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15 páginas, 5 figuras, 1 tabla. Dispone de información suplementaria de libre acceso en: https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00337, We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatus bilineatus 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., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovacion y Universidades, ́ Journal of Proteome Research pubs.acs.org/jpr Article https://dx.doi.org/10.1021/acs.jproteome.0c00337 J. Proteome Res. 2020, 19, 3518−3532 3528 Madrid (Spain) to J.J.C. and by Vicerrectori ́ a de Investigacion, ́ Universidad de Costa Rica to B.L. The authors wish to express their gratitude to Conselho Nacional de Desenvolvimento Cienti ́ fico e Tecnologico (CNPq/MCTIC), Coordenac ́ a̧ o de ̃ Aperfeicoamento de Pessoal de Ni ̧ ́ vel Superior (CAPES/ MEC), Fundaca̧ o de Amparo a ̃ ́ Pesquisa do Estado de Saõ Paulo (FAPESP), and Fundaca̧ o Rondo ̃ ̂nia de Amparo ao Desenvolvimento das Aco̧ ̃es Cienti ́ ficas e Tecnologicas de ́ Pesquisa do Estado de Rondônia (FAPERO) for financial support. Authors gratefully thank Dr. Paulo Sergio Bernarde, ́ Universidade Federal do Acre, Campus Floresta, Laboratorió de Herpetologia, Cruzeiro do Sul, Acre, Brazil, for the kind donation of the pictures of B. b. smaragdinus and B. b. bilineatus shown in Figure 1. The venom of Peruvian B. b. smaradignus was used in this study as part of a research agreement signed between the Instituto Nacional de Salud (Peru) and the ́ Universidad Peruana Cayetano Heredia

16 páginas, 7 figuras. Snakebite envenoming is a neglected tropical disease that may claim over 100,000 human lives annually worldwide. Snakebite occurs as the result of an interaction between a human and a snake that elicits either a defensive response from the snake or, more rarely, a feeding response as the result of mistaken identity. Snakebite envenoming is therefore a biological and, more specifically, an ecological problem. Snake venom itself is often described as a "cocktail", as it is a heterogenous mixture of molecules including the toxins (which are typically proteinaceous) responsible for the pathophysiological consequences of envenoming. The primary function of venom in snake ecology is pre-subjugation, with defensive deployment of the secretion typically considered a secondary function. The particular composition of any given venom cocktail is shaped by evolutionary forces that include phylogenetic constraints associated with the snake's lineage and adaptive responses to the snake's ecological context, including the taxa it preys upon and by which it is predated upon. In the present article, we describe how conceptual frameworks from ecology and evolutionary biology can enter into a mutually enlightening relationship with clinical toxinology by enabling the consideration of snakebite envenoming from an "ecological stance". We detail the insights that may emerge from such a perspective and highlight the ways in which the high-fidelity descriptive knowledge emerging from applications of -omics era technologies - "venomics" and "antivenomics" - can combine with evolutionary explanations to deliver a detailed understanding of this multifactorial health crisis. Studies by JJC’s research group cited in this review were partially funded by grants from the Ministerio de Ciencia e Innovacion, ´ Madrid, Spain (BMC 2004-01432, BFU 2007-61563, BFU 2010-173730, BFU 2013-42833-P, and BFU 2017-89103-P). JJC wants to acknowledge and heartly thank all the researchers and collaborators of these projects who contributed laboratory work and numerous hours of scientific discussions. EABU was supported by a Norwegian Research Council FRIPROYRT Fellowship no. 287462. TNWJ was supported by National Health and Medical Research Grant 13/093/002 AVRU. KS was supported by DBT / Wellcome Trust India Alliance Fellowship (IA/I/19/2/504647). Support by Vicerrectoría de Investigacion ´ (University of Costa Rica) to work performed at Instituto Clodomiro Picado is also gratefully acknowledged.

11 páginas, 4 figuras. 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. This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain).Open access for this article was enabled by the participation of Instituto de Biomedicina de Valencia in an allinclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CSIC.

