ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING.

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 (TPP) 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 TPP 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.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

A plethora of rodents: Rattlesnake predators generate unanticipated patterns of venom resistance in a grassland ecosystem.

Predation has the potential to impart strong selective pressures on organisms within their environments, resulting in adaptive changes in prey that minimize risk of predation. Pressures from venomous snakes present an exceptional challenge to prey, as venom represents a unique chemical arsenal evolutionarily tailored to incapacitate prey. In response, venom resistance has been detected in various snake prey species, and to varying degrees. This study analyzes venom resistance in an eastern Colorado grassland habitat, where the Prairie Rattlesnake (Crotalus viridis) and Desert Massasauga Rattlesnake (Sistrurus tergeminus edwardsii) co-occur with a suite of grassland rodents. We test for venom resistance across rodent and snake pairings using two geographically distant field sites to determine the role of 1) predation pressure and trophic ecology, and 2) sympatric and allopatric patterns of venom resistance. Resistance was measured using serum-based metalloproteinase inhibition assays to determine potential inhibition of proteolytic activity, augmented by median lethal dose (LD50) assays on rodent species to assess toxicity of crude venoms. Resistance is present in several rodent species, with strong resistance present in populations of Eastern Woodrat (Neotoma floridana), Ord's Kangaroo Rat (Dipodomys ordii), and Northern Grasshopper Mouse (Onychomys leucogaster). Resistance is less developed in other species, including the House Mouse (Mus musculus) and Plains Pocket Mouse (Perognathus flavescens). An unexpected differential is present, where Lincoln County Kangaroo Rats are highly resistant to venom of co-occurring Prairie Rattlesnakes yet are sensitive to an allopatric population of Prairie Rattlesnakes in Weld County. Lincoln Co. Northern Grasshopper Mice also demonstrate extremely elevated resistance to Weld Co. Prairie Rattlesnake venoms, and they may possess resistance mechanisms for myotoxin a, an abundant component of Weld Co. C. v viridis venoms. This study illustrates the complexity of venom resistance in biological communities that can exist when incorporating multiple species interactions. Future studies aimed at characterizing resistance mechanisms at the molecular level will provide a more detailed physiological context for understanding mechanisms by which resistance to venoms occurs.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

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.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

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.

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Feasibility of detecting snake envenomation biomarkers from dried blood spots.

Biofluid proteomics is a sensitive and high throughput technique that provides vast amounts of molecular data for biomarker discovery. More recently, dried blood spots (DBS) have gained traction as a stable, noninvasive, and relatively cheap source of proteomic data for biomarker identification in disease and injury. Snake envenomation is responsible for significant morbidity and mortality worldwide; however, much remains unknown about the systemic molecular response to envenomation and acquiring biological samples for analysis is a major hurdle. In this study, we utilized DBS acquired from a case of lethal rattlesnake envenomation to determine the feasibility of discovering biomarkers associated with human envenomation. We identified proteins that were either unique or upregulated in envenomated blood compared to non-envenomated blood and evaluated if physiological response pathways and protein markers that correspond to the observed syndromes triggered by envenomation could be detected. We demonstrate that DBS provide useful proteomic information on the systemic processes that resulted from envenomation in this case and find evidence for a massive and systemic inflammatory cascade, combined with coagulation dysregulation, complement system activation, hypoxia response activation, and apoptosis. We also detected potential markers indicative of lethal anaphylaxis, cardiac arrest, and brain death. Ultimately, DBS proteomics has the potential to provide stable and sensitive molecular data on envenomation syndromes and response pathways, which is particularly relevant in low-resource areas which may lack the materials for biofluid processing and storage.

Evidence that genomic incompatibilities and other multilocus processes impact hybrid fitness in a rattlesnake hybrid zone.

Hybrid zones provide valuable opportunities to understand the genomic mechanisms that promote speciation by providing insight into factors involved in intermediate stages of speciation. Here, we investigate introgression in a hybrid zone between two rattlesnake species (Crotalus viridis and Crotalus oreganus concolor) that have undergone historical allopatric divergence and recent range expansion and secondary contact. We use Bayesian genomic cline models to characterize genomic patterns of introgression between these lineages and identify loci potentially subject to selection in hybrids. We find evidence for a large number of genomic regions with biased ancestry that deviate from the genomic background in hybrids (i.e., excess ancestry loci), which tend to be associated with genomic regions with higher recombination rates. We also identify suites of excess ancestry loci that show highly correlated allele frequencies (including conspecific and heterospecific combinations) across physically unlinked genomic regions in hybrids. Our findings provide evidence for multiple multilocus evolutionary processes impacting hybrid fitness in this system.

Origins, genomic structure and copy number variation of snake venom myotoxins.

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous ß-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

The roles of balancing selection and recombination in the evolution of rattlesnake venom.

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

The Rattlesnake W Chromosome: A GC-Rich Retroelement Refugium with Retained Gene Function Across Ancient Evolutionary Strata.

Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited Y and W chromosomes contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes are lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a "refugium" for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

Convergent evolution of toxin resistance in animals.

