- Email: [email protected]
A bright future for integrative venomics
Toxicon xxx (2015) 1e4
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Editorial
A bright future for integrative venomics
Venomous secretions are produced by a myriad of animal species, from invertebrates to vertebrates. As a general rule, peptides and proteins represent the most abundant and functionally relevant components of these dangerous “cocktails”. It may be argued that the first and indispensable requirement to understand a particular venom is to know its composition, and, to this end, the combination of -omics technologies have emerged as the most powerful tools available to date (Eichberg et al., 2015). The application of -omics methodologies to the study of venoms of diverse origins has grown steadily in recent years, and promises to open the door to a landscape where the protein composition of a large proportion of biologically and clinically relevant venoms will be known in deep detail. In combination with a wide variety of experimental approaches, the availability of such comprehensive basic information will pave the way to a profound understanding on the emergence and evolution of venom toxins, including their structural and functional diversification during millions of years of natural selection. The ever-increasing capabilities of the -omics analytical platforms and their associated bioinformatic algorithms can provide information, which required years or months of work in earlier times, in hours or even minutes today, using just minuscule amounts of samples. Locus-resolution for individual venom components will increasingly be the goal of proteomic analyses (Calvete, 2014; Eichberg et al., 2015), in parallel growth with the availability of highquality transcriptomic and genomic data on venomous organisms, a major limiting factor at present. The quantitative profiling of venom proteins is also increasingly being pursued, since it adds a further dimension to the informative value provided by their mere qualitative cataloguing. Indeed, understanding the molecular basis of complex adaptive traits, such as venoms, demands both qualitative and quantitative comparisons of the temporal and spatial patterns of venom variation. Other complementary strategies related to -omics analyses of venoms (‘venomics’) involve the immunorecognition of their components by antibodies present in therapeutical antivenoms ('antivenomics') (Calvete et al., 2014; nchez et al., 2015)- and the functional assessment of toxic venom Sa components in experimental models (‘toxicovenomics’) (Laustsen et al., in this issue). Altogether, these complementary strategies, in combination with genomic, transcriptomic, and proteomic insights on venoms, highlight the dynamic nature of the original ‘venomics’ concept and its evolution into a new era of ‘integrative venomics’, providing a holistic, expanded view of venoms. Besides serving to deepen our understanding of the evolution of venoms and the essentially untapped potential of their toxins as sources
of chemical and pharmacological novelty, integrative venomics is also of utmost importance for understanding the pathological processes that underlie snakebite envenomings, for the rational design of novel broad-range polyspecific antivenoms to mitigate the deficit of antivenom supply in certain regions of the world, and nchez et al., 2015). for the optimized use of existing antivenoms (Sa This Special Issue on Toxinomics presents a collection of studies that focus along the above-mentioned lines, analyzing diverse aspects of venoms and toxins by the application of omic tools. Hence, Viala and co-workers report venomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom (Viala, Hildebrand, Fucase, et al., in this issue; Viala, Hildebrand, Trusch, et al., in this issue). Despite this species exhibits restricted ecological niche and geographical distribution, it has a raising medical importance as this rattlesnake population is increasing. The venomics analysis revealed a broad arsenal of toxins in C. vegrandis venom: PIII- and PII-metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, L-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins. In another paper from the same laboratory, Viala, Hildebrand, Fucase et al. (in this issue), Viala, Hildebrand, Trusch et al. (in this issue) describe the venom arsenal of the eastern brown snake, Pseudonaja textilis, the predominant cause of snakebites in mainland Australia, by combining high throughput proteomics and transcriptomics. This venom induces defibrination coagulopathy, renal failure and microangiopathic hemolytic anemia, and cardiovascular collapse has been described as an early cause of death in patients (White, 1999). However, so far, the mechanisms involved have not been fully identified. The combined proteomic and transcriptomic results revealed that the complex toxin repertoire of P. textilis venom includes, among other, protrombinase coagulation factors, neurotoxic textilotoxin PLA2 subunits and other PLA2s, 3FTxs, the Kunitz-type protease inhibitor textilinin, PIII-SVMPs, C-type lectins, cysteine rich secretory proteins, calreticulin, dipeptidase 2, a new transcript variant of venom coagulation factor 5a, and the existence of splicing variants of PLA2 modifying the UTR and signal peptide from a same mature protein. A quantitative proteomic analysis of the venoms of other medically important snakes, the vietnamese kraits Bungarus multicinctus and Bungarus fasciatus revealed the presence in these venoms of the same toxin families, but in completely different relative abundances, than found in venoms of congeneric species from other geographic regions (Ziganshin et al., in this issue). In another paper included in this Special Issue, Maitreyee Sharma and co-workers describe the first venomic analysis of Indian Russell's viper (Daboia
http://dx.doi.org/10.1016/j.toxicon.2015.10.024 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
2
Editorial / Toxicon xxx (2015) 1e4
russelii russelii) venom, a medically important snake and member of “Big Four” snakes of India, which was done by combination of gel filtration chromatography and tandem mass spectrometry (Sharma et al., in this issue). The strength of performing joint protein compositional and toxicity profiles is illustrated in studies on venoms of Micrurus ndez snake species from the Caribbean region of Costa Rica (Ferna et al., in this issue), the olive sea snake, Aipysurus laevis (Laustsen et al., in this issue), and the social wasp Polybia paulista (Dias ndez and co-workers show that the et al., in this issue). Ferna venoms of two sympatric monadal coral snakes, Micrurus alleni and Micrurus mosquitensis, display highly divergent compositions: the former dominated by three-finger toxins (3FTx) and the latter by phospholipases A2 (PLA2), and that protein family abundances correlated with enzymatic and toxic characteristics of the venoms. In addition, venoms from species of the M. mosquitensis clade, but not those within the M. alleni group, contain PLA2-like/Kunitztype inhibitor complex(es) that resemble the ASIC1a/2-activating MitTx heterodimeric toxin isolated from M. tener venom (Bohlen et al., 2011). Future genus-wide venomic investigation is necessary to assess the evolutionary origin and adaptive relevance of the phenotypic variability of Micrurus venoms and whether the reported venom 3FTx/PLA2 dichotomy represents a general trend across New World elapids. On the other hand, the venom of A. laevis appeared to be remarkably simple, consisting of phospholipases A2 (71.2%), three-finger toxins (3FTx; 25.3%), cysteine-rich secretory proteins (CRISP; 2.5%), and traces (0.2%) of a complement control module protein. Using a Toxicity Score Laustsen et al. (2015a), Laustsen et al. (in this issue) determined that short neurotoxins represent the most lethal components of the olive sea snake venom. Scorpion venoms include a number of peptides with different pharmacological activities. Toxins