38 páginas, 6 figuras Venoms are integrated phenotypes used by a wide range of organisms for predatory and defensive purposes. The study of venoms is of great interest in diverse fields, such as evolutionary ecology and biotechnology. Omics technologies have contributed to understanding the evolutionary mechanisms that molded snake venoms to their present-day structural and functional variability landscape. Areas covered: This review article reflects on two recent implementations in venomics: absolute quantification of intact proteins by elemental mass spectrometry, and top-down molecular mass spectrometry. Expert commentary: Leveraging on a new way of polyatomic interference removal, a triple quadrupole inductively coupled plasma mass spectrometry configuration has proven feasible for the absolute quantification of venom toxins via sulfur detection. A major advantage of this approach over quantitative molecular mass spectrometry techniques is that only a generic S-standard is required to quantify all the chromatographically separated sulfur-containing fractions. Top-down venomics is in its infancy but, due to recent hardware and software developments, is gaining momentum. Proteoform-resolved venom proteomes are needed to understand the spatio-temporal variability landscape underlying the adaptations that drive intraspecific venom evolution. Integrating top-down venomics and absolute proteoform quantification into a novel elemental and molecular mass spectrometry configuration will represent a quantitative leap in the study of individual venoms. This paper was supported by grant BFU2013-42833-P, from the Ministerio de Economía y Competitividad, Madrid, Spain.

10 páginas, 4 figuras, 1 tabla en material suplementario. El documento de transparencia asociado a este artículo, se puede encontrar en la versión online., BACKGROUND: Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. METHODS: A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. RESULTS: Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A2 (PLA2); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. CONCLUSIONS: The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. GENERAL SIGNIFICANCE: This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt., This study was supported by grants BFU2013-42833-P (Ministerio de Economía y Competitividad, Madrid, Spain) and MR/L01839X/1 (Medical Research Council, UK). NRC was supported by a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and the Royal Society.

Understanding the origin and maintenance of phenotypic variation, particularly across a continuous spatial distribution, represents a key challenge in evolutionary biology. For this, animal venoms represent ideal study systems: they are complex, variable, yet easily quantifiable molecular phenotypes with a clear function. Rattlesnakes display tremendous variation in their venom composition, mostly through strongly dichotomous venom strategies, which may even coexist within a single species. Here, through dense, widespread population-level sampling of the Mojave rattlesnake, Crotalus scutulatus, we show that genomic structural variation at multiple loci underlies extreme geographical variation in venom composition, which is maintained despite extensive gene flow. Unexpectedly, neither diet composition nor neutral population structure explain venom variation. Instead, venom divergence is strongly correlated with environmental conditions. Individual toxin genes correlate with distinct environmental factors, suggesting that different selective pressures can act on individual loci independently of their co-expression patterns or genomic proximity. Our results challenge common assumptions about diet composition as the key selective driver of snake venom evolution and emphasize how the interplay between genomic architecture and local-scale spatial heterogeneity in selective pressures may facilitate the retention of adaptive functional polymorphisms across a continuous space.

Comunicaciones a congresos

Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. Over the last decade, several studies have shown that peroxidases isolated from the leaves of different kinds of palm trees such as the royal palm (Roystonea regia), the date palm (Phoenix dactylifera), the African oil palm (Elaeis guineensis), the ruffle palm (Aiphanes cariotifolia) and the windmill palms (Trachycarpus fortunei and Chamaerops excelsa) exhibit higher activity and stability than commercially available peroxidases isolated from, for example, horseradish roots (Armoracia rusticana) and soybean (Glycine max). Here, the structure, thermal denaturation, and the catalytic cycle of peroxidases from palm trees are reviewed and compared with those of other plant peroxidases. In addition, we report the biotechnological potential of palm peroxidases and their implications in cellular aging and diseases, such as Refsum’s and Alzheimer’s diseases. This paper summarizes the main characteristics of the palm peroxidases studied.

Comunicaciones a congresos

Póster original presentado Joint Meeting: 2nd Congress of the Spanish Proteomics Society and 1st Meeting of the European Proteomics Association, celebrado en Valencia (España), en 2007, Retinoic acid (RA), the active form of vitamin A, induces neuroblastoma cells SH-SY5Y to differentiate. In addition to its classical transcriptional actions regulating the expression of specific genes, RA acts in an extra-genomic way, modulating the activity of relevant signalling cascades. In particular, RA treatment activates PI3K/Akt signalling pathway, and this activation is an essential requirement for neural differentiation (López-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002). Western blots using an antibody which specifically recognizes the phosphorylation site of Akt have demonstrated that Akt activation in response to RA treatment produced a rapid phosphorylation of downstream substrates. Moreover, RA treatment resulted in rapid phosphorylation of well-known targets of Akt, such as mTOR and the ribosomal kinase p70S6, and other proteins, as CREB and histone H3, which are likely targets of RA non-genomic actions according to the scientific literature. We have carried out a proteomic approach in order to identify novel nuclear proteins whose phosphorylation state is modified by these extra-genomic actions of RA. Nuclei from control and RA-treated (1mM, 30 min) neuroblastoma cells were obtained, and nuclear phospho-proteins were purified by affinity chromatrography. The phosph-oproteins were separated by 2-D gel electrophoresis (IEF/SDS-PAGE), and comparison of 2-D protein patterns using a bioinformatic software package allowed the selection of a series of spots that have undergone changes on their phosphorylation state. These spots with differential expression could be identified by mass spectrometry analysis as the proteins nucleophosmin/B23, hnRNP-C1/C2, hnRNP-K, HMGB1, PABP and histone H1.5. All those proteins have a nuclear distribution and phosphorylation sites. Finally, the observed RA-induced phosphorylation changes in these proteins were confirmed in 2-D western blots and immunoprecipitation experiments., E. J. Laserna is recipient of a predoctoral fellowship from the Generalitat Valenciana. This work was supported by PNI+D+I (SAF 2003-00311).

