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A useful approach to deepen our knowledge about the origin and evolution of venom systems in Reptilia has been exploring the vast biodiversity of this clade of vertebrates in search of orally produced proteins with toxic actions, as well as their corresponding delivery systems. The occurrence of toxins in anguimorph lizards has been demonstrated experimentally or inferred from reports of the toxic effects of the oral secretions of taxa within the Varanidae and Helodermatidae families. In the present study, we have focused on two alligator lizards of the Anguidae family, the Mexican alligator lizard, Abronia graminea, and the red-lipped arboreal alligator lizard, A. lythrochila. In addition, the fine morphology of teeth of the latter species is described. The presence of a conserved set of proteins, including B-type natriuretic peptides, cysteine-rich secretory proteins, group III phospholipase A2, and kallikrein, in submandibular gland extracts was demonstrated for both Abronia species. These proteins belong to toxin families found in oral gland secretions of venomous reptile species. This finding, along with previous demonstration of toxin-producing taxa in both paleo- and neoanguimorpha clades, provides further support for the existence of a handful of conserved toxin families in oral secretions across the 100+ million years of Anguimorpha cladogenesis.

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Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.

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For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.

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Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

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We report the first proteomics analyses of the venoms of two poorly studied snakes, the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to South Pacific Costa Rica and western Panamá. These venom proteomes share a conserved compositional pattern reported in four other congeneric species within the clade of South American Porthidium species, P. nasutum, P. lansbergii, P. ophryomegas, and P. porrasi. The paraspecific immunorecognition profile of antivenoms produced in Costa Rica (ICP polyvalent), Perú (Instituto Nacional de Salud) and Brazil (soro antibotrópico pentavalente, SAB, from Instituto Butantan) against the venom of P. arcosae was investigated through a third-generation antivenomics approach. The maximal venom-binding capacities of the investigated antivenoms were 97.1 mg, 21.8 mg, and 25.7 mg of P. arcosae venom proteins per gram of SAB, ICP, and INS-PERU antibody molecules, respectively, which translate into 28.4 mg, 13.1 mg, and 15.2 mg of total venom proteins bound per vial of SAB, ICP, and INS-PERU AV. The antivenomics results suggest that 21.8%, 7.8% and 6.1% of the SAB, ICP, and INS-PERU antibody molecules recognized P. arcosae venom toxins. The SAB antivenom neutralized P. arcosae venom's lethality in mice with an ED50 of 31.3 mgV/g SAB AV. This preclinical neutralization paraspecificity points to Brazilian SAB as a promising candidate for the treatment of envenomings by Ecuadorian P. arcosae. BIOLOGICAL SIGNIFICANCE: Assessing the preclinical efficacy profile of antivenoms against homologous and heterologous medically relevant snake venoms represents an important goal towards defining the biogeographic range of their clinical utility. This is particularly relevant in regions, such as Mesoamerica, where a small number of pharmaceutical companies produce antivenoms against the venoms of a small number of species of maximum medical relevance among the local rich herpetofauna, leaving a wide range of snakes of secondary medical relevance, but also causing life-threatening human envenomings without nominal clinical coverage. This work is part of a larger project aiming at mapping the immunological characteristics of antivenoms generated in Latin American countries towards venoms of such poorly studied snakes of the local and neighboring countries' herpetofauna. Here we report the proteomics characterization of the Manabi hognosed pitviper Porthidium arcosae endemic to the western coastal province of Manabí (Ecuador), and the Costa Rican hognosed pitviper P. volcanicum with distribution restricted to southwestern Costa Rica, the antivenomics assessment of three bothropoid commercial antivenoms produced in Costa Rica, Perú, and Brazil against the venom components of P. arcosae, and the in vivo capacity of the Brazilian soro antibotrópico pentavalente (SAB) from Instituto Butantan to neutralize the murine lethality of P. arcosae venom. The preclinical paraspecific ED50 of 31.3 mg of P. arcosae venom per gram of antivenom points to Brazilian SAB as a promising candidate for the treatment of envenomings by the Manabi hognosed pitviper P. arcosae.