affecting ion channels ~ ez and Possani (in this are among the most studied. Santiban issue) review the structural classification and the proposed evolution of the more than 800 Knottin toxin-like peptide structures from different scorpions available in the Pfam and InterPro databases. Their results suggest that Naþ and Kþ ion-channel antagonists have evolved independently, suggesting that the current classification needs revision. A number of venomic studies included in this Special Issue involve investigations of venoms from poorly studied non-reptile organisms. Thus, the crude venom of Polybia paulista was analyzed by Dias and co-workers (Dias et al., in this issue) using HPLC-ITTOF/MS and MSn. Fourteen major peptides were detected and sequenced, and all the peptides were synthesized on solid-phase and submitted to a series of bioassays. Zhang et al. present in this Special Issue the first survey of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses (Zhang et al., in this issue). L. vittata is a medium-sized spider mainly distributed in the southwest of China. This study shows that L. vittata venom mainly consists of peptides exhibiting inhibitory effects on voltage-gated ion channels in rat dorsal root ganglia neurons, with a strongest inhibition on tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated Naþ channels. In addition, transcriptomic and bioinformatic analyses suggest that the venom may represent an untapped source of novel bioactive neurotoxins. In another paper included in this Special Issue, a mass spectrometry analysis of the venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported (RodríguezRavelo et al., in this issue). 200 individual molecular masses were identified in both venoms, from which 63 are identical in males and females, although the relative abundance of identical components appeared to be different among the genders. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom, and two new
putative Kþ-channel peptides were sequenced by Edman degradation. Finally, a conceptually novel method developed to investigate functions of venom components, using venom gene RNA interference knockdown in the venomous animal coupled with RNA sequencing in the envenomated host animal, is also published in this Special Issue on Toxinomics (Siebert et al., in this issue). A combined analysis of the venom gland transcriptome and venom proteome of Gloydius intermedius (Yang et al., in this issue) underpins the existence of hemorrhagic and neurotoxic venom phenotypes among Gloydius species. This finding sheds light on the origin and the evolution of type-II neurotoxic venoms in viperid venoms. Type-II venom represents a recent innovation in certain New World species of the genera Crotalus (Mackessy, 2008; Calvete et al., 2010) and Bothriechis (Lomonte et al., 2015). Gloydius intermedius is the only known Old World taxon containing type-II venom (Yang et al., 2015), and may thus represent the extant Asian descendant of the ancestral lineage that brought the neurotoxic venom phenotype across Beringia into the New Word. To date bottom-up venomics is the most widely used strategy to characterize the composition of venoms; however, locus resolution is rarely achieved through peptide-centric approaches. A major reason for this limitation is that the pre-MS separation steps do not separate from each other different toxin proteoforms or isoforms having high sequence similarity and/or overlapping molecular masses. Analysis of intact proteins using top-down mass spectrometry has the potential to eliminate this shortcoming (Petras et al., 2015). This Special Issue on Toxinomics opens with the report on the characterization of the venom proteome, and the bioactivity screening of the Anatolian Meadow viper, Vipera €çmen et al., in this issue). The minute amount of anatolica (Go venom available from this rare and small-sized species was profiled by a combination of bottom-up and top-down mass spectrometry, unveiling the complexity of the venom which would have remained undetected with conventional venomic approaches. Although acquiring a complete “parts list” of extant venoms is at the reach of proteomics, it is clear that we are unable to come back to the past to reconstruct the natural histories of venoms and the organisms that produce them. Notwithstanding, advancements in mass spectrometry and next-generation nucleic acid sequencing, the pillars of state-of-the-art venomics, coupled with the increasing emphasis on a combination of proteomic, transcriptomic, and genomic approaches, have resulted in the ability of venomic analyses to put forth clear, testable and falsifiable hypotheses about unknown aspects of venom biology that only future research will clarify. Venom gland transcriptome analyses are powerful for determining venom transcript expression profiles that potentially explain the translation from the genotype to an adaptive phenotype in the species that produces the venom (Margres et al., 2014). Transcriptome sequencing is now also widely adopted as an efficient means to aid in proteomic investigations of the chemical diversity of venoms (Brahma et al., 2015). To improve the efficiency of analysis of these large datasets, Prashanth and Lewis report in this Special Issue an efficient transcriptome analysis pipeline to accelerate the discovery and characterisation of cone snail venom peptides (Prashanth and Lewis, in this issue). On the other hand, research on genomes of venomous organisms is in its infancy. However, comparative genomic studies would provide the clues for constructing gene trees for understanding the origins and evolution of venoms. The genome includes the full repertoire of genes, not only those that are actively transcribed, but also the many additional genetic features such as introns, intergenic regions and cisand trans-transcriptional regulatory elements that have pivotal roles in the control of expression and the evolution of genes. At least in snakes, the evolution of venom genes is thought to involve the duplication of non-toxic physiological protein-coding genes
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
Editorial / Toxicon xxx (2015) 1e4
that are selective expressed in the venom gland, where they are neofunctionalized to become venom toxins (Casewell et al., 2013; Reyes-Velasco et al., 2015). The recent release of a draft genome sequence for the King Cobra, Ophiophagus hannah, has provided important support for the tandem duplication model of the gene duplication and neofunctionalization for venom locus evolution (Vonk et al., 2013). Snake venom PLA2 genes have been shown to often occur in duplicated tandem arrays (Ikeda et al., 2010). Studies of multi-gene protein families are crucial for understanding the role of gene duplication in generating protein diversity. However, many evolutionary analyses of gene families are based on coding sequences, and do not take into account many potentially confounding evolutionary factors, such as recombination and convergence due to selection. In this issue, Malhotra and co-workers use novel genomic PLA2 sequences from 20 species of understudied Asian pitvipers alongside available genomic PLA2 sequences from another four crotaline and several viperine species, to illustrate the importance of using non-coding and coding genomic information to elaborate models of toxin multi-gene family evolution (Malhotra et al., in this issue). In the same line, Yamaguchi and colleagues discuss genomic evidence for interisland variegation of venom Lys49 phospholipase A2 isozyme genes in Protobothrops snake species inhabiting the southwestern islands of Japan (Yamaguchi et al., in this issue). Despite the small number of articles represented in this Special Issue, their thematic and the experimental approaches used in conducting these studies allow us to predict, without any fear of contradiction, that next-generation venomics, in the broadest sense of this concept, promises a bright future to the field of venom research.