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10 págs, 5 figuras. Información suplementaria en: https://doi.org/10.1016/j.toxicon.2020.07.001, The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom., This work was financially supported by Fundaç~ao de Amparo �a Pesquisa do Estado de S~ao Paulo – FAPESP (grant numbers: 2017/ 01890–0, 2018/25786–0; 2018/25899–0, 2017/26533–6 and 2017/ 16908–2), Conselho Nacional de Desenvolvimento Científico e Tec-nol�ogico (CNPq) (405399/2018–9) and partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain).

2 páginas y 1 figura. Corrigendum del registro: http://hdl.handle.net/10261/256298, 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., This study was partly supported by grant EFP-00021744 financed by Fundaç ̃ao Rondˆonia de Amparo ao Desenvolvimento das Aç ̃oes Cien- tíficas e Tecnol ́ogicas (FAPERO, Rondˆonia, Brazil). Research performed at IBV-CSIC was partially funded by grant BFU2017-89103-P from the Ministerio de Ciencia e Innovaci ́on, Madrid, Spain. Work performed at Instituto Clodomiro Picado was supported by Vicerrectoría de Inves- tigaci ́on (741-C0-071) and Vicerrectoría de Acci ́on Social (Proyecto ED- 3248) of Universidad de Costa Rica. The excellent bench work assistance of Sarai Quesada-Bernat with antivenomics, and Yania Rodríguez Jim ́enez with determination of the murine LD50 for P. arcosae venom and neutralization of its lethal effect by the Brazilian pentavalent bothropic antivenom, are gratefully acknowledged

14 páginas, 9 figuras, 3 tablas, 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., This study was financially supported by DGAPA-PAPIIT (project IN211621), CONACYT (project264255); FORDECYT PRONACE (project 1715618/2020), FORDECYT (project 303045), Clemson University and the National Science Foundation (DEB 1822417) to CLP.

51 páginas, incluye 5 figuras y 2 tablas, 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 Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination. Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability., IA, FL, and FM-F are supported by FCT - Fundação para a Ciência e a Tecnologia, Portugal (ref. SFRH/BD/137797/2018, SFRH/BD/131722/2017, and DL57/2016/CP1440/CT0010, respectively). Analyses at the Evolutionary and Translational Venomics Laboratory of the Institute of Biomedicine of Valencia were partially financed by Ministerio de Ciencia e Innovación, Spain (grant BFU2020_PID2020-119593GB-I00).

11 páginas, 3 figuras, 3 tablas, Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia e Innovaci ́on, Madrid (Spain), Project 741-B7-108 from the Vicerrectoría de Investigaci ́on of Universidad de Costa Rica, and Beca del Mill ́on, SEP-484-2019, from the Sistema de Estudios de Posgrado, Universidad de Costa Rica. The collaboration of our col- leagues at Instituto Clodomiro Picado and Laboratorio de Ven ́omica Evolutiva y Traslacional, Instituto de Biomedicina de Valencia, CSIC, is gratefully acknowledged. This work was carried out in partial fulfill- ment of the requirements for the M.Sc. degree for A. S ́anchez at the University of Costa Rica

6 páginas, 3 figuras., Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense., This work was funded from a studentship supported by Elizabeth Artin Kazandjian to T.D.K.; grant PE 2600/1 from the German Research Foundation (DFG) to D.P.; grant OPUS 1354156 from the U.S. National Science Foundation to H.W.G.; grants FAPESP 2017/18922-2 and 2019/05026-4 from the São Paulo Research Foundation to R.R.d.S.; grants RPG-2012-627 and RFG-10193 from the Leverhulme Trust to R.A.H. and W.W.; grant MR/L01839X/1 from the U.K. Medical Research Council to J.M.G., R.A.H., J.J.C., and N.R.C.; Career Development Fellowships APP1162503 and DP190103787 from the Australian National Health and Medical Research Council to I.V.; fellowship DE160101142 and grant DP160104025 from the Australian Research Council, and fellowship FRIPRO-YRT #287462 from the Research Council of Norway to E.A.B.U.; and a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and Royal Society to N.R.C.

