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New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Anthropogenic activities are the main sources of soil, air, and water pollution by metals, including cadmium (Cd), lead (Pb), chromium (Cr), the metalloid arsenic (As), magnesium (Mg), zinc (Zn), and copper (Cu). The goal of this study was to assess the presence and concentration of toxic (As, Cd, Pb, and Cr) and essential metals (Mg, Zn, and Cu) in the liver and kidneys from 96 free-ranging rattlesnakes (Crotalus durissus) from Minas Gerais (Brazil). Bioaccumulation of Cd and Pb were significantly higher in males and heavier rattlesnakes (those with body weight above the average of the study population). Average ± standard deviations of Cd, Pb, Cr, Cu, Mg, Zn, and As in the general population (n = 96) were 3.19 ± 2.52; 5.98 ± 8.49; 0.66 ± 1.97; 3.27 ± 2.85; 776.14 ± 2982.92; 27.44 ± 29.55; and 0.32 ± 1.46; respectively. Bioaccumulation of some metals correlated positively with changes in hematologic and serum biochemical parameters. Results of this study were contrasted with previous studies assessing metal bioaccumulation in other species of terrestrial or aquatic snakes. Considering their position in the food chain and the broad range of bioaccumulation of both toxic and essential metals observed in this study, rattlesnakes may function as highly relevant biological sentinels for environmental pollution., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

AbstractTemperate reptiles are often considered to be low-energy systems, with their discrete use of time and energy making them model systems for the study of time-energy budgets. However, the semifrequent replacement and sloughing of the epidermis is a ubiquitous feature of squamate reptiles that is often overlooked when accounting for time and energy budgets in these animals. We used open-flow respirometry to measure both the energetic effort of ecdysis and the duration of the associated metabolic upregulation (likely related to behavioral changes often reported for animals in shed) in wild-caught timber rattlesnakes ( Crotalus horridus ). We hypothesized that total effort of skin biosynthesis and physical removal would be related to body mass and expected the duration of the process to remain static across individuals at a fixed temperature (25°C). We provide both the first measurements of the cost of skin biosynthesis and physical removal in a reptile and the highest-resolution estimate of process duration recorded to date. We found that skin biosynthesis, but not the cost of physical removal of the epidermis, was related to body mass. Shed cycle duration was consistent across individuals, taking nearly 4 wk from process initiation to physical removal of the outermost epidermal layer. Total energetic effort of ecdysis was of sizeable magnitude, requiring ∼3% of the total annual energy budget of a timber rattlesnake. Energetic effort for a 500-g snake was equivalent to the amount of metabolizable energy acquired from the consumption of approximately two adult mice. Ecdysis is a significant part of the time-energy budgets of snakes, necessitating further attention in studies of reptilian energetics.

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.

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA 2 . Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms., 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 B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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.

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.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

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.)

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA 4 . These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA 4 is a key mediator of the immunosuppressive effects of CTX., 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.)

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.

The growing problem of antibiotic resistance among bacteria requires searching for new therapeutic agents with bacteriostatic and/or bactericidal properties. Crotoxin is a β-neurotoxin from the venom of the Crotalus durissus terrificus. It is composed of two subunits: CA (non-active) and CB (with phospholipase A 2 activity). It has already been shown that the isolated CB, but not the CA, subunit of crotoxin exhibits an antibacterial activity towards a variety of Gram-positive and Gram-negative bacterial species. However, no studies on the whole crotoxin complex have been carried out so far. We tested the antibacterial properties of crotoxin, as well as its isolated CB subunit, towards Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6535, Micrococcus luteus ATCC 10240, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 10145. Both toxins exhibited antibacterial properties only against Micrococcus luteus ATCC 10240. Crotoxin showed only bacteriostatic activity with a MIC of 46 µM, while the CB subunit acted as both a bacteriostatic and bactericidal agent with a MIC = MBC = 0.21 μM. The bacteriostatic effect of the toxins was independent of the enzymatic activity of the CB subunit. Bactericidal properties, however, require phospholipase A 2 activity. Both toxins reduced bacteria viability at the MIC by 72% and 85% for crotoxin- and CB-treated bacteria, respectively. The membrane permeability increased approximately three times within the first hour of incubation with toxins; afterwards, either no significant changes or a decrease of membrane permeability, compared to the control cells, were observed. We isolated a single, approximately 30 kDa bacterial wall protein which belongs to the NlpC/P60 family that interacts with crotoxin leading to the inhibition of bacterial growth. Neither crotoxin nor the CB subunit showed any cytotoxic properties to human fibroblasts at the MIC during the three-day incubation.