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BACKGROUND: Bothrops asper represents the clinically most important snake species in Central America and Northern South America, where it is responsible for an estimated 50-80% of snakebites. Compositional variability among the venom proteomes of B. asper lineages across its wide range mirrors clinical differences in their envenomings. Bothropic antivenoms generated in a number of Latin American countries commonly exhibit a certain degree of paraspecific effectiveness in the neutralization of congeneric venoms. Defining the phylogeographic boundaries of an antivenom's effectivity has implications for optimizing its clinical use. However, the molecular bases and impact of venom compositions on the immune recognition and neutralization of the toxic activities of across geographically disparate populations of B. asper lineages has not been comprehensively studied. METHODOLOGY/PRINCIPAL FINDINGS: Third-generation antivenomics was applied to quantify the cross-immunorecognizing capacity against the individual components of venoms of three B. asper lineages (B. asper (sensu stricto), B. ayerbei and B. rhombeatus) distributed in south-western (SW) Colombia, of six Latin American antivenoms, produced against homologous (Colombia, INS-COL and PROBIOL) and Costa Rica (ICP)), and heterologous (Argentina (BIOL), Perú (INS-PERU) and Venezuela (UCV)) bothropic venoms. In vivo neutralization assays of the lethal, hemorrhagic, coagulant, defibrinogenating, myotoxic, edematogenic, indirect hemolytic, and proteolytic activities of the three SW Colombian B. asper lineage venoms were carried to compare the preclinical efficacy of three (Colombian INS-COL and PROBIOL, and Costa Rican ICP) antivenoms frequently used in Colombia. Antivenomics showed that all the six antivenom affinity matrices efficiently immunoretained most of the B. asper lineages venom proteins and exhibited impaired binding towards the venoms' peptidomes. The neutralization profile of the INS-COL, PROBIOL and ICP antivenoms towards the biological activities of the venoms of SW Colombian B. asper (sensu stricto), B. ayerbei and B. rhombeatus lineages was coherent with the antivenomics outcome. In addition, the combination of in vitro (antivenomics) and in vivo neutralization results allowed us to determine their toxin-specific and venom neutralizing antibody content. Noteworthy, heterologous INS-PERU, BIOL, and UCV bothropic antivenoms had equal or higher binding capacity towards the venoms components of SW Colombian B. asper lineages that the homologous Colombian and Costa Rican antivenoms. CONCLUSIONS/SIGNIFICANCE: The combined in vitro and in vivo preclinical outcome showed that antivenoms manufactured in Colombia and Costa Rica effectively neutralize the major toxic activities of SW Colombian B. asper lineage venoms. The antivenomics profiles of the heterologous antivenoms manufactured in Argentina, Venezuela, and Perú strongly suggests their (pre)clinical adequacy for the treatment of B. asper lineage envenomings in SW Colombia. However, their recommendation in the clinical setting is pending on in vivo neutralization testing and clinical testing in humans. Bothrops asper is a highly adaptable snake species complex, which is considered the most dangerous snake throughout much of its distribution range from the Atlantic lowland of eastern México to northwestern Perú. Antivenoms are the only scientifically validated treatment of snakebite envenomings. Venom variation is particularly common in wide ranging species, such as B. asper, and may result in variable clinical presentations of envenomings, as is the case for the B. asper species complex, potentially undermining the efficacy of snakebite treatments depending on the immunization mixture used in the generation of the antivenom. Conversely, phylogenetic conservation of antigenic determinants confers an unpredictable degree of paraspecificity to homologous antivenoms produced for a geographic area, but also to heterologous congeneric antivenoms, towards the venom components of allopatric conspecific populations. This work aimed at comparing the preclinical profile of a panel of Latin American homologous and heterologous antivenoms against the venoms of B. asper lineages distributed in SW Colombia. The outcome of this study strongly suggests the suitability of considering the heterologous antivenoms BIOL (Argentina), UCV (Venezuela) and INS-PERU (Perú) as alternatives to homologous Colombian INS-COL and PROBIOL and Costa Rican ICP antivenoms for the treatment of envenomings by B. asper (sensu stricto) in W Colombia and Ecuador, B. ayerbei in Cauca and Nariño (Colombia), and B. rhombeatus in Cauca river valley, SW Colombia.

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This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

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Bothrops asper is a venomous pitviper that is widely distributed and of clinical importance in Mesoamerica and northern South America, where it is responsible for 50-80% of all envenomations by Viperidae species. Previous work suggests that B. asper has a complex phylogeographic structure, with the existence of multiple evolutionarily distinct lineages, particularly in the inter-Andean valleys of north South America. To explore the impact of the evolutionary history of B. asper on venom composition, we have investigated geographic variation in the venom proteome of this species from the populations from the Pacific side of Ecuador and south-western Colombia. Among the 21 classes of venom components identified, proteins from mainly four major toxin families, snake venom metalloproteases (PI- and PII-SVMP), phospholipases A2 (K49- and D49-PLA2s), serine proteinases (SVSP), and C-type lectins-like (CTL) proteins are major contributors to the geographic variability in venom. Principal component analyses demonstrate significant differences in venom composition between B. asper lineages previously identified through combination of molecular, morphological and geographical data, and provide additional insights into the selection pressures modulating venom phenotypes on a geographic scale. In particular, altitudinal zonation within the Andean mountain range stands out as a key ecological factor promoting diversification in venom. In addition, the pattern of distribution of PLA2 molecules among B. asper venoms complements phylogenetic analysis in the reconstruction of the dispersal events that account for the current biogeographic distribution of the present-day species' phylogroups. Ontogenic variation was also evident among venoms from some Ecuadorian lineages, although this age-related variation was less extreme than reported in B. asper venoms from Costa Rica. The results of our study demonstrate a significant impact of phylogenetic history on venom composition in a pitviper and show how analyses of this variation can illuminate the timing of the cladogenesis and ecological events that shaped the current distribution of B. asper lineages. BIOLOGICAL SIGNIFICANCE: Bothrops asper, called "the ultimate pitviper" due to its defensive behavior, large body size, and medical importance, represents a species complex that is widely distributed from southern México southwards across north-western South America to north-western Perú. This work reports the characterization of the venom proteomes of B. asper lineages from the Pacific sides of Ecuador and south-western Colombia. Multivariate analyses indicate that variability in venom composition among the B. asper lineages is driven by proteins from four major toxin families, presumably in response to selection pressures created by recent and historical ecological conditions created by geological and climatic events from the Pliocene-Pleistocene to the present along the Central and South American Continental Divide. The emerging biogeographic pattern of venom variation, interpreted in the context of the current phylogenetic hypotheses, support and complement previously proposed evolutionary Plio-Pleistocene dispersal events that shaped the present-day distribution range of B. asper lineages. In addition, our venomics data indicate the occurrence of genetic exchange between Colombian and Pacific Costa Rican populations, which may have occurred during the second wave of B. asper migration into Mesoamerica. Our work represents a foundation for a future broader sampling and more complete "-omics" analyses to deepen our understanding of the patterns and causes of venom variation in this medically important pitviper.

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We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.

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