19 páginas, 8 figuras, 2 tablas. Información suplementaria disponible en: https://doi.org/10.1016/j.jprot.2020.103945., 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., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia e Innovación, Madrid (Spain). The authors gratefully acknowledge the excellent benchwork by Yania Rodríguez, Sarai Quesada, Jordi Durban, Libia Sanz and Alicia Pérez, and the Ceiba Foundation (Pasto-Nariño, Colombia) for the doctoral training scholarship to D. M-O. Funding to D. S-V. was provided by a FulbrightSenescyt grant, and funds from The Ohio State University Graduate School and Universidad Tecnológica Indoamérica. We are grateful to A. Loaiza-Lange, O. Torres-Carvajal, D. Núñez, C. Tapia, P. SantacruzOrtega, M. Bolaños, A. Freire-Lascano, I. Tapia, E. Arbeláez, A. Arteaga, M. Salazar, and R. Valenzuela for assistance in the laboratory and the field. We also thank the help provided by the staff of Jama-Coaque Ecological Reserve and Bosque Protector Cerro Blanco. Samples used in this study were obtained under permission of Autoridad Nacional de Licencias Ambientales-ANLA (Permiso Marco de Recolección de Especímenes de Especies Silvestres de la Diversidad Biológica con fines de Investigación Científica No comercial, Resolución 0152, 12 de Febrero de 2015) from Ministerio de Ambiente y Desarrollo Sostenible (Colombia), and collection permit 008-09 IC-FAU-DNB/MA from Ministerio del Ambiente, Ecuador and specimens deposited at Museo de Zoología (QCAZ), Pontificia Universidad Católica del Ecuador. This study was performed as a partial requirement for the PhD. degree of Diana Mora-Obando at the University of Valencia, Spain.

18 pages, 5 fig, 3 tables.Data that support the findings of this study are available in the Dryad Data Repository (https://doi.org/10.5061/dryad.wm37pvmjz), Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long-sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins ("venom interactive proteins" [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum-based resistance is both population- and species-specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum "resistome" involves broad-based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co-evolutionary interactions between species at the molecular level., HLG was supported by NSF Grant 1638872 during preparation of the manuscript. Research in the laboratory of J.J.C. was partly financed by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain). Fieldwork for serum and venom col-lection was funded by the American Museum of Natural History's Theodore Roosevelt Grant and American Society of Naturalist Graduate Student Research Grant, both to M.L.H. M.L.H. was sup-ported by an NSF Postdoctoral Research Fellowship 1711141 during preparation of the manuscript

14 páginas, 3 figuras, 2 tablas. The datasets generated during the current study are available in Additional file 1 (Table S1) and Additional file 2 (Table S2). Raw mass spectrometric data are available from the corresponding authors on reasonable request., BACKGROUND: The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. METHODS: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. RESULTS: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. CONCLUSION: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain) to JJC. The authors wish to express their gratitude to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCTIC), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC) and Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas de Pesquisa do Estado de Rondônia (FAPERO) for financial support.

10 páginas, 3 figuras, 1 tabla. Dispone de material suplementario en: https://doi.org/10.1016/j.jprot.2020.103882, The Duvernoy's gland secretory proteome of the false coral snake Rhinobothryum bovallii (Costa Rica), unveiled applying bottom-up venomics, comprises a handful of toxins belonging to only three protein families, three-finger toxin (3FTx), cysteine-rich secretory protein (CRISP), and snake venom metalloprotease (PIII-SVMP). Except for small differences in the relative abundance of the PIII-SVMPs, which may be due to individual variability, no evidence of geographic variability or ontogenetic changes was found among the venom proteomes of the juvenile and adult R. bovallii snakes sampled. Major monomeric (86.5%mol) and minor dimeric (2.8%mol) 3FTxs dominate the toxin arsenal of the Costa Rican false coral snake. The remaining 10.7% of the venom proteome comprises CRISP (8.2%) and PIII-SVMP (2.4%) molecules. In vivo lethality assays showed that R. bovallii produces venom that is non-toxic towards mammalian prey, and which exerts a different toxic effect on domestic chicken chicks and baby green iguana. Toxicovenomic analysis of R. bovallii venom in the iguana model identified two 3FTx RP-HPLC fractions that faithfully mimicked the irreversible immobilizing effect of the whole venom. BIOLOGICAL SIGNIFICANCE: With more than 2200 species in family Colubridae (sensu lato), rear-fanged snakes comprise approximately two-thirds of the extant species of advanced snakes. Snakebites from venomous snakes that are of medical concern are predominantly from front-fanged snakes of families Viperidae and Elapidae. On the other hand, rear-fanged snakes have been conventionally considered non-venomous, and thus their venoms have remained a largely untapped area of venomics. However, increasing documentation of life-threadening, even fatal, envenomings from rear-fanged snakes has sparked interest in their venoms. Appying bottom-up venomics we have revealed that the Duvernoy's gland secretory proteome of R. bovallii comprises a handful of toxins belonging to only three protein families, with slow-acting three-finger toxins (3FTx) that are non-toxic towards mammalian prey and show preference towards diapsid taxa representing the dominant structural and functional proteins. Our work documents for the first time 3FTxs exerting different effect in an avian model than in a reptile model. Besides, the 3FTx fractions that faithfully mimicked the irreversible iguana-immobilizing effect of the whole venom were identified through toxicovenomic analysis of R. bovallii venom on Iguana iguana. Our work underscores the importance of using biologically-relevant animal toxicity models for investigating the biological roles of venoms in an evolutionary-ecological context., This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain) to JJC. The authors gratefully acknowledge the excellent bench assistance of Stephanie Chaves and Julián Fernández, Instituto Clodomiro Picado, UCR, and Andres Vega for sharing his R. bovalli photographs.

