the lipid constituents of venoms of the snake species Crotalus durissus terrificus and Bothrops moojeni. Phosphatidylcholines (PC), Lyso-PCs, phosphatidylethanolamines (PE), Lyso-PE, phosphatidylserine (PS), phosphatidylinositol (PI), ceramides (Cer), and sphingomyelin (SM) species were detected in the analyzed snake venoms. The identified lipids included bioactive compounds such as platelet-activating factor (PAF) precursor, PAF-like molecules, plasmalogens, ceramides, and sphingomyelins with long fatty acid chain lengths, which may be associated with the systemic responses triggered by C. d. terrificus and B. moojeni envenomation. These responses include platelet aggregation, activation of intercellular adhesion molecule 1 (ICAM1), apoptosis, as well as the production of pro-inflammatory lipid mediators, cytokines, and reactive species. The newly proposed lipidomics strategy provided valuable information regarding the lipid profiles of viperid venoms, which could lead to increased understanding of the complex pathology promoted by snakebite envenomation.

Phospholipase A 2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA 2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA 2 inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA 2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA 2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA 2 activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the extensive body of research on snake venom, many facets of snake venom systems, such as the physiology and regulation of the venom gland itself, remain virtually unstudied. Here, we use time series gene expression analyses of the rattlesnake venom gland in comparison with several non-venom tissues to characterize physiological and cellular processes associated with venom production and to highlight key distinctions of venom gland cellular and physiological function. We find consistent evidence for activation of stress response pathways in the venom gland, suggesting that mitigation of cellular stress is a crucial component of venom production. Additionally, we demonstrate evidence for an unappreciated degree of cellular and secretory activity in the steady state venom gland relative to other secretory tissues and identify vacuolar ATPases as the likely mechanisms driving acidification of the venom gland lumen during venom production and storage.

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

Bothrops brazili is a pitviper from Amazonian region, responsible for many accidents in Peru. Despite its relevance, its venom has not been extensively characterized. In the present work, Bothrops brazili venom (BbV) components were analyzed by RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Approximately 37 proteins were identified, belonging to 7 families. Snake venom metalloproteinases (SVMPs) were the most abundant proteins of the venom (33.05%), followed by snake venom serine proteinases (SVSPs, 26.11%), phospholipases A 2 (PLA 2 , 25.57%), snake C-type lectins (CTLs, 9.61%), L-aminoacid oxidase (LAAO, 3.80%), cystein-rich secretory proteins (CRISP, 1.67%) and Bradykinin-potentiating peptide (BPP, 0.20%). In vitro enzymatic activities of BbV showed high levels of SVMP activity and reduced Hyal activity in comparison with other bothropic venoms. Furthermore, BbV reduced VERO cells viability. ELISA and Western Blotting showed that both Peruvian and Brazilian bothropic antivenoms were able to recognize BbV components. This work provides an overview of BbV venom content and indicates a potential efficiency of Peruvian and Brazilian antivenoms to treat accidents with this species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients ( n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding ( n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding ( n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Investigations into the nature of platelet functional variety and consequences for homeostasis require new methods for resolving single platelet phenotypes. Here we combine droplet microfluidics with flow cytometry for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin > TRAP-14>ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level responses in health and disease.