Crotoxin (CTX), the major toxin of Crotalus durissus terrificus snake venom, induces an inhibitory effect on tumor development and modulates the functions of macrophages (MØs), which play a key role as a defense mechanism against tumor growth. In early tumor progression stage, MØs are avidly phagocytic (inflammatory cell), releasing reactive nitrogen intermediates-RNI/ROI and cytokines TNF-α, IL-1β, and IL-6. However, when the tumor has been developed, tumor-associated MØ (angiogenic cell) presents a decrease in the mentioned activities. We reported that CTX stimulates H 2 O 2 release, NO production and secretion of cytokines by peritoneal MØs obtained from non-tumor-bearing rats. Considering that the mentioned mediators control tumor growth, it is mandatory to investigate whether CTX stimulates the production of these mediators by MØs obtained from tumor-bearing animals. The aim of this work was then to evaluate the CTX effect on metabolism and functions of peritoneal MØs obtained from Walker 256 tumor-bearing rats. For this purpose, male Wistar rats were subcutaneously inoculated in the right flank with 1 mL sterile suspension of 2 × 10 7 Walker 256 tumor cells. CTX (18 μg per animal) was subcutaneously administered in two protocols: a) on the 1st day of tumor cell injection and b) on the 4th day of tumor cell inoculation. In both protocols, MØs were obtaining on the 14th day of tumor cell inoculation to evaluate the release of H 2 O 2, NO, and pro-inflammatory cytokines (IL-1β, TNFα, and IL-6); maximal activity of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase, and 14 CO 2 production from [U- 14 C]-glucose and [U- 14 C]-glutamine. The treatment with CTX stimulated the release of NO, H 2 O 2 , and cytokines, and glucose and glutamine metabolism. Metabolic and functional changes induced by CTX were accompanied by a decrease of tumor growth as indicated by tumor fresh weight and diameter. These results indicate CTX not only as a scientific tool to investigate changes in metabolism and functions of peritoneal MØs but also for a better understanding of the mechanisms involved in tumor growth., 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.)

Objective: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis., Methods: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated., Results: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile., Conclusion: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Phosphodiesterases (PDEs) constitute an enzyme group able to hydrolyze nucleic acids as well as some second messengers. Due to this ability and their expression in several human tissues and organs, PDEs can control a gamut of physiological processes. They are also involved in some pathological conditions, such as Alzheimer's disease and erectile dysfunction. PDEs are also expressed in snake venom glands, being called snake venoms phosphodiesterases, or simply svPDEs. The occurrence of these enzymes has already been reported in crotalid, elapid and viperid venoms, such as Crotalus, Naja and Trimeresurus, respectively, but not all of them have been characterized concerning their structure, activity and function. In this review, we are addressing general characteristics of svPDEs, in addition to their structural, biochemical and functional characteristics, and we also report some potential applications of svPDEs., 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.)