11 páginas, 6 figuras y 2 yablas. Se puede consultar información complementaria en: https:// doi.org/10.1016/j.jprot.2020.103865., We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to México comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel β-neurotoxic heterodimeric PLA2, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 μg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 μg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD50s, Birmex; 114 LD50s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s. BIOLOGICAL SIGNIFICANCE: This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to México. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel β-neurotoxic heterodimeric PLA2, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s., This research was partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain), grants PAPIIT-DGAPA IN207218 and CONACyT-FORjDECYT No. 303045 (“Venenos y Antivenenos”).

Artículo editorial. 1 página.

12 Páginas, 5 Figuras, Facultative parthenogenesis (FP) is asexual reproduction in plant and animal species that would otherwise reproduce sexually. This process in vertebrates typically results from automictic development (likely terminal fusion) and is phylogenetically widespread. In squamate reptiles and chondrichthyan fishes, FP has been reported to occur in nature and can result in the production of reproductively viable offspring; suggesting that it is of ecological and evolutionary significance. However, terminal fusion automixis is believed to result in near genome-wide reductions in heterozygosity; thus, FP seems likely to affect key phenotypic characters, yet this remains almost completely unstudied. Snake venom is a complex phenotypic character primarily used to subjugate prey and is thus tightly linked to individual fitness. Surprisingly, the composition and function of venom produced by a parthenogenetic pitviper exhibits a high degree of similarity to that of its mother and conspecifics from the same population. Therefore, the apparent loss of allelic diversity caused by FP appears unlikely to have a significant impact on the prey-capturing ability of this snake. Accordingly, the pitviper offspring produced by FP retained complex phenotypic characteristics associated with fitness. This result reinforces the potential ecological and evolutionary importance of FP and questions our understanding of the inheritance of venom-associated genes., NRC was supported by a Sir Henry Dale Fellowship (200517/Z/16/Z) jointly funded by the Wellcome Trust and the Royal Society. None of the authors of this manuscript have a financial interest related to this work

69 páginas, 9 figuras, 3 tablas, Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation. Biological significance: The mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation., This study was supported by grants BFU2013-42833-P (Ministerio de Economía y Competitividad, Madrid, Spain), MR/L01839X/1 (Medical Research Council, UK), NNF13OC0005613 (the Novo Nordisk Foundation), Knud Højgaards Fond, Augustinusfonden, PE 2600/1 and the Cluster of Excellence UniCat (Deutsche Forschungsgemeinschaft, Bonn, Germany).

18 páginas, 8 figuras, 1 tabla, 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.

5 páginas, 3 figuras, 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., This study was partly supported by grant BFU2020_PID2020-119593GB-I00 from the Ministerio de Ciencia e Innovacion, ´ Madrid (Spain).