The genetic origins of novelty are a central interest of evolutionary biology. Most new proteins evolve from preexisting proteins but the evolutionary path from ancestral gene to novel protein is challenging to trace, and therefore the requirements for and order of coding sequence changes, expression changes, or gene duplication are not clear. Snake venoms are important novel traits that are comprised of toxins derived from several distinct protein families, but the genomic and evolutionary origins of most venom components are not understood. Here, we have traced the origin and diversification of one prominent family, the snake venom metalloproteinases (SVMPs) that play key roles in subduing prey in many vipers. Genomic analyses of several rattlesnake ( Crotalus ) species revealed the SVMP family massively expanded from a single, deeply conserved adam28 disintegrin and metalloproteinase gene, to as many as 31 tandem genes in the Western Diamondback rattlesnake ( Crotalus atrox ) through a number of single gene and multigene duplication events. Furthermore, we identified a series of stepwise intragenic deletions that occurred at different times in the course of gene family expansion and gave rise to the three major classes of secreted SVMP toxins by sequential removal of a membrane-tethering domain, the cysteine-rich domain, and a disintegrin domain, respectively. Finally, we show that gene deletion has further shaped the SVMP complex within rattlesnakes, creating both fusion genes and substantially reduced gene complexes. These results indicate that gene duplication and intragenic deletion played essential roles in the origin and diversification of these novel biochemical weapons., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Pit viper venom commonly causes venom-induced consumptive coagulopathy (VICC), which can be complicated by life-threatening hemorrhage. VICC has a complex pathophysiology affecting multiple steps of the coagulation pathway. Early detection of VICC is challenging because conventional blood tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) are unreliable for early-stage monitoring of VICC progress. As the effects on the coagulation cascade may differ, even in the same species, the traditional coagulation pathways cannot fully explain the mechanisms involved in VICC or may be too slow to have any clinical utility. Antivenom should be promptly administered to neutralize the lethal toxins, although its efficacy remains controversial. Transfusion, including fresh frozen plasma, cryoprecipitate, and specific clotting factors, has also been performed in patients with bleeding. The effectiveness of viscoelastic monitoring in the treatment of VICC remains poorly understood. The development of VICC can be clarified using thromboelastography (TEG), which shows the procoagulant and anticoagulant effects of snake venom. Therefore, we believe that TEG may be able to be used to guide hemostatic resuscitation in victims of VICC. Here, we aim to discuss the advantages of TEG by comparing it with traditional coagulation tests and propose potential treatment options for VICC.

Background: Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A 2 (PLA 2 ) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis nigroviridis) and Talamancan Palm-Pitvipers (B. nubestris)., Results: We assembled 1517 coding sequences, including 43 toxins for B. nigroviridis and 1787 coding sequences including 42 toxins for B. nubestris. The venom gland transcriptomes were extremely divergent between these two species with one B. nigroviridis exhibiting a primarily neurotoxic pattern of expression, both B. nubestris expressing primarily hemorrhagic toxins, and a second B. nigroviridis exhibiting a mixed expression phenotype. Weighted gene coexpression analyses identified six submodules of transcript expression variation, one of which was highly associated with SVMPs and a second which contained both subunits of the neurotoxic PLA 2 complex. The sub-module association of these toxins suggest common regulatory pathways underlie the variation in their expression and is consistent with known patterns of inheritance of similar haplotypes in other species. We also find evidence that module associated toxin families show fewer gene duplications and transcript losses between species, but module association did not appear to affect sequence diversification., Conclusion: Sub-modular regulation of expression likely contributes to the diversification of venom phenotypes within and among species and underscores the role of modularity in facilitating rapid evolution of complex traits.

This work reports the purification and biochemical and functional characterization of ACP-TX-I and ACP-TX-II, two phospholipases A 2 (PLA 2 ) from Agkistrodon contortrix pictigaster venom. Both PLA 2 s were highly purified by a single chromatographic step on a C 18 reverse phase HPLC column. Various peptide sequences from these two toxins showed similarity to those of other PLA 2 toxins from viperid snake venoms. ACP-TX-I belongs to the catalytically inactive K49 PLA 2 class, while ACP-TX-II is a D49 PLA 2 , and is enzymatically active. ACP-TX-I PLA 2 is monomeric, which results in markedly diminished myotoxic and inflammatory activities when compared with dimeric K49 PLA 2 s, confirming the hypothesis that dimeric structure contributes heavily to the profound myotoxicity of the most active viperid K49 PLA 2 s. ACP-TX-II exhibits the main pharmacological actions reported for this protein family, including in vivo local myotoxicity, edema-forming activity, and in vitro cytotoxicity. ACP-TX-I PLA 2 is cytotoxic to A549 lung carcinoma cells, indicating that cytotoxicity to these tumor cells does not require enzymatic activity.

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom 'Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species., (Copyright © 2019 Elsevier B.V. All rights reserved.)