Rationale: The endocannabinoid modulation of fear and anxiety due to the on-demand synthesis and degradation is supported by a large body of research. Although it has been proposed that anandamide (AEA) in the substantia nigra pars reticulata (SNpr) seems to be important for the organisation of innate fear-related behaviours, a role for endogenous AEA has yet to be clarified., Methods: Mice were treated with the fatty acid amide hydrolase (FAAH) selective inhibitor URB597 at different concentrations (0.01, 0.1, 1 nmol/0.1 µL) in the SNpr and confronted by rattlesnakes (Crotalus durissus terrificus). The most effective dose of URB597 (1 nmol) was also preceded by microinjections of the CB 1 receptor antagonist AM251 (0.1 nmol) into the SNpr, and mice were then confronted by the venomous snake., Results: URB597 (0.1 and 1 nmol) in the SNpr decreased the expression of defensive behaviours such as defensive attention, escape, and time spent inside the burrow of mice confronted by rattlesnakes. Moreover, pretreatment of SNpr with AM251 suppressed these antiaversive effects of URB597 in this midbrain structure., Conclusion: Overall, these data clearly indicate that the panicolytic consequences of endogenous AEA enhancement in the SNpr are mediated by CB1 receptor signalling., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Snake venom L-Amino-acid oxidase (svLAAO) has become a critical research target in molecular biology and medical science since its widespread presence and diverse biological roles, including antitumor application. Our research confirmed that Crotalus adamanteus (C. adamanteus) venom LAAO exhibited potential anti-ovarian cancer activity both in vivo and in vitro. C. adamanteus venom LAAO significantly reduced viability of ovarian cancer cells and caused morphological changes that preceded cell death. The results of molecular biology experiments showed that C. adamanteus venom LAAO caused expression changes of genes related to apoptotic pathways either intrinsically or extrinsically in ovarian cancer cells. Animal experiments and histological analysis also proved that C. adamanteus venom LAAO could effectively inhibit the tissue damage caused by ovarian cancer, and animals treated with C. adamanteus venom LAAO showed higher survival time. Catalase blocked the major apoptosis induction of C. adamanteus venom LAAO on ovarian cancer cells, suggesting that the cytotoxicity of C. adamanteus venom LAAO on ovarian cancer cells was mainly mediated by H 2 O 2 . These results infer that C. adamanteus venom LAAO will have some advantages in new drug research and antitumor drug development in future., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 envenomation is a common but neglected disease that affects millions of people around the world annually. Among venomous snake species in Brazil, the tropical rattlesnake (Crotalus durissus terrificus) accounts for the highest number of fatal envenomations and is responsible for the second highest number of bites. Snake venoms are complex secretions which, upon injection, trigger diverse physiological effects that can cause significant injury or death. The components of C. d. terrificus venom exhibit neurotoxic, myotoxic, hemotoxic, nephrotoxic, and cardiotoxic properties which present clinically as alteration of central nervous system function, motor paralysis, seizures, eyelid ptosis, ophthalmoplesia, blurred vision, coagulation disorders, rhabdomyolysis, myoglobinuria, and cardiorespiratory arrest. In this study, we focused on proteomic characterization of the cardiotoxic effects of C. d. terrificus venom in mouse models. We injected venom at half the lethal dose (LD50) into the gastrocnemius muscle. Mouse hearts were removed at set time points after venom injection (1 h, 6 h, 12 h, or 24 h) and subjected to trypsin digestion prior to high-resolution mass spectrometry. We analyzed the proteomic profiles of >1300 proteins and observed that several proteins showed noteworthy changes in their quantitative profiles, likely reflecting the toxic activity of venom components. Among the affected proteins were several associated with cellular deregulation and tissue damage. Changes in heart protein abundance offer insights into how they may work synergistically upon envenomation. SIGNIFICANCE: Venom of the tropical rattlesnake (Crotalus durissus terririficus) is known to be neurotoxic, myotoxic, nephrotoxic and cardiotoxic. Although there are several studies describing the biochemical effects of this venom, no work has yet described its proteomic effects in the cardiac tissue of mice. In this work, we describe the changes in several mouse cardiac proteins upon venom treatment. Our data shed new light on the clinical outcome of the envenomation by C. d. terrificus, as well as candidate proteins that could be investigated in efforts to improve current treatment approaches or in the development of novel therapeutic interventions in order to reduce mortality and morbidity resulting from envenomation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.)

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1β induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1β secretion. Overall, this work helps elucidate the function of CVX in immune system cells., (© 2022. The Author(s).)