Tesis doctoral 326P. Imágenes y tablas, Bothrops asper es un complejo de especies de importancia clínica en Latinoamérica causante de aproximadamente entre el 50 a 80% de los envenenamientos ofídicos de tipo botrópico. La abundancia de individuos, los hábitats crípticos que frecuenta este grupo y su amplia distribución desde el Sur de México hasta el norte de Perú, explican la alta incidencia de accidentes ocasionados por estas serpientes. La integración de datos moleculares, morfológicos y geográficos sugieren una estructura filogeográfica compleja de la especie en Centro y norte de Suramérica, con la existencia de diferentes linajes, algunos con procesos de divergencia reciente, especialmente en los valles interandinos al suroccidente de Colombia y Ecuador. La comparación de los venenos también puede contribuir al entendimiento de la historia evolutiva de B. asper; por lo tanto, analizamos los proteomas de 12 grupos de ejemplares adultos procedentes de cinco linajes de B. asper del suroccidente colombiano y Ecuador. Los resultados reflejan variación intraespecífica entre los grupos, específicamente en cuatro de los componentes mayoritarios: metaloproteinasas, serina proteasas, fosfolipasas A2 y proteínas similares a lectinas tipo C. Análisis de componentes principales construidos a partir de la abundancia de proteínas y péptidos presentes en los venenos, así como la comparación de masas moleculares de PLA2 recuperadas de los venenos de Mesoamérica y Suramérica, son congruentes con los análisis filogeográficos y filogenéticos previos, y argumentan a favor de las hipótesis que interpretan la diversificación del grupo. La cordillera de los Andes, los gradientes altitudinales en hábitats de montaña, las diferencias ecosistémicas y los nichos ecológicos en los valles interandinos han sido factores determinantes en los procesos de divergencia no solo de serpientes, sino también de otros grupos taxonómicos en Colombia y Ecuador. Adicionalmente, se observó variación ontogénica entre juveniles y adultos de algunos linajes de B. asper del Ecuador, siendo la abundancia relativa de PLA2 de tipo K49 una de las características distintivas entre los grupos. La variabilidad composicional entre los proteomas del veneno de los linajes de B. asper distribuidos en el suroccidente colombiano, ha reflejado diferencias a nivel de los cuadros clínicos presentados por las víctimas de envenenamiento. A la fecha, estos casos son tratados con antivenenos disponibles en el país, principalmente de producción nacional; no obstante, los antivenenos botrópicos generados en varios países de América Latina suelen mostrar cierto grado de eficacia paraespecífica en la neutralización de venenos congenéricos. Con base en lo anterior, y teniendo en cuenta que Colombia ha atravesado por periodos de escases de este medicamento esencial, la inmunoreactividad y capacidad neutralizante de sueros producidos en Colombia, y otros países de Latinoamérica contra los venenos de tres linajes de B. asper del suroccidente colombiano fueron comparadas. Los resultados demostraron que los antivenenos homólogos producidos en Colombia y Costa Rica son efectivos en la neutralización in vivo de la mayoría de las actividades biológicas producidas por estos venenos, y que los antivenenos heterólogos producidos en Argentina, Venezuela y Perú fueron capaces de reconocer in vitro la mayoría de los componentes tóxicos de los venenos, por lo que tendrían un gran potencial para el tratamiento clínico del ofidismo botrópico en esta región del país., Este trabajo fue realizado gracias al apoyo económico de los siguientes proyectos de investigación: BFU2017-89103-P del Ministerio de Ciencia, Innovación y Universidades, Madrid, España (Investigador Principal. Juan José Calvete Chornet). La Fundación Centro de Estudios Interdisciplinarios Básicos y Aplicados - CEIBA otorgó un crédito educativo condonable a Diana Lorena Mora Obando en el marco del proyecto “Fortalecimiento de capacidades regionales en investigación, desarrollo tecnológico e innovación en el Departamento de Nariño” del año 2017, para la realización de sus estudios en el programa de Doctorado en Biomedicina y Biotecnología de la Universidad de Valencia, el cual incluyó una estancia de investigación en el Instituto Clodomiro Picado de la Universidad de Costa Rica.

Tesis doctoral, 311 p. y figuras, Este trabajo se ha realizado con el soporte económico de los proyectos de investigación que se enumeran a continuación: BFU2007-61563: Enfoque “top-down” aplicado a la venómica. Evolución de la familia de las disintegrinas y evaluación del uso de la disintegrina jerdostatina para la visualización in vivo de angiogénesis dependiente de la integrina α1β1. Ministerio de Ciencia e Innovación (IP. Juan José Calvete Chornet). BFU2010-17373: Venómica Traslacional. Ministerio de Ciencia e Innovación (IP. Juan José Calvete Chornet). PROMETEO 2010/005. Generalitat Valenciana (IP. Juan José Calvete Chornet). Gema Bolás González-Lliberós ha disfrutado de una beca predoctoral JAE-PreDoctoral (07_00680) otorgada por el Consejo Superior de Investigaciones Científicas (CSIC).