References Bohlen, C.J., Chesler, A.T., Sharif-Naeini, R., Medzihradszky, K.F., Zhou, S., King, D., S anchez, E.E., Burlingame, A.L., Basbaum, A.I., Julius, D., 2011. A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain. Nature 479, 410e414. Brahma, R.K., McCleary, R.J., Kini, R.M., Doley, R., 2015. Venom gland transcriptomics for identifying, cataloging, and characterizing venom proteins in snakes. Toxicon 93, 1e10. Calvete, J.J., Sanz, L., Cid, P., de la Torre, P., Flores-Díaz, M., Dos Santos, M.C., n, A., Gutie rrez, J.M., Borges, A., Bremo, A., Angulo, Y., Lomonte, B., Alape-Giro 2010. Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. J. Proteome Res. 9, 528e544. Calvete, J.J., 2014. Next-generation snake venomics: protein-locus resolution through venom proteome decomplexation. Expert Rev. Proteom. 11, 315e329. rrez, J.M., 2014. Omics meets biology: Calvete, J.J., Sanz, L., Pla, D., Lomonte, B., Gutie application to the design and preclinical assessment of antivenoms. Toxins 6, 3388e3405. Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A., Fry, B.G., 2013. Complex cocktails: the evolutionary novelty of venoms. Trends Ecol. Evol. 28, 219e229. Dias, N.B., de Souza, B.M., Gomes, P.C., Brigatte, P., Palma, M.S., 2015. Peptidome profiling of venom from the social wasp Polybia paulista. Toxicon (in this issue). Eichberg, S., Sanz, L., Calvete, J.J., Pla, D., 2015. Constructing comprehensive venom proteome reference maps for integrative venomics. Expert Rev. Proteom. 12, 557e573. ndez, J., Vargas-Vargas, N., Pla, D., Sasa, M., Rey-Su Ferna arez, P., Sanz, L., rrez, J.M., Calvete, J.J., Lomonte, B., 2015. Snake venomics of Micrurus alleni Gutie and Micrurus mosquitensis from the Caribbean region of Costa Rica reveals two divergent compositional patterns in New World elapids. Toxicon (in this issue). € Goçmen, B., Heiss, P., Petras, D., Nalbantsoy, A., Süssmuth, R.D., 2015. Mass spectrometry guided venom profiling and bioactivity screening of the Anatolian Meadow Viper, Vipera anatolica. Toxicon (in this issue). Ikeda, N., Chijiwa, T., Matsubara, K., Oda-Ueda, N., Hattori, S., Matsuda, Y., Ohno, M., 2010. Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake. Gene 461, 15e25. rrez, J.M., 2015a. Selecting Laustsen, A.H., Lohse, B., Lomonte, B., Engmark, M., Gutie
3
key toxins for focused development of elapid snake antivenoms and inhibitors guided by a toxicity score. Toxicon 104, 43e45. rrez, J.M., Rasmussen, A.R., Engmark, M., Gravlund, P., Laustsen, A.H., Gutie Sanders, K.L., Lohse, B., Lomonte, B., 2015b. Danger in the reef: proteome, toxicity, and neutralization of the venom of the olive sea snake, Aipysurus laevis. Toxicon. http://dx.doi.org/10.1016/j.toxicon.2015.07.008 (in this issue). ndez, J., Sanz, L., Pla, D., Gutie rrez, J.M., Lomonte, B., Mora-Obando, D., Ferna Calvete, J.J., 2015. First crotoxin-like phospholipase A2 complex from a New World non-rattlesnake species: nigroviriditoxin, from the arboreal neotropical snake Bothriechis nigroviridis. Toxicon 93, 144e154. Mackessy, S.P., 2008. Venom composition in rattlesnakes: trends and biological significance. In: Hayes, W.K., Beaman, K.R., Cardwell, M.D., Bush, S.P. (Eds.), The Biology of Rattlesnakes. University Press, Loma Linda, CA, pp. 495e510. Malhotra, A., Creer, S., Harris, J.B., Thorpe, R.S., 2015. The importance of being genomic: non-coding and coding sequences suggest different models of toxin multi-gene family evolution. Toxicon (in this issue). Margres, M.J., McGivern, J.J., Wray, K.P., Seavy, M., Calvin, K., Rokyta, D.R., 2014. Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). J. Proteom. 96, 145e158. Petras, D., Heiss, P., Süssmuth, R.D., Calvete, J.J., 2015. Venom proteomics of Indonesian King Cobra, Ophiophagus hannah: integrating top-down and bottom-up approaches. J. Proteome Res. 14, 2539e2556. Prashanth, J.R., Lewis, R.J., 2015. An efficient transcriptome analysis pipeline to accelerate venom peptide discovery and characterisation. Toxicon (in this issue). Reyes-Velasco, J., Card, D.C., Andrew, A.L., Shaney, K.J., Adams, R.H., Schield, D.R., Casewell, N.R., Mackessy, S.P., Castoe, T.A., 2015. Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom. Mol. Biol. Evol. 32, 173e183. Rodríguez-Ravelo, R., Batista, C.V., Coronas, F.I., Zamudio, F.Z., Hern andez pez, G., Ruiz-Urquiola, A., Possani, L.D., 2015. ComparOrihuela, L., Espinosa-Lo ative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus. Toxicon (in this issue). rrez, J.M., 2015. S anchez, L.V., Pla, D., Herrera, M., Chippaux, J.P., Calvete, J.J., Gutie Evaluation of the preclinical efficacy of four antivenoms, distributed in subSaharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria. Toxicon 106, 97e107. ~ ez-Lo pez, C.E., Possani, L.D., 2015. Overview of the Knottin scorpion toxinSantib an like peptides in scorpion venoms: insights on their classification and evolution. Toxicon (in this issue). Siebert, A.L., Wheeler, D., Werren, J.H., 2015. A new approach for investigating venom function applied to venom calreticulin in a parasitoid wasp. Toxicon (in this issue). Sharma, M., Das, D., Iyer, J.K., Kini, R.M., Doley, R., 2015. Unveiling the complexities of Daboia russelii venom, a medically important snake of India, by tandem mass spectrometry. Toxicon in this issue. Viala, V.L., Hildebrand, D., Fucase, T.M., Sciani, J.M., Prezotto-Neto, J.P., Riedner, M., Sanches, L., Nishimura, P.J., Oguiura, N., Pimenta, D.C., Schlüter, H., Betzel, C., Arni, R.K., Spencer, P.J., 2015a. Proteomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom: evidence of a broad arsenal of toxins. Toxicon (in this issue). Viala, V.L., Hildebrand, D., Trusch, M., Fucase, T.M., Sciani, J.M., Pimenta, D.C., Arni, R.K., Schlüter, H., Betzel, C., Mirtschin, P., Dunstan, N., Spencer, P.J., 2015b. Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection of new venom proteins and splicing variants. Toxicon (in this issue). Vonk, F.J., Casewell, N.R., Henkel, C.V., Heimberg, A.M., Jansen, H.J., McCleary, R.J., Kerkkamp, H.M., Vos, R.A., Guerreiro, I., Calvete, J.J., Wüster, W., Woods, A.E., Logan, J.M., Harrison, R.A., Castoe, T.A., de Koning, A.P., Pollock, D.D., Yandell, M., Calderon, D., Renjifo, C., Currier, R.B., Salgado, D., Pla, D., Sanz, L., Hyder, A.S., Ribeiro, J.M., Arntzen, J.W., van den Thillart, G.E., Boetzer, M., Pirovano, W., Dirks, R.P., Spaink, H.P., Duboule, D., McGlinn, E., Kini, R.M., Richardson, M.K., 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. U. S. A. 110, 20651e20656. White, J., 1999. Envenomation, prevention and treatment in Australia. In: Mackessy, S.P. (Ed.), Handbook of Venoms and Toxins of Reptiles. CRC Press, Boca Raton, p. 521. Yamaguchi, K., Chijiwa, T., Yamamura, T., Ikeda, N., Yatsui, T., Hayama, S., Hattori, S., Oda-Ueda, N., Ohno, M., 2015. Interisland variegation of venom [Lys49]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan. Toxicon (in this issue). Yang, Z.M., Guo, Q., Ma, Z.-R., Chen, Y., Wang, Z.-Z., Wang, X.-M., Wang, Y.-M., Tsai, I.H., 2015a. Structures and functions of crotoxin-like heterodimers and acidic phospholipases A2 from Gloydius intermedius venom: insights into the origin of neurotoxic-type rattlesnakes. J. Proteom. 112, 210e223. Yang, Z.M., Yang, Y.E., Chen, Y., Cao, J., Zhang, C., Liu, L.L., Wang, Z.Z., Wang, X.M., Wang, Y.M., Tsai, I.H., 2015b. Transcriptome and proteome of the highly neurotoxic venom of Gloydius intermedius. Toxicon (in this issue). Zhang, F., Liu, C., Tan, H., Wang, H., Jiang, Y., Liang, S., Zhang, F., Liu, Z., 2015. A survey
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
4
Editorial / Toxicon xxx (2015) 1e4
of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses. Toxicon (in this issue). Ziganshin, R.H., Kovalchuk, S.I., Arapidi, G.P., Starkov, V.G., Hoang, A.N., Thi Nguyen, T.T., Nguyen, K.C., Shoibonov, B.B., Tsetlin, V.I., Utkin, Y.N., 2015. Quantitative proteomic analysis of Vietnamese krait venoms: neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. Toxicon (in this issue). *
Juan J. Calvete Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain
Bruno Lomonte** Instituto Clodomiro Picado, Universidad de Costa Rica, Costa Rica *
Corresponding author.
** Corresponding author. E-mail address: [email protected] (J.J. Calvete). E-mail address: [email protected] (B. Lomonte).