To characterise the effect of two common induction agents, propofol and alfaxalone, on mean arterial blood pressure (MAP) and heart rate (HR), we equipped 19 adult South American rattlesnakes (Crotalus durissus) with an indwelling arterial catheter approximately 24 h prior to recording of baseline resting values. Then, seven snakes received alfaxalone (15 mg kg -1 ) intravascularly (IV) through the catheter, while groups two and three (both n = 6) received propofol (15 mg kg -1 IV). The first two groups were not handled, while the group 3 was manually restrained for 2 min for a mock injection of 0.2 ml saline into the ventral tail vein. Baseline HR was similar in all groups and handling caused a significant tachycardia (p = 0.031) in group three. When given IV to undisturbed animals, both propofol and alfaxalone induced a significant increase in HR (p = 0.0022 and p = 0.0045, respectively) lasting approximately 30 min, but with values only significantly exceeding baseline for the first 5 min for propofol and the first 10 min with alfaxalone. Handling caused a significant increase in MAP (p = 0.0313). Propofol did not affect MAP (p = 0.1064), while alfaxalone caused a marked hypertension (although only significant at 2 min; p = 0.031). Manual restraint significantly increases both HR and MAP, which may lead to a masking of true cardiovascular effects of anaesthetic agents., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Ctn[15-34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15-34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15-34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

l-Amino acid oxidase (LAAO) is widely distributed in nature and shows important biological activity. It induces cell apoptosis and has antibacterial properties. This study was designed to investigate the effect of methyl substituent on its activity as methylated derivatives of l-tyrosine, labelled with short-lived B+ emitters, have been used in oncological diagnostics. To study isotope effects in the oxidative deamination of O-methyl-l-tyrosine, the deuterated isotopomer, i.e. O-methyl-[2-2H]-l-tyrosine, was synthesized by isotope exchange, catalyzed enzymatically by tryptophanase. Isotope effects were determined using the spectrophotometric non-competitive method. The values of isotope effects indicate that the α-C-H bond cleavage occurs in the rate determining step of the investigated reaction and α-hydrogen plays a role in the substrate binding process at the enzyme active site. The inhibitory effect on LAAO activity was studied with α-methyl-l-tyrosine and N-methyl-l-tyrosine. The mode of inhibition was determined based on Lineweavear-Burk plots intersections. α-Methyl-l-tyrosine has been found a mixed type inhibitor of the investigated enzyme, whereas N-methyl-l-tyrosine is a non-competitive inhibitor of LAAO., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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.

The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A 2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom., (Copyright © 2020. Published by Elsevier Ltd.)

Using venom for predation often leads to the evolution of resistance in prey. Understanding individual variation in venom resistance is key to unlocking basic mechanisms by which antagonistic coevolution can sustain variation in traits under selection. For prey, the opposing challenges of predator avoidance and resource acquisition often lead to correlated levels of risk and reward, which in turn can favor suites of integrated morphological, physiological and behavioral traits. We investigate the relationship between risk-sensitive behaviors, physiological resistance to rattlesnake venom, and stress in a population of California ground squirrels. For the same individuals, we quantified foraging decisions in the presence of snake predators, fecal corticosterone metabolites (a measure of "stress"), and blood serum inhibition of venom enzymatic activity (a measure of venom resistance). Individual responses to snakes were repeatable for three measures of risk-sensitive behavior, indicating that some individuals were consistently risk-averse whereas others were risk tolerant. Venom resistance was lower in squirrels with higher glucocorticoid levels and poorer body condition. Whereas resistance failed to predict proximity to and interactions with snake predators, individuals with higher glucocorticoid levels and in lower body condition waited the longest to feed when near a snake. We compared alternative structural equation models to evaluate alternative hypotheses for the relationships among stress, venom resistance, and behavior. We found support for stress as a shared physiological correlate that independently lowers venom resistance and leads to squirrels that wait longer to feed in the presence of a snake, whereas we did not find evidence that resistance directly facilitates latency to forage. Our findings suggest that stress may help less-resistant squirrels avoid a deadly snakebite, but also reduces feeding opportunities. The combined lethal and non-lethal effects of stressors in predator-prey interactions simultaneously impact multiple key traits in this system, making environmental stress a potential contributor to geographic variation in trait expression of toxic predators and resistant prey.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes ( Crotalus ornatus , C. molossus nigrescens , C. scutulatus, and C. atrox ) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Transient receptor potential ankyrin 1 from rattlesnakes (rsTRPA1) and boas (bTRPA1) was previously proposed to underlie thermo-sensitive infrared sensing based on transcript enrichment in infrared-sensing neurons and hyper-thermosensitivity expressed in Xenopus oocytes. It is unknown how these TRPA1s show thermosensitivities that overwhelm other thermoreceptors, and why rsTRPA1 is more thermosensitive than bTRPA1. Here, we show that snake TRPA1s differentially require Ca 2+ for hyper-thermosensitivity and that predisposition to cytosolic Ca 2+ potentiation correlates with superior thermosensitivity. Extracellularly applied Ca 2+ upshifted the temperature coefficients (Q10s) of both TRPA1s, for which rsTRPA1, but not bTRPA1, requires cytosolic Ca 2+ . Intracellular Ca 2+ chelation and substitutive mutations of the conserved cytosolic Ca 2+ -binding domain lowered rsTRPA1 thermosensitivity comparable to that of bTRPA1. Thapsigargin-evoked Ca 2+ or calmodulin little affected rsTRPA1 activity or thermosensitivity, implying the importance of precise spatiotemporal action of Ca 2+ . Remarkably, a single rattlesnake-mimicking substitution in the conserved but presumably dormant cytosolic Ca 2+ -binding domain of bTRPA1 substantially enhanced thermosensitivity through cytosolic Ca 2+ like rsTRPA1, indicating the capability of this single site in the determination of both cytosolic Ca 2+ dependence and thermosensitivity. Collectively, these data suggest that Ca 2+ is essential for the hyper-thermosensitivity of these TRPA1s, and cytosolic potentiation by permeating Ca 2+ may contribute to the natural variation of infrared senses between rattlesnakes and boas.