211 páginas, figuras y tablas

Tesis doctoral. 213 páginas. Figuras, bibliografía y anexos., [EN] This thesis aims to provide more evidence, not only in characterizing the variability interspecific observed in the venom composition of the South American snakes Crotalus simus, Atropoides picadoi, Atropoides mexicanus, Bothriechis lateralis, Bothriechis schlegelii, Cerrophidion Sasai (previously known as Cerrophidion Godmani) and Bothrops asper, but try explain biological phenomena responsible for the intraspecific geographical and ontogenetic variability observed in certain species of snakes. In this sense, this thesis has attempted to study transcriptomic and proteomic data from organisms without prior genomic information in order to find out if such variability could answer a genomic divergence or, on the other hand, certain regulatory elements such as miRNA could play a key role in the diversity of observed phenotypes. The knowledge of the molecular mechanisms that underlie this variability could open a new path in the treatment of snakebite and the evaluation of the current therapies. To address these objectives, the development of the thesis needed to study the phylogenetic relationships of the members of suborder Serpentes, the knowledge of venom protein families and the knowledge of the recent massive DNA sequencing techniques. Our results suggest a potential post-transcriptional regulatory mechanisms that could play a not described role in toxin expression regulation., [CAT] El treball desenvolupat durant la present tesi doctoral pretén aportar més evidències, no només en la caracterització de la variabilitat interespecífica observada en la composició del verí dels cròtals sud- americans Crotalus simus, Atropoides picadoi, Atropoides mexicanus, Bothriechis lateralis, Bothriechis schlegelii, Cerrophidion sasai (abans Cerrophidion godmani) i Bothrops asper, sinó també intentar explicar els fenòmens biològics responsables de la variabilitat intraespecífica observada en determinades espècies de serps tant a nivell geogràfic com ontogenètic. En aquest sentit, la feina desenvolupada durant la present tesi doctoral ha intentat estudiar de forma conjunta les dades transcriptòmiques i les dades proteòmiques per a organismes sense informació genòmica prèvia, en el que hem volgut anomenar “proteotranscriptòmica”, per tal d’esbrinar si l’esmentada variabilitat podria respondre a una divergència genòmica o, per altra banda, determinats elements reguladors com els miRNA podrien jugar un paper clau en la diversitat de fenotips observats. Amb això es pretén avançar en el coneixement dels mecanismes de regulació que fan possible aquesta variabilitat, la comprensió dels quals podrien obrir un nou camí en el tractament de les patologies associades en els accidents per mossegada de serp i a l’avaluació de les teràpies aplicades. Per abordar aquests objectius, el desenvolupament de la tesi doctoral va necessitar de l’estudi de les relacions filogenètiques dels membres del subordre Serpentes així com del coneixement de les famílies de proteïnes existents als verins i de les tècniques de seqüenciació massiva de DNA, coneixements que intentaran ser explicats en els diferents apartats de la manera més entenedora possible. Els nostres resultats van possibilitar proposar potencials mecanismes de regulació post-transcripcional que podrien jugar un paper fins ara no descrit en la regulació de l’expressió de les toxines i van ajudar a la caracterització ‘de novo’ de determinades proteïnes del verí no descrites anteriorment., Aquest treball ha estat realitzat amb el suport econòmic dels projectes d’investigació que s’enumeren a continuació: BFU2007-61563: Enfoque "top-down" aplicado a la venómica. Evolución de la familia de las disintegrinas y evaluación del uso de la disintegrina jerdostatina para la visualización in vivo de angiogénesis dependiente de la integrina alpha1beta1. Ministerio de Educación y Ciencia (I.P. J.J. Calvete) BFU2010-17373: Venómica Traslacional Ministerio de Ciencia e Innovación (IP. J.J. Calvete) Jordi Durban i Sanchez ha gaudit d’una beca predoctoral FPI atorgada pel Ministerio de Ciencia e Innovación associada al projecte BFU2007-61563

Tesis doctoral del Departamento de Bioquímica y Biología Molecular, Universitat de València

