Proteomic analysis and Lethality of the venom of Aegaeobuthus nigrocinctus, a scorpion of medical significance in the Middle East.

The scorpion Aegaeobuthus nigrocinctus inhabits areas in Turkey and the Levant region of the Middle East where severe/lethal envenomings have been reported. Previous research indicated its extreme venom lethality to vertebrates and distinct envenomation syndrome. We report on the composition of A. nigrocinctus venom from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality in mice was also assessed (LD50 = 1.05 (0.19-1.91) mg/kg, i.p), confirming A. nigrocinctus venom toxicity from Levantine populations. Forty-seven peaks were resolved using RP-HPLC, 25 of which eluted between 20-40% acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 19.6, 26.1, 46.3 and 57.7 kDa. MALDI-TOF venom fingerprinting detected 20 components within the 1,000-12,000 m/z range. Whole venom 'shotgun' bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 67 different components belonging to 15 venom families, with ion channel-active components (K+ toxins (23); Na+ toxins (20); Cl- toxins (2)) being predominant. The sequence of a peptide (named α-KTx9.13) ortholog to Leiurus hebraeus putative α-KTx9.3 toxin was fully determined, which exhibited 81-96% identity to other members of the α-KTx9 subfamily targeting Kv1.x and Ca2+-activated K+ channels. Chlorotoxin-like peptides were also identified. Our study underscores the medical significance of A. nigrocinctus in the region and reveals the potential value of its venom components as lead templates for biomedical applications. Future work should address whether available antivenoms in the Middle East are effective against A. nigrocinctus envenoming in the Levant area.

Venom-microbiomics of eight species of Neotropical spiders from the Theraphosidae family.

AIM: Tarantulas are one of the largest predatory arthropods in tropical regions. Tarantulas though not lethal to humans, their venomous bite kills small animals and insect upon which they prey. To understand the abiotic and biotic components involved in Neotropical tarantula bites, we conducted a venom-microbiomics study in eight species from Costa Rica. METHODS AND RESULTS: We determined that the toxin profiles of tarantula venom are highly diverse using shotgun proteomics; the most frequently encountered toxins were ω-Ap2 toxin, neprilysin-1, and several teraphotoxins. Through culture-independent and culture-dependent methods, we determined the microbiota present in the venom and excreta to evaluate the presence of pathogens that could contribute to primary infections in animals, including humans. The presence of opportunistic pathogens with hemolytic activity was observed, with a prominence of Stenotrophomonas in the venoms. Other bacteria found in venoms and excreta with hemolytic activity included members of the genera Serratia, Bacillus, Acinetobacter, Microbacterium, and Morganella. CONCLUSIONS: Our data shed light on the venom- and gut-microbiome associated with Neotropical tarantulas. This information may be useful for treating bites from these arthropods in both humans and farm animals, while also providing insight into the toxins and biodiversity of this little-explored microenvironment.

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.

Antibody-dependent enhancement of toxicity of myotoxin II from Bothrops asper.

Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper, in a mouse model of envenoming that mimics a snakebite. While clinical ADET related to snake venom has not yet been reported in humans, this report of ADET of a toxin from the animal kingdom highlights the necessity of assessing even well-known antibody formats in representative preclinical models to evaluate their therapeutic utility against toxins or venoms. This is essential to avoid potential deleterious effects as exemplified in the present study.

Venomics of Leiurus abdullahbayrami, the most lethal scorpion in the Levant region of the Middle East.

The scorpion Leiurus abdullahbayrami has been associated with severe/lethal envenomings throughout the Levant region of the Middle East, encompassing Turkey, Syria, and Lebanon, and only scarce information is available on its venom composition, activity, and antigenicity. We report on the composition of L. abdullahbayrami venom collected from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality, through LD50 determination in mice (intraperitoneal), was also assessed (0.75 (0.16-1.09) mg/kg), confirming L.abdullahbayrami venom vertebrate toxicity. Fifty-four peaks were detected using RP-HPLC, half of which eluted in the gradient region between 20 and 40% acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 24.4, 43.1, and 48.9 kDa. Venom mass fingerprint by MALDI-TOF detected 21 components within the 1000-12,000 m/z range. Whole venom 'shotgun' bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 113 different components belonging to 15 venom families and uncharacterized proteins, with ion channel-active components (K+ channel toxins (28); Na+ channel toxins (42); Cl- channel toxins (4); Ca+2 toxins (2)) being predominant. A single match for a L. adbullahbayrami NaTx was found in the UniProt database with other congeneric species, toxin h3.1 from Leiurus hebraeus, suggesting this might be an indication of venom divergence within Leiurus, eventhough this warrants further investigation involving venom proteomics and transcriptomics of relevant species. Considering such potential interspecific venom variation, future work should address whether preparation of a specific anti-L. abdullahbayrami antivenom is justified.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Toxinological profile and histopathological alterations induced by Bothrocophias campbelli venom from Colombia.