Available online xxx
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Editorial
A bright future for integrative venomics
Venomous secretions are produced by a myriad of animal species, from invertebrates to vertebrates. As a general rule, peptides and proteins represent the most abundant and functionally relevant components of these dangerous “cocktails”. It may be argued that the first and indispensable requirement to understand a particular venom is to know its composition, and, to this end, the combination of -omics technologies have emerged as the most powerful tools available to date (Eichberg et al., 2015). The application of -omics methodologies to the study of venoms of diverse origins has grown steadily in recent years, and promises to open the door to a landscape where the protein composition of a large proportion of biologically and clinically relevant venoms will be known in deep detail. In combination with a wide variety of experimental approaches, the availability of such comprehensive basic information will pave the way to a profound understanding on the emergence and evolution of venom toxins, including their structural and functional diversification during millions of years of natural selection. The ever-increasing capabilities of the -omics analytical platforms and their associated bioinformatic algorithms can provide information, which required years or months of work in earlier times, in hours or even minutes today, using just minuscule amounts of samples. Locus-resolution for individual venom components will increasingly be the goal of proteomic analyses (Calvete, 2014; Eichberg et al., 2015), in parallel growth with the availability of highquality transcriptomic and genomic data on venomous organisms, a major limiting factor at present. The quantitative profiling of venom proteins is also increasingly being pursued, since it adds a further dimension to the informative value provided by their mere qualitative cataloguing. Indeed, understanding the molecular basis of complex adaptive traits, such as venoms, demands both qualitative and quantitative comparisons of the temporal and spatial patterns of venom variation. Other complementary strategies related to -omics analyses of venoms (‘venomics’) involve the immunorecognition of their components by antibodies present in therapeutical antivenoms ('antivenomics') (Calvete et al., 2014; nchez et al., 2015)- and the functional assessment of toxic venom Sa components in experimental models (‘toxicovenomics’) (Laustsen et al., in this issue). Altogether, these complementary strategies, in combination with genomic, transcriptomic, and proteomic insights on venoms, highlight the dynamic nature of the original ‘venomics’ concept and its evolution into a new era of ‘integrative venomics’, providing a holistic, expanded view of venoms. Besides serving to deepen our understanding of the evolution of venoms and the essentially untapped potential of their toxins as sources
of chemical and pharmacological novelty, integrative venomics is also of utmost importance for understanding the pathological processes that underlie snakebite envenomings, for the rational design of novel broad-range polyspecific antivenoms to mitigate the deficit of antivenom supply in certain regions of the world, and nchez et al., 2015). for the optimized use of existing antivenoms (Sa This Special Issue on Toxinomics presents a collection of studies that focus along the above-mentioned lines, analyzing diverse aspects of venoms and toxins by the application of omic tools. Hence, Viala and co-workers report venomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom (Viala, Hildebrand, Fucase, et al., in this issue; Viala, Hildebrand, Trusch, et al., in this issue). Despite this species exhibits restricted ecological niche and geographical distribution, it has a raising medical importance as this rattlesnake population is increasing. The venomics analysis revealed a broad arsenal of toxins in C. vegrandis venom: PIII- and PII-metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, L-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins. In another paper from the same laboratory, Viala, Hildebrand, Fucase et al. (in this issue), Viala, Hildebrand, Trusch et al. (in this issue) describe the venom arsenal of the eastern brown snake, Pseudonaja textilis, the predominant cause of snakebites in mainland Australia, by combining high throughput proteomics and transcriptomics. This venom induces defibrination coagulopathy, renal failure and microangiopathic hemolytic anemia, and cardiovascular collapse has been described as an early cause of death in patients (White, 1999). However, so far, the mechanisms involved have not been fully identified. The combined proteomic and transcriptomic results revealed that the complex toxin repertoire of P. textilis venom includes, among other, protrombinase coagulation factors, neurotoxic textilotoxin PLA2 subunits and other PLA2s, 3FTxs, the Kunitz-type protease inhibitor textilinin, PIII-SVMPs, C-type lectins, cysteine rich secretory proteins, calreticulin, dipeptidase 2, a new transcript variant of venom coagulation factor 5a, and the existence of splicing variants of PLA2 modifying the UTR and signal peptide from a same mature protein. A quantitative proteomic analysis of the venoms of other medically important snakes, the vietnamese kraits Bungarus multicinctus and Bungarus fasciatus revealed the presence in these venoms of the same toxin families, but in completely different relative abundances, than found in venoms of congeneric species from other geographic regions (Ziganshin et al., in this issue). In another paper included in this Special Issue, Maitreyee Sharma and co-workers describe the first venomic analysis of Indian Russell's viper (Daboia
http://dx.doi.org/10.1016/j.toxicon.2015.10.024 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
2
Editorial / Toxicon xxx (2015) 1e4
russelii russelii) venom, a medically important snake and member of “Big Four” snakes of India, which was done by combination of gel filtration chromatography and tandem mass spectrometry (Sharma et al., in this issue). The strength of performing joint protein compositional and toxicity profiles is illustrated in studies on venoms of Micrurus ndez snake species from the Caribbean region of Costa Rica (Ferna et al., in this issue), the olive sea snake, Aipysurus laevis (Laustsen et al., in this issue), and the social wasp Polybia paulista (Dias ndez and co-workers show that the et al., in this issue). Ferna venoms of two sympatric monadal coral snakes, Micrurus alleni and Micrurus mosquitensis, display highly divergent compositions: the former dominated by three-finger toxins (3FTx) and the latter by phospholipases A2 (PLA2), and that protein family abundances correlated with enzymatic and toxic characteristics of the venoms. In addition, venoms from species of the M. mosquitensis clade, but not those within the M. alleni group, contain PLA2-like/Kunitztype inhibitor complex(es) that resemble the ASIC1a/2-activating MitTx heterodimeric toxin isolated from M. tener venom (Bohlen et al., 2011). Future genus-wide venomic investigation is necessary to assess the evolutionary origin and adaptive relevance of the phenotypic variability of Micrurus venoms and whether the reported venom 3FTx/PLA2 dichotomy represents a general trend across New World elapids. On the other hand, the venom of A. laevis appeared to be remarkably simple, consisting of phospholipases A2 (71.2%), three-finger toxins (3FTx; 25.3%), cysteine-rich secretory proteins (CRISP; 2.5%), and traces (0.2%) of a complement control module protein. Using a Toxicity Score Laustsen et al. (2015a), Laustsen et al. (in this issue) determined that short neurotoxins represent the most lethal components of the olive sea snake venom. Scorpion venoms include a number of peptides with different pharmacological activities. Toxins affecting ion channels ~ ez and Possani (in this are among the most studied. Santiban issue) review the structural classification and the proposed evolution of the more than 800 Knottin toxin-like peptide structures from different scorpions available in the Pfam and InterPro databases. Their results suggest that Naþ and Kþ ion-channel antagonists have evolved independently, suggesting that the current classification needs revision. A number of venomic studies included in this Special Issue involve investigations of venoms from poorly studied non-reptile organisms. Thus, the crude venom of Polybia paulista was analyzed by Dias and co-workers (Dias et al., in this issue) using HPLC-ITTOF/MS and MSn. Fourteen major peptides were detected and sequenced, and all the peptides were synthesized on solid-phase and submitted to a series of bioassays. Zhang et al. present in this Special Issue the first survey of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses (Zhang et al., in this issue). L. vittata is a medium-sized spider mainly distributed in the southwest of China. This study shows that L. vittata venom mainly consists of peptides exhibiting inhibitory effects on voltage-gated ion channels in rat dorsal root ganglia neurons, with a strongest inhibition on tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated Naþ channels. In addition, transcriptomic and bioinformatic analyses suggest that the venom may represent an untapped source of novel bioactive neurotoxins. In another paper included in this Special Issue, a mass spectrometry analysis of the venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported (RodríguezRavelo et al., in this issue). 200 individual molecular masses were identified in both venoms, from which 63 are identical in males and females, although the relative abundance of identical components appeared to be different among the genders. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom, and two new
putative Kþ-channel peptides were sequenced by Edman degradation. Finally, a conceptually novel method developed to investigate functions of venom components, using venom gene RNA interference knockdown in the venomous animal coupled with RNA sequencing in the envenomated host animal, is also published in this Special Issue on Toxinomics (Siebert et al., in this issue). A combined analysis of the venom gland transcriptome and venom proteome of Gloydius intermedius (Yang et al., in this issue) underpins the existence of hemorrhagic and neurotoxic venom phenotypes among Gloydius species. This finding sheds light on the origin and the evolution of type-II neurotoxic venoms in viperid venoms. Type-II venom represents a recent innovation in certain New World species of the genera Crotalus (Mackessy, 2008; Calvete et al., 2010) and Bothriechis (Lomonte et al., 2015). Gloydius intermedius is the only known Old World taxon containing type-II venom (Yang et al., 2015), and may thus represent the extant Asian descendant of the ancestral lineage that brought the neurotoxic venom phenotype across Beringia into the New Word. To date bottom-up venomics is the most widely used strategy to characterize the composition of venoms; however, locus resolution is rarely achieved through peptide-centric approaches. A major reason for this limitation is that the pre-MS separation steps do not separate from each other different toxin proteoforms or isoforms having high sequence similarity and/or overlapping molecular masses. Analysis of intact proteins using top-down mass spectrometry has the potential to eliminate this shortcoming (Petras et al., 2015). This Special Issue on Toxinomics opens with the report on the characterization of the venom proteome, and the bioactivity screening of the Anatolian Meadow viper, Vipera €çmen et al., in this issue). The minute amount of anatolica (Go venom available from this rare and small-sized species was profiled by a combination of bottom-up and top-down mass spectrometry, unveiling the complexity of the venom which would have remained undetected with conventional venomic approaches. Although acquiring a complete “parts list” of extant venoms is at the reach of proteomics, it is clear that we are unable to come back to the past to reconstruct the natural histories of venoms and the organisms that produce them. Notwithstanding, advancements in mass spectrometry and next-generation nucleic acid sequencing, the pillars of state-of-the-art venomics, coupled with the increasing emphasis on a combination of proteomic, transcriptomic, and genomic approaches, have resulted in the ability of venomic analyses to put forth clear, testable and falsifiable hypotheses about unknown aspects of venom biology that only future research will clarify. Venom gland transcriptome analyses are powerful for determining venom transcript expression profiles that potentially explain the translation from the genotype to an adaptive phenotype in the species that produces the venom (Margres et al., 2014). Transcriptome sequencing is now also widely adopted as an efficient means to aid in proteomic investigations of the chemical diversity of venoms (Brahma et al., 2015). To improve the efficiency of analysis of these large datasets, Prashanth and Lewis report in this Special Issue an efficient transcriptome analysis pipeline to accelerate the discovery and characterisation of cone snail venom peptides (Prashanth and Lewis, in this issue). On the other hand, research on genomes of venomous organisms is in its infancy. However, comparative genomic studies would provide the clues for constructing gene trees for understanding the origins and evolution of venoms. The genome includes the full repertoire of genes, not only those that are actively transcribed, but also the many additional genetic features such as introns, intergenic regions and cisand trans-transcriptional regulatory elements that have pivotal roles in the control of expression and the evolution of genes. At least in snakes, the evolution of venom genes is thought to involve the duplication of non-toxic physiological protein-coding genes
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
Editorial / Toxicon xxx (2015) 1e4
that are selective expressed in the venom gland, where they are neofunctionalized to become venom toxins (Casewell et al., 2013; Reyes-Velasco et al., 2015). The recent release of a draft genome sequence for the King Cobra, Ophiophagus hannah, has provided important support for the tandem duplication model of the gene duplication and neofunctionalization for venom locus evolution (Vonk et al., 2013). Snake venom PLA2 genes have been shown to often occur in duplicated tandem arrays (Ikeda et al., 2010). Studies of multi-gene protein families are crucial for understanding the role of gene duplication in generating protein diversity. However, many evolutionary analyses of gene families are based on coding sequences, and do not take into account many potentially confounding evolutionary factors, such as recombination and convergence due to selection. In this issue, Malhotra and co-workers use novel genomic PLA2 sequences from 20 species of understudied Asian pitvipers alongside available genomic PLA2 sequences from another four crotaline and several viperine species, to illustrate the importance of using non-coding and coding genomic information to elaborate models of toxin multi-gene family evolution (Malhotra et al., in this issue). In the same line, Yamaguchi and colleagues discuss genomic evidence for interisland variegation of venom Lys49 phospholipase A2 isozyme genes in Protobothrops snake species inhabiting the southwestern islands of Japan (Yamaguchi et al., in this issue). Despite the small number of articles represented in this Special Issue, their thematic and the experimental approaches used in conducting these studies allow us to predict, without any fear of contradiction, that next-generation venomics, in the broadest sense of this concept, promises a bright future to the field of venom research.