La composición proteica de los venenos de serpiente refleja el hecho de que éstos se originaron en etapas tempranas de la evolución de los reptiles escamosos, por reclutamiento y transformación por evolución acelerada de un número reducido de proteínas endógenas. Los venenos de los vipéridos poseen un arsenal de proteínas capaces de degradar la matriz extracelular e interferir en la cascada de coagulación, el sistema hemostático y la reparación de tejidos. Los proteomas de las serpientes Viperinae caracterizados hasta la fecha están constituidos por isoformas de unas pocas familias de proteínas, cuya abundancia relativa presenta gran variación interespecífica. Las proteínas mayoritarias de los venenos de las víboras y serpientes de cascabel se agrupan en enzimas (proteasas dependientes de serina, metaloproteasas dependientes de Zn2+ del grupo de las reprolisinas, isoenzimas del grupo II de fosfolipasas A2, L-amino acido oxidasa) y proteínas sin actividad enzimática (proteínas similares a lectinas dependientes de Ca2+ y disintegrinas). Las disintegrinas (40-100 AAs) son liberadas al veneno por procesamiento proteolítico de las metaloproteasas PII y actúan inhibiendo la adhesión de las integrinas a sus ligandos. Se clasifican en función de su longitud y número de enlaces disulfuro en monoméricas largas (~90 AAs, 7 SS), medias (~70 AAs, 6 SS) y cortas (~ 40 AAs, 4 SS) y diméricas (subunidades de ~ 63 AAs, 2 SS Inter.- y 4 SS intracatenarios). El objetivo general de esta Tesis Doctoral ha sido profundizar en el estudio de las bases moleculares de la diversificación estructural y funcional de la familia de las disintegrinas. Partimos de un esquema evolutivo basado en la pérdida sucesiva de enlaces disulfuro ("ingeniería de enlaces disulfuro") y la acumulación de mutaciones ("química combinatorial") en el bucle de inhibición de integrinas. La aplicación de técnicas proteómicas nos ha permitido determinar la composición proteica del veneno crudo de Sistrurus m. barbouri, Echis ocellatus y Bitis arietans. Para ello los venenos se fraccionaron por RP-HPLC y cada una de estas fracciones fue analizada por SDS-PAGE, secuenciación N-terminal, determinación de masa molecular, contenido en cisteínas libres, enlaces disulfuro y huella peptídica por espectrometría de masas MALDI-TOF, además de secuenciación de novo por espectrometría de masas en tándem (MS/MS). Así pudimos adjudicar cada pico obtenido tras la separación a 10-12 familias de proteínas conocidas. Con el objeto de comparar proteoma y transcriptoma e investigar la posible presencia de intermediarios evolutivos de las familias de disintegrinas no expresados en el veneno se han analizado librerías de ADNc de glándulas de venenos de Bitis arietans (Ba) y Echis ocellatus (Eo). En el caso de Ba identificamos un producto que presenta un dominio tipo disintegrina ECD con 16 cisteínas, conservando el enlace entre las CysXIII-CysXVI, pero que carece del dominio rico en cisteínas típico de las metaloproteasas PIII, lo cual indica que la deleción del dominio rico en Cys ocurrió con anterioridad a la pérdida del enlace disulfuro C13-C16. En Eo amplificamos precursores de disintegrinas diméricas, los cuales pertenecen a la clase "mensajeros cortos" (carecen del dominio metaloproteasa). La existencia de mensajeros que codifican para disintegrina no expresados en el veneno podría indicar la existencia de "fondos de reserva genómicos" de eventual relevancia para la adaptación a ecosistemas cambiantes. Además, mediante el análisis de la organización genómica de disintegrinas de las diferentes familias estructurales, hemos demostrado que la pérdida sucesiva de intrones forma parte de un mecanismo evolutivo de diversificación de las disintegrinas que conlleva una evolución acelerada y una minimización de las estructuras génica y proteica., Venoms of Viperidae and Crotalidae snakes contain proteins that interfere with the coagulation cascade, the haemostatic system and tissue repair. The general aim of this thesis was elucidate the molecular details of the mechanisms leading to snake venom toxin diversification. We describe the application of the proteomic characterization of Echis ocellatus (Eo), Bitis arietans (Ba) and Sistrurus m. barbouri (Smb) venoms. Their protein composition was analyzed by RP-HPLC and SDS-PAGE of each separated protein fraction. The molecular mass and the number of cysteine residues/disulphide bonds were determined by MALDI-TOF mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion, peptide mass fingerprinting and de novo sequencing by MS/MS. Our results show that snake venoms are composed of proteins belonging to a few protein families, including enzymes (serine proteinases, Zn2+-dependent PI-PIV metalloproteases, group II phospholipase A2 isoenzymes) and proteins with no enzymatic activity as disintegrins, which are potent antagonist of integrins. To understand the genomic basis of the accelerated evolution of disintegrins we have undertaken the analysis of cDNAs encoding disintegrins from venom gland libraries of Eo and Ba and the genomic organization of disintegrin genes. The cDNA sequences coding for dimeric disintegrins lack the metalloproteinase domain and belong thus to the short-coding class. Analysis of two distinct messengers coding for the short disintegrin ocellatusin strongly argues for a common ancestry of short- and dimeric disintegrin subunits. We also report the cloning and sequence analysis of a novel and unique ECD-disintegrin-like domain from Ba. It contains the 16 cysteine residues that are conserved in all PIII disintegrin-like domains but lacks the cysteine-rich domain. On the other hand, comparison of the exon-intron organization revealed that the evolutionary pathway leading to the diversification of disintegrins also involved the miniminization of the gene organization by the successive removal of introns.