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.)

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox . Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

A global public health crisis has emerged with the extensive dissemination of multidrug-resistant microorganisms. Antimicrobial peptides (AMPs) from plants and animals have represented promising tools to counteract those resistant pathogens due to their multiple pharmacological properties such as antimicrobial, anticancer, immunomodulatory and cell-penetrating activities. In this review, we will focus on crotamine and crotalicidin, which are two interesting examples of membrane active peptides derived from the South America rattlesnake Crotalus durrisus terrificus venom. Their full-sequences and structurally-minimized fragments have potential applications, as anti-infective and anti-proliferative agents and diagnostics in medicine and in pharmaceutical biotechnology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A 2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE 2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE 2 , PGD 2 , and TXB 2 ). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE 2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Ana Eduarda Zulim de Carvalho et al.)

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies., (© 2019 Wiley Periodicals, Inc.)

Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498 amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Traits can evolve rapidly through changes in gene expression or protein-coding sequences. However, these forms of genetic variation can be correlated and changes to one can influence the other. As a result, we might expect traits lacking differential expression to preferentially evolve through changes in protein sequences or morphological adaptation. Given the lack of differential expression across the distribution of sidewinder rattlesnakes ( Crotalus cerastes), we tested this hypothesis by comparing the coding regions of genes expressed in the venom gland transcriptomes and fang morphology. We calculated Tajima's D and F ST across four populations comparing toxin and nontoxin loci. Overall, we found little evidence of directional selection or differentiation between populations, suggesting that changes to protein sequences do not underlie the evolution of sidewinder venom or that toxins are under extremely variant selection pressures. Although low-expression toxins do not have higher sequence divergence between populations, they do have more standing variation on which selection can act. Additionally, we found significant differences in fang length among populations. The lack of differential expression and sequence divergence suggests sidewinders-given their generalist diet, moderate gene flow and environmental variation-are under stabilizing selection which functions to maintain a generalist phenotype. Overall, we demonstrate the importance of examining the relationship between gene expression and protein-coding changes to understand the evolution of complex traits.

Ellagitannins constitute the largest group of hydrolyzable tannins of plants, and, from this group, casuarictin (Casu) was identified in some plant species. However, to our knowledge, no investigation of secretory phospholipase A2 (sPLA2) inhibition by Casu has been performed yet. Casuarictin was isolated by chromatography n-butanol (n-BuOH) partition of Laguncularia racemosa leaves. The pharmacological and biological effects of Casu were evaluated on isolated sPLA2 from the rattlesnake ( Crotalus durissus terrificus ) and using a plant bacterial strain. The compound was able to form a protein complex consisting of a stable sPLA2 + Casu complex. Analyses carried out with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) revealed that the molecular mass of sPLA2 increased from 14,425.62 to 15,362.74 Da. The enzymatic activity of the sPLA2 + Casu complex was significantly lower than that of native sPLA2. Besides, molecular interactions of Casu with sPLA2 were able to virtually abolish the native edematogenic effect as well as myonecrosis induced by the protein when injected 10 min after sPLA2. Therefore, Casu may be considered a potential anti-inflammatory that can be used to treat edema and myonecrosis induced by serine-secreting phospholipase A2. In addition, the compound also showed great antimicrobial potential.