Convergence is the phenomenon whereby similar phenotypes evolve independently in different lineages. One example is resistance to toxins in animals. Toxins have evolved many times throughout the tree of life. They disrupt molecular and physiological pathways in target species, thereby incapacitating prey or deterring a predator. In response, molecular resistance has evolved in many species exposed to toxins to counteract their harmful effects. Here, we review current knowledge on the convergence of toxin resistance using examples from a wide range of toxin families. We explore the evolutionary processes and molecular adaptations driving toxin resistance. However, resistance adaptations may carry a fitness cost if they disrupt the normal physiology of the resistant animal. Therefore, there is a trade-off between maintaining a functional molecular target and reducing toxin susceptibility. There are relatively few solutions that satisfy this trade-off. As a result, we see a small set of molecular adaptations appearing repeatedly in diverse animal lineages, a phenomenon that is consistent with models of deterministic evolution. Convergence may also explain what has been called 'autoresistance'. This is often thought to have evolved for self-protection, but we argue instead that it may be a consequence of poisonous animals feeding on toxic prey. Toxin resistance provides a unique and compelling model system for studying the interplay between trophic interactions, selection pressures and the molecular mechanisms underlying evolutionary novelties.

Venom production and secretion in reptiles.

The venom glands of reptiles, particularly those of front-fanged advanced snakes, must satisfy conflicting biological demands: rapid synthesis of potentially labile and highly toxic proteins, storage in the gland lumen for long periods, stabilization of the stored secretions, immediate activation of toxins upon deployment and protection of the animal from the toxic effects of its own venom. This dynamic system could serve as a model for the study of a variety of different phenomena involving exocrine gland activation, protein synthesis, stabilization of protein products and secretory mechanisms. However, these studies have been hampered by a lack of a long-term model that can be propagated in the lab (as opposed to whole-animal studies). Numerous attempts have been made to extend the lifetime of venom gland secretory cells, but only recently has an organoid model been shown to have the requisite qualities of recapitulation of the native system, self-propagation and long-term viability (>1 year). A tractable model is now available for myriad cell- and molecular-level studies of venom glands, protein synthesis and secretion. However, venom glands of reptiles are not identical, and many differ very extensively in overall architecture, microanatomy and protein products produced. This Review summarizes the similarities among and differences between venom glands of helodermatid lizards and of rear-fanged and front-fanged snakes, highlighting those areas that are well understood and identifying areas where future studies can fill in significant gaps in knowledge of these ancient, yet fascinating systems.

Insights on the inhibition properties of Jatromollistatin (a cyclic heptapeptide) against Crotalus adamanteus metalloendopeptidase using molecular docking analysis.

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites.

Integration of transcriptomic and proteomic approaches for snake venom profiling.

INTRODUCTION: Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. AREAS COVERED: In this review, we highlight several examples, from both published and unpublished work in our lab, demonstrating how a combined venom gland transcriptome and proteome methodology allows for comprehensive characterization of venoms, including those from understudied rear-fanged snake species, and we provide recommendations for using these approaches. EXPERT OPINION: When characterizing venoms, peptide mass fingerprinting using databases built predominately from protein sequences originating from model organisms can be disadvantageous, especially when the intention is to document protein diversity. Therefore, the use of species-specific venom gland transcriptomes corrects for the absence of these unique peptide sequences in databases. The integration of transcriptomics and proteomics improves the accuracy of either approach alone for venom profiling.

Prevention and improvement of clinical management of snakebite in Southern Asian countries: A proposed road map.

In the Southern Asian countries, snakebite takes a substantial toll in terms of human life, inflicts acute morbidity and long term disability both physical and psychological, and therefore represents a neglected socio-economic problem and severe health issue that requires immediate medical attention. The 'Big Four' venomous snakes, viz. Daboia russelii, Naja naja, Bungarus caeruleus and Echis carinatus, are prominent, medically important species and are the most dangerous snakes of this region; therefore, the commercial polyvalent antivenom (PAV) contains antibodies against the venoms of these snakes. However, envenomations by species other than the 'Big Four' snakes are grossly neglected, and PAV is only partially effective in neutralizing the venom of these snakes. Many issues confounding effective treatment of snakebite are discussed in this review, and these hurdles preventing successful treatment of snakebite must be addressed. However, in South Asian countries, the pre-hospital treatment and appropriate first aid are equally important to mitigate the problem of snakebite and therefore, these issues are also highlighted here. Further, this review suggests a roadmap and guidelines for the prevention of snakebite and improvement of hospital management of snakebite in these Southern Asian countries.

Drought, desertification and poverty: A geospatial analysis of snakebite envenoming in the Caatinga biome of Brazil.

Epidemiological data on snakebite in the Brazilian state of Ceará are scarce, as the only report on this subject was last published in 1997. However, according to the Brazilian system of recording disease incidents (Sistema de Informação de Agravos de Notificação [SINAN]), more than 13,000 snakebites have been registered since 2001 in the state of Ceará, making this disease an important public health issue. In the present study, we evaluate the influence of environmental changes, including drought and desertification, on the risk of snakebite envenoming in the Brazilian northeastern state of Ceará. We compare public data on snakebites from Brazilian Epidemiological Surveillance System (DATASUS), rainfall records, advanced desertification maps, pastures and socioeconomic information of the 184 municipals in Ceará between 2001 and 2017. During the period of investigation, 8,945 snakebites were recorded, the majority (93.8%) of which involved venomous snakes. Almost half of the municipals (48%) had 100 incidences or more per 100,000 inhabitants. Data collected also highlight month-to-month occurrences of snakebites, with trends to rise shortly after the onset of precipitation, peaking in July and then trending downward as rainfall decreases, reaching the lowest level in December. We deduce an inverse relationship between Human Development Index (HDI) and snakebites per area. Spearman correlation and principal component analysis support the hypothesis that water scarcity and desertification are linked to increased risk of snakebite envenoming. Our study indicates that besides poverty, dry and desertified areas represent risk factors associated with increased incidence of snakebite envenoming in the state of Ceará.