Snakebite envenomings most frequently reported in Colombia are caused by snakes of the genera Bothrops, Bothriechis, Bothrocophias, and Porthidium. Their venoms induce local and systemic pathophysiological effects, sometimes leading to permanent sequelae such as reduced mobility of the limbs, amputations, besides the risk of death. The genus Bothrocophias includes nine species, among which B. campbelli has a distribution restricted to the department of Nariño in Colombia. In this work we determined the toxinological profile its venom, by performing assays for the lethal, hemorrhagic, edematogenic, and myotoxic activities in mouse models, as well as for in vitro coagulant activity on human plasma. The lethal toxicity of the venom was 142.7 µg venom/mouse (111.4-179.8 µg/mouse; 6.6-10.6 µg/g body weight) by intraperitoneal route. Its hemorrhagic activity (minimum hemorrhagic dose: 12.7 ± 2.3 µg) is generally weaker compared to other South American vipers, but edematogenic (minimum edematogenic dose 1.0 ± 0.3 µg), and myotoxic (minimum myotoxic dose 3.9 ± 2.5 µg) activities are very potent. Histopathological examination of the injected mouse gastrocnemius muscle showed prominent disorganization of the myofibrils, myonecrosis, and an intense inflammatory leukocyte infiltrate. In vitro, the minimal coagulant dose was 12.3 ± 0.5 µg. Overall, this toxinological profile would predict that the clinical picture of envenomings by B. campbelli might be characterized by moderate disturbances in the coagulation cascade, mild local hemorrhage, and, conversely, severe myonecrosis and edema, which could potentially lead to compartment syndrome and gangrene.

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review.

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

Proteomic profile of the venom of three dark-colored Tityus (Scorpiones: Buthidae) from the tropical rainforests of Costa Rica.

OBJECTIVE: We aimed to elucidate the potential differences in the venom peptide sequences of three Tityus species from Costa Rican rainforests: T. jaimei, T. championi and T. dedoslargos, compared to T. cf. asthenes from Colombia, which could explain the low level of scorpionism in Costa Rica, evidenced by the lack of epidemiological data. METHODOLOGY: We applied venom proteomics of peptides purified by RP-HPLC and compared the obtained sequences from venoms of these Tityus species to the sequences previously identified from Tityus inhabiting other Central and South American regions. RESULTS: Venom proteome analysis evidences that most of the putative peptide toxins identified in Costa Rican dark-colored Tityus are very similar to those present in other T. (Atreus) from the region. CONCLUSIONS: Our study suggests that, in the case of potential envenomation by Tityus in Costa Rica, the same level of toxicity should be observed, compared to other cases caused by members of the subgenus from other geographical localities. On the other hand, compared to countries with more accelerated urban expansion, Costa Rican Tityus still inhabit secondary rainforests and do not commonly share the same spaces with humans, so the lack of epidemiological evidence of medical emergencies caused by envenoming by this scorpion group could be more related to ecological and demographic factors and less attributed to the characteristics of the venom.

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals.

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats.

Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

Development of a novel angiotensin converting enzyme 2 stimulator with broad implications in SARS-CoV2 and type 1 diabetes.

Angiotensin-converting enzyme 2 (ACE2) is protective in cardiovascular disease, lung injury and diabetes yet paradoxically underlies our susceptibility to SARs-CoV2 infection and the fatal heart and lung disease it can induce. Furthermore, diabetic patients have chronic, systemic inflammation and altered ACE2 expression resulting in increased risk of severe COVID-19 and the associated mortality. A drug that could increase ACE2 activity and inhibit cellular uptake of severe acute respiratory syndrome coronavirus 2 (SARs-CoV2), thus decrease infection, would be of high relevance to cardiovascular disease, diabetes and SARs-CoV2 infection. While the need for such a drug lead was highlighted over a decade ago receiving over 600 citations,1 to date, no such drugs are available.2 Here, we report the development of a novel ACE2 stimulator, designated '2A'(international PCT filed), which is a 10 amino acid peptide derived from a snake venom, and demonstrate its in vitro and in vivo efficacy against SARs-CoV2 infection and associated lung inflammation. Peptide 2A also provides remarkable protection against glycaemic dysregulation, weight loss and disease severity in a mouse model of type 1 diabetes. No untoward effects of 2A were observed in these pre-clinical models suggesting its strong clinical translation potential.

Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes.

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

Proteomic analysis of the mandibular glands from the Chinese crocodile lizard, Shinisaurus crocodilurus - Another venomous lizard?

Based on its phylogenetic relationship to monitor lizards (Varanidae), Gila monsters (Heloderma spp.), and the earless monitor Lanthanotus borneesis, the Chinese crocodile lizard, Shinisaurus crocodilurus, has been assigned to the Toxicofera clade, which comprises venomous reptiles. However, no data about composition and biological activities of its oral secretion have been reported. In the present study, a proteomic analysis of the mandibular gland of S. crocodilurus and, for comparison, of the herbivorous Solomon Island skink Corucia zebrata, was performed. Scanning electron microscopy (SEM) of the teeth from S. crocodilurus revealed a sharp ridge on the anterior surface, but no grooves, whereas those of C. zebrata possess a flattened crown with a pointed cusp. Proteomic analysis of their gland extracts provided no evidence of venom-derived peptides or proteins, strongly supporting the non-venomous character of these lizards. Data are available via ProteomeXchange with identifier PXD039424.