References Bohlen, C.J., Chesler, A.T., Sharif-Naeini, R., Medzihradszky, K.F., Zhou, S., King, D., S anchez, E.E., Burlingame, A.L., Basbaum, A.I., Julius, D., 2011. A heteromeric Texas coral snake toxin targets acid-sensing ion channels to produce pain. Nature 479, 410e414. Brahma, R.K., McCleary, R.J., Kini, R.M., Doley, R., 2015. Venom gland transcriptomics for identifying, cataloging, and characterizing venom proteins in snakes. Toxicon 93, 1e10. Calvete, J.J., Sanz, L., Cid, P., de la Torre, P., Flores-Díaz, M., Dos Santos, M.C., n, A., Gutie rrez, J.M., Borges, A., Bremo, A., Angulo, Y., Lomonte, B., Alape-Giro 2010. Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. J. Proteome Res. 9, 528e544. Calvete, J.J., 2014. Next-generation snake venomics: protein-locus resolution through venom proteome decomplexation. Expert Rev. Proteom. 11, 315e329. rrez, J.M., 2014. Omics meets biology: Calvete, J.J., Sanz, L., Pla, D., Lomonte, B., Gutie application to the design and preclinical assessment of antivenoms. Toxins 6, 3388e3405. Casewell, N.R., Wüster, W., Vonk, F.J., Harrison, R.A., Fry, B.G., 2013. Complex cocktails: the evolutionary novelty of venoms. Trends Ecol. Evol. 28, 219e229. Dias, N.B., de Souza, B.M., Gomes, P.C., Brigatte, P., Palma, M.S., 2015. Peptidome profiling of venom from the social wasp Polybia paulista. Toxicon (in this issue). Eichberg, S., Sanz, L., Calvete, J.J., Pla, D., 2015. Constructing comprehensive venom proteome reference maps for integrative venomics. Expert Rev. Proteom. 12, 557e573. ndez, J., Vargas-Vargas, N., Pla, D., Sasa, M., Rey-Su Ferna arez, P., Sanz, L., rrez, J.M., Calvete, J.J., Lomonte, B., 2015. Snake venomics of Micrurus alleni Gutie and Micrurus mosquitensis from the Caribbean region of Costa Rica reveals two divergent compositional patterns in New World elapids. Toxicon (in this issue). € Goçmen, B., Heiss, P., Petras, D., Nalbantsoy, A., Süssmuth, R.D., 2015. Mass spectrometry guided venom profiling and bioactivity screening of the Anatolian Meadow Viper, Vipera anatolica. Toxicon (in this issue). Ikeda, N., Chijiwa, T., Matsubara, K., Oda-Ueda, N., Hattori, S., Matsuda, Y., Ohno, M., 2010. Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake. Gene 461, 15e25. rrez, J.M., 2015a. Selecting Laustsen, A.H., Lohse, B., Lomonte, B., Engmark, M., Gutie
3
key toxins for focused development of elapid snake antivenoms and inhibitors guided by a toxicity score. Toxicon 104, 43e45. rrez, J.M., Rasmussen, A.R., Engmark, M., Gravlund, P., Laustsen, A.H., Gutie Sanders, K.L., Lohse, B., Lomonte, B., 2015b. Danger in the reef: proteome, toxicity, and neutralization of the venom of the olive sea snake, Aipysurus laevis. Toxicon. http://dx.doi.org/10.1016/j.toxicon.2015.07.008 (in this issue). ndez, J., Sanz, L., Pla, D., Gutie rrez, J.M., Lomonte, B., Mora-Obando, D., Ferna Calvete, J.J., 2015. First crotoxin-like phospholipase A2 complex from a New World non-rattlesnake species: nigroviriditoxin, from the arboreal neotropical snake Bothriechis nigroviridis. Toxicon 93, 144e154. Mackessy, S.P., 2008. Venom composition in rattlesnakes: trends and biological significance. In: Hayes, W.K., Beaman, K.R., Cardwell, M.D., Bush, S.P. (Eds.), The Biology of Rattlesnakes. University Press, Loma Linda, CA, pp. 495e510. Malhotra, A., Creer, S., Harris, J.B., Thorpe, R.S., 2015. The importance of being genomic: non-coding and coding sequences suggest different models of toxin multi-gene family evolution. Toxicon (in this issue). Margres, M.J., McGivern, J.J., Wray, K.P., Seavy, M., Calvin, K., Rokyta, D.R., 2014. Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). J. Proteom. 96, 145e158. Petras, D., Heiss, P., Süssmuth, R.D., Calvete, J.J., 2015. Venom proteomics of Indonesian King Cobra, Ophiophagus hannah: integrating top-down and bottom-up approaches. J. Proteome Res. 14, 2539e2556. Prashanth, J.R., Lewis, R.J., 2015. An efficient transcriptome analysis pipeline to accelerate venom peptide discovery and characterisation. Toxicon (in this issue). Reyes-Velasco, J., Card, D.C., Andrew, A.L., Shaney, K.J., Adams, R.H., Schield, D.R., Casewell, N.R., Mackessy, S.P., Castoe, T.A., 2015. Expression of venom gene homologs in diverse python tissues suggests a new model for the evolution of snake venom. Mol. Biol. Evol. 32, 173e183. Rodríguez-Ravelo, R., Batista, C.V., Coronas, F.I., Zamudio, F.Z., Hern