While ectothermic organisms often experience considerable circadian variation in body temperature under natural conditions, the study of the effects of temperature on metabolic rates are traditionally based on subjecting animals to constant temperature regimes. Whether data resulting from constant-temperature experiments accurately predicts temperature effects under more natural fluctuating temperature regimes remains uncertain. To address such possibility, we measured the resting metabolic rates of the South American rattlesnakes (Crotalus durissus) under constant and circadian fluctuating thermal regimes in a range of temperatures. Metabolic rates measured at constant 20 °C and 25 °C did not differ from the rates measured at fluctuating regimes with corresponding mean temperatures. However, the difference between thermal regimes increased with temperature, with the metabolic rate measured at constant 30 °C being greater than that measured at the fluctuating thermal regime with corresponding mean temperature. Therefore, our results indicate that thermal regime effects on rattlesnakes' metabolism is dependent on temperature range. Broadly, our results highlight the importance of considering multi-factorial attributes of temperature variation in the exam of its effects over functional traits. Such approach provides a more solid support for inferences about temperature effects on the life history, ecology and conservation of ectothermic organisms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Endothelium is the main source of catecholamine release in the electrical-field stimulation (EFS)-induced aortic contractions of the non- venomous snake Panterophis guttatus. However, adrenergic vasomotor control in venomous snakes such as Crotalus durissus terrificus and Bothrops jararaca has not yet been investigated. Crotalus and Bothrops aortic rings were mounted in an organ bath system. EFS-induced aortae contractions were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) or tetrodotoxin (1 μM). Frequency-induced contractions were also performed in aortae with endothelium removed. Immunohistochemical localization of both tyrosine hydroxylase (TH) and S-100 protein in snake aortic rings and brains, as well as in human tissue (paraganglioma tumour) were carried out. EFS (4 to 16 Hz) induced frequency-dependent aortic contractions in both Crotalus and Bothrops. The EFS-induced contractions were significantly reduced in the presence of either guanethidine or phentolamine in both snakes (p<0.05), whereas tetrodotoxin had no effect in either. Removal of the endothelium abolished the EFS-induced contractions in both snakes aortae (p<0.05). Immunohistochemistry revealed TH localization in endothelium of both snake aortae and human vessels. Nerve fibers were not observed in either snake aortae. In contrast, both TH and S100 protein were observed in snake brains and human tissue. Vascular endothelium is the main source of catecholamine release in EFS-induced contractions in Crotalus and Bothrops aortae. Human endothelial cells also expressed TH, indicating that endothelium- derived catecholamines possibly occur in mammalian vessels., Competing Interests: The authors have declared that no competing interests exist.

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.

Proteins constitute almost 95% of snake venom's dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA 2 , LAAO, 5'nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Oxidation of low-density lipoproteins (LDLs) induces development of cardiovascular disease. Recently, reports of studies using atomic force microscopy (AFM) have described that the elastic modulus of metal-induced oxidized LDLs is lower than the modulus before oxidation. However, the mechanisms of change of the elastic modulus have not been well investigated. We postulated that disorder of the LDL structure might decrease the elastic modulus. This study measured the elastic modulus of LDLs before and after enzyme treatment with V8 protease, α-chymotrypsin, and phospholipase A 2 . After LDLs were obtained from serum by ultracentrifugation, LDLs or enzyme-treated LDLs were physically absorbed. They were crowded on a mica surface. Although V8 protease and α-chymotrypsin did not induce the elastic modulus change, treatment with PLA 2 decreased the elastic modulus. The LDL particle size did not change during the enzyme treatment. Results suggest that disordering of the lipid structure of the LDL might contribute to the elastic modulus change. Results show that AFM might be a useful tool to evaluate disorders of complex nanoscale particle structures from lipids and proteins such as lipoproteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence., (Copyright © 2017 Elsevier B.V. All rights reserved.)