Stable production of recombinant SARS-CoV-2 receptor-binding domain in mammalian cells with co-expression of a fluorescent reporter and its validation as antigenic target for COVID-19 serology testing.

SARS-CoV-2 receptor binding domain (RBD) recognizes the angiotensin converting enzyme 2 (ACE2) receptor in host cells that enables infection. Due to its antigenic specificity, RBD production is important for development of serological assays. Here we have established a system for stable RBD production in HEK 293T mammalian cells that simultaneously express the recombinant fluorescent protein dTomato, which enables kinetic monitoring of RBD expression by fluorescence microscopy. In addition, we have validated the use of this recombinant RBD in an ELISA assay for the detection of anti-RBD antibodies in serum samples of COVID-19 convalescent patients. Recombinant RBD generated using this approach can be useful for generation of antibody-based therapeutics against SARS-CoV-2, as well serological assays aimed to test antibody responses to this relevant virus.

Lys49 myotoxins, secreted phospholipase A2-like proteins of viperid venoms: A comprehensive review.

Muscle necrosis is a potential clinical complication of snakebite envenomings, which in severe cases can lead to functional or physical sequelae such as disability or amputation. Snake venom proteins with the ability to directly damage skeletal muscle fibers are collectively referred to as myotoxins, and include three main types: cytolysins of the "three-finger toxin" protein family expressed in many elapid venoms, the so-called "small" myotoxins found in a number of rattlesnake venoms, and the widespread secreted phospholipase A2 (sPLA2) molecules. Among the latter, protein variants that conserve the sPLA2 structure, but lack such enzymatic activity, have been increasingly found in the venoms of many viperid species. Intriguingly, these sPLA2-like proteins are able to induce muscle necrosis by a mechanism independent of phospholipid hydrolysis. They are commonly referred to as "Lys49 myotoxins" since they most often present, among other substitutions, the replacement of the otherwise invariant residue Asp49 of sPLA2s by Lys. This work comprehensively reviews the historical developments and current knowledge towards deciphering the mechanism of action of Lys49 sPLA2-like myotoxins, and points out main gaps to be filled for a better understanding of these multifaceted snake venom proteins, to hopefully lead to improved treatments for snakebites.

Antibodies against a single fraction of Micrurus dumerilii venom neutralize the lethal effect of whole venom.

The coralsnake Micrurus dumerilii (Elapidae) is reported to cause envenomings of medical importance. Previous studies characterized the protein composition of its venom, with phospholipase A2 (PLA2) proteins the most abundant. However, it is unknown which venom components are responsible for its lethal toxicity. Fractionation of M. dumerilii venom from Colombia was carried out using RP-HPLC and each fraction was screened for lethal effect in mice at a dose of 20 µg by intraperitoneal route. Results showed that only one fraction, F9, was lethal. This fraction displayed PLA2 activity, induced indirect hemolysis in vitro, as well as edema and myotoxicity in vivo. SDS-PAGE of unreduced F9 evidenced two bands of 8 and 15 kDa, respectively, consistent with the detection of proteins with masses of 13,217.77 Da, 7144.06 Da, and 7665.55 Da. Tryptic digestion of F9 followed by nESI-MS/MS revealed peptide sequences matching proteins of the three-finger toxin (3FTx) and PLA2 families. Immunization of a rabbit with F9 proteins elicited antibody titers up to 1:10,000 by ELISA. After serum fractionation with caprylic acid, the obtained IgG was able to neutralize the lethal effect of the complete venom of M. dumerilii using a challenge of 2 ×LD50 at the IgG/venom ratio of 50:1 (w/w). In conclusion, present results show that the lethal effect of M. dumerilii venom in mice is mainly driven by one fraction which contains 3FTx and PLA2 proteins. The antibodies produced against this fraction cross-recognized other PLA2s and neutralized the lethal effect of whole M. dumerilii venom, pointing out to the potential usefulness of F9 as a relevant antigen for improving current coral snake antivenoms.

Half a century of research on Bothrops asper venom variation: biological and biomedical implications.

Snake venoms are a complex biological mixture of proteins with or without enzymatic activity, peptides, and nucleotides, among other components. It is produced in specialized secretory glands located in the maxillary region, being the result of millions of years of evolution and whose biological functions are defense, immobilization, and digestion of prey. Venoms present intraspecific (i.e., individual, ontogenetic, geographical) and interspecific (i.e., between sympatric and allopatric species) variation, and the study of this variability has become the focus of toxinological research. Bothrops asper is responsible for highest incidence, morbimortality and severe cases of envenoming in Mesoamerica and northern South America. Given its clinical importance, its venom has been characterized and compared qualitatively and quantitatively across the species range. More than 50 years of research show that B. asper venom is endowed with an interesting intraspecific variability. Knowing this variation has allowed advances in the elucidation of the biological role of the venom, a better understanding of the clinical signs and symptoms in patients envenomed by B asper, the immunological implications in the context of antivenoms production, and the generation of new ideas that could be useful to solve different biological and evolutionary questions of one of the venomous snakes with the greatest distribution and strongest public health impact in Latin America.