andez pez, G., Ruiz-Urquiola, A., Possani, L.D., 2015. ComparOrihuela, L., Espinosa-Lo ative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus. Toxicon (in this issue). rrez, J.M., 2015. S anchez, L.V., Pla, D., Herrera, M., Chippaux, J.P., Calvete, J.J., Gutie Evaluation of the preclinical efficacy of four antivenoms, distributed in subSaharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria. Toxicon 106, 97e107. ~ ez-Lo pez, C.E., Possani, L.D., 2015. Overview of the Knottin scorpion toxinSantib an like peptides in scorpion venoms: insights on their classification and evolution. Toxicon (in this issue). Siebert, A.L., Wheeler, D., Werren, J.H., 2015. A new approach for investigating venom function applied to venom calreticulin in a parasitoid wasp. Toxicon (in this issue). Sharma, M., Das, D., Iyer, J.K., Kini, R.M., Doley, R., 2015. Unveiling the complexities of Daboia russelii venom, a medically important snake of India, by tandem mass spectrometry. Toxicon in this issue. Viala, V.L., Hildebrand, D., Fucase, T.M., Sciani, J.M., Prezotto-Neto, J.P., Riedner, M., Sanches, L., Nishimura, P.J., Oguiura, N., Pimenta, D.C., Schlüter, H., Betzel, C., Arni, R.K., Spencer, P.J., 2015a. Proteomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom: evidence of a broad arsenal of toxins. Toxicon (in this issue). Viala, V.L., Hildebrand, D., Trusch, M., Fucase, T.M., Sciani, J.M., Pimenta, D.C., Arni, R.K., Schlüter, H., Betzel, C., Mirtschin, P., Dunstan, N., Spencer, P.J., 2015b. Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection of new venom proteins and splicing variants. Toxicon (in this issue). Vonk, F.J., Casewell, N.R., Henkel, C.V., Heimberg, A.M., Jansen, H.J., McCleary, R.J., Kerkkamp, H.M., Vos, R.A., Guerreiro, I., Calvete, J.J., Wüster, W., Woods, A.E., Logan, J.M., Harrison, R.A., Castoe, T.A., de Koning, A.P., Pollock, D.D., Yandell, M., Calderon, D., Renjifo, C., Currier, R.B., Salgado, D., Pla, D., Sanz, L., Hyder, A.S., Ribeiro, J.M., Arntzen, J.W., van den Thillart, G.E., Boetzer, M., Pirovano, W., Dirks, R.P., Spaink, H.P., Duboule, D., McGlinn, E., Kini, R.M., Richardson, M.K., 2013. The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. U. S. A. 110, 20651e20656. White, J., 1999. Envenomation, prevention and treatment in Australia. In: Mackessy, S.P. (Ed.), Handbook of Venoms and Toxins of Reptiles. CRC Press, Boca Raton, p. 521. Yamaguchi, K., Chijiwa, T., Yamamura, T., Ikeda, N., Yatsui, T., Hayama, S., Hattori, S., Oda-Ueda, N., Ohno, M., 2015. Interisland variegation of venom [Lys49]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan. Toxicon (in this issue). Yang, Z.M., Guo, Q., Ma, Z.-R., Chen, Y., Wang, Z.-Z., Wang, X.-M., Wang, Y.-M., Tsai, I.H., 2015a. Structures and functions of crotoxin-like heterodimers and acidic phospholipases A2 from Gloydius intermedius venom: insights into the origin of neurotoxic-type rattlesnakes. J. Proteom. 112, 210e223. Yang, Z.M., Yang, Y.E., Chen, Y., Cao, J., Zhang, C., Liu, L.L., Wang, Z.Z., Wang, X.M., Wang, Y.M., Tsai, I.H., 2015b. Transcriptome and proteome of the highly neurotoxic venom of Gloydius intermedius. Toxicon (in this issue). Zhang, F., Liu, C., Tan, H., Wang, H., Jiang, Y., Liang, S., Zhang, F., Liu, Z., 2015. A survey
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024
4
Editorial / Toxicon xxx (2015) 1e4
of the venom of the spider Lycosa vittata by biochemical, pharmacological and transcriptomic analyses. Toxicon (in this issue). Ziganshin, R.H., Kovalchuk, S.I., Arapidi, G.P., Starkov, V.G., Hoang, A.N., Thi Nguyen, T.T., Nguyen, K.C., Shoibonov, B.B., Tsetlin, V.I., Utkin, Y.N., 2015. Quantitative proteomic analysis of Vietnamese krait venoms: neurotoxins are the major components in Bungarus multicinctus and phospholipases A2 in Bungarus fasciatus. Toxicon (in this issue). *
Juan J. Calvete Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain
Bruno Lomonte** Instituto Clodomiro Picado, Universidad de Costa Rica, Costa Rica *
Corresponding author.
** Corresponding author. E-mail address: [email protected] (J.J. Calvete). E-mail address: [email protected] (B. Lomonte).
Available online xxx
Please cite this article in press as: Calvete, J.J., Lomonte, B.A bright future for integrative venomics, Toxicon (2015), http://dx.doi.org/10.1016/ j.toxicon.2015.10.024