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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.

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.

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.

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.

The application in recent years of the high-throughput methodologies constituting snake venomics has provided snake venom research with renewed interest and enthusiasm, leading to the rapid production of a remarkable amount of information on a multitude of snake venoms, and providing a comprehensive view of their compositions and properties. This has allowed to obtain a detailed insight into some of the processes underlying snake venom origin and evolution, and has led to the identification of compounds of potential medicinal relevance, and to the development of powerful therapeutic tools to mitigate the global burden of snakebite. Nonetheless, considerable knowledge gaps exist in snake venom research, especially concerning the attention paid to certain topics and snake species. The main goal of this thesis is to contribute to this research field, increasing the current knowledge on the venoms produced by European vipers, focusing on species of the genus Vipera from the Iberian Peninsula. Aiming to provide the first qualitative and quantitative estimate of the trends and biases in snake venom research, we reviewed a total of 267 snake venom studies published between 1964 and 2021. While our analysis showed an overall positive trend in the number of articles published yearly, it also underlined severe neglect of snake families of supposedly minor medical relevance (e.g., Atractaspididae), underrepresentation of some of the areas most impacted by snakebite (i.e., Indomalayan and Afrotropic realms), and limited interest in the ecological and functional context of snake venom. Indeed, snakes of the family Viperidae were significantly more represented than any other snake taxon retrieved, the Neotropics were the most represented biogeographic realm for number of studied species, and the vast majority of the publications focused on snake venom characterisation, whereas more ecology-related topics were rarely considered. Although snakes of the family Viperidae were the mos

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SIGNIFICANCE: Lataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern

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.

Snakebite envenoming is a major issue in certain developing parts of the world, with tens of thousands of deaths and a multitude of lingering permanent medical conditions on a yearly basis as a result. Venoms consist of a plethora of biologically active compounds, mainly peptides and proteins, that can induce these effects. Snake venom characterisation, in terms of identifying and quantifying the venom toxin proteins in crude venoms, can aid in pinpointing the venom toxins responsible for pathologies observed in snakebite victims. It can also assist in developing a new generation of efficient antivenoms. To facilitate this characterisation process, several approaches have been developed, of which venomics, toxicovenomics and antivenomics are the most important ones and aim to map venom toxin composition, their toxic effects, and antivenom efficacy, respectively. Most venomics approaches include peptide-centric analyses of protein digests in combination with advanced separation and detection hardware and involve the use of generic and dedicated software tools for data processing. However, a shift towards protein-level, and even proteoform-level, analysis can be observed over the last few years. A variety of hyphenated intact protein separation techniques, including gel-eluted liquid fraction entrapment electrophoresis, OFFGEL, gel filtration and several (ultra)high pressure and nanoscale chromatographic separations can be named, which allow retention of intact or native states of toxin proteins with ever increasing resolutions. Similarly, rapid detection hardware advances on the mass spectrometry side, and labelling techniques, allow for increasingly sensitive and quantitative venom analysis and characterisation. Most venomics research includes several complementary analytical techniques performed in parallel, with each technique often leading to exclusive protein identifications but also showing significant overlap. The original venomics approach, developed by the C

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 m