Posters and presentation information
Thank you for considering to present your work as a poster at Venoms and Toxins 2020 Virtual.
Digital poster submission deadline: Prepare your poster as you would normally do for printing, and submit your final poster as both PDF and JPG/PNG files via the link below no later than 07th September 2020. Late posters may not be included in the conference programme. Please DO NOT send your poster files by email.
Naming your poster files: Name your poster files as follows: <your surname>-Ven20V-Poster.pdf | <your surname>-Ven20V-Poster.png | <your surname>-Ven20V-Poster.jpg, etc. For example, for David Jones, name your file as Jones-Ven20V-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, venoms2020v, Oxford-venoms-poster. Such files will be automatically rejected.
Poster presentation: Posters will be made available via a secure page to the symposium participants before the meeting. There will be two ways to interact with the poster presenters:
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- the participants can post their questions on Twitter at any time using the meeting hashtag #VenOx20V, as well as the poster specific hashtag (given under each poster abstract) – do tag @LPMHealthcare in your tweets.
The poster presenters should regularly check Twitter for any questions about their posters before, during and after the meeting, and post their answers on Twitter using appropriate hashtags, as above.
Any further information about the poster presentations at this digital meeting will be available in the future.
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Accepted posters
(Presenters in Bold)
Accepted poster abstracts (Unedited) will be published below. If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by email at VenomsOxford@gmail.com.
Discovery of broadly-neutralizing recombinant human monoclonal antibodies against cobra cytotoxins using cross-panning phage display
(Poster)
Twitter: #VenOx20V & #SAhmadi
Shirin Ahmadi, Christoffer Sorensen, Melissa Hale, Tulika, Andreas H. Laustsen
Department of Bioengineering and Biomedicine, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark
Snakebite envenoming is a Category A Neglected Tropical Disease that affects the lives of hundreds of thousands of people, predominantly those who live in the tropical and sub-tropical regions of the world. The only standard treatment for the victims is administration of the antivenoms that are mixtures of polyclonal antibodies obtained from the sera of hyper-immunized animals. Although antivenoms have been effective in saving numerous lives, they have some drawbacks including, but not limited to, the propensity of inducing adverse immune responses in victims due to their heterologous nature, as well as low efficacy in neutralizing small venom toxins such as cytotoxins. Therefore, different research avenues are pursued to overcome the drawbacks of the animal-derived antivenoms. One of the promising alternatives is recombinant antivenom which is a cocktail of recombinant human monoclonal antibodies, where each monoclonal antibody is specifically selected to neutralize one medically relevant venom component. However, the number of monoclonal antibodies included in the formulation of a recombinant antivenom must be limited to make them feasible and cost-competitive to manufacture. In this relation, developing broadly-neutralizing antibodies can be a rational approach for decreasing the number of different monoclonal antibodies that are needed for producing recombinant antivenoms. Here, we demonstrate that by using a cross-panning phage display strategy, we were able to discover a recombinant human antibody in single chain variable fragment (scFv) format that could broadly bind to different cytotoxin-containing venom fractions obtained from several different African cobra snakes. This monoclonal scFv was converted to IgG format, expressed in CHO cells and demonstrated to retain the broadly-binding capability of the parental monoclonal scFv in ELISA. In the future, the IgG molecules will be tested in in vitro and in vivo neutralization assays.
Sponge-in-a-tube: a simple and practical technique for fish venom extraction – Protein content analysis in scorpionfish (Scorpaenidae) and weever fish (Trachinidae)
(Poster)
Twitter: #VenOx20V & #FAlmada
Frederico Almada1, José Neto1, Américo G. Duarte2, Francisca Rodrigues1, Luisa Maia3, José J.G. Moura3, Stephane Besson3, Juan Calvete4
1 MARE—Marine and Environmental Sciences Centre, ISPA Instituto Universitário, Rua Jardim do Tabaco 34, 1149-041 Lisboa, Portugal
2 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
3 LAQV-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa 2829-516 Caparica, Portugal
4 Evolutionary and Translational Venomics Laboratory, CSIC, Jaume Roig 11, 46010 Valencia, Spain
Prospective research fields targeting new marine compounds have a wide range, from life sciences and drug discovery to engineering and biomimetics. Surprisingly, bioactive compounds such as the ones obtained from thousands of venomous fish species have been largely overlooked. The major limitations on the study of fish venoms are: 1) passive venom delivery apparatus makes its extraction difficult; 2) venom thermal lability makes its preservation hard; 3) effective cryptobenthic camouflage with many species hiding among rocks, algae and sediments on the sea floor; 4) challenging taxonomic identifications due to morphological similarities; 5) neglected number of accidents with humans and 6) assumption that fish venoms are conserved across species because they evolved as a defensive strategy and only aim to stun their predators. However, no comparative studies are available to demonstrate the complexity of these natural cocktails. With the goal to facilitate research in this field, we describe a new protocol for fish venom extraction. Sponge-in-a-tube consists in a synthetic sponge within a collection tube that is used to apply pressure on the venomous spines and absorb the venom. This technique is easy to apply during field work, facilitates swift sample preservation and allows immediate release of the fish. Sponge-in-a-tube tackles the disadvantages of former methods, namely sacrifice the fish or cripple its defensive apparatus. SDS-PAGE analysis revealed that it does not affect venom electrophoretic profile and yields crude venom extracts with less contaminants. Recent results also show that sponge-in-a-tube maintains venom functional properties namely their haemolytic effects. It was tested for weever fish (from 9cm) and scorpion fish (to 45cm) and is easy to apply even by non-specialist researchers. In the future, comparative studies on fish venoms will allow to prioritize research in fish toxinology and pave the way for potential biotechnological applications.
A comparative study between the flow properties of spitting and non-spitting cobra venom
(Poster)
Twitter: #VenOx20V & #EBLedesma
Edgar Barajas-Ledesma1, Ignazio Avella2, Nicholas R. Casewell3, Robert A. Harrison3, Paul Rowley3, Edouard Crittenden3, Wolfgang Wüster4, Riccardo Castiglia5, Chris Holland1 and Arie van der Meijden2
1Department of Materials Science and Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK
2CIBIO/InBIO – Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Rua Padre Armando Quintas 7, 4485–661, Vairão, Portugal
3Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK
4Molecular Ecology and Fisheries Genetics Laboratory, School of Natural Sciences, Bangor University, Bangor, LL57 2UW, UK
5Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Università di Roma “La Sapienza”, CAP 00151, via A. Borelli 50, Rome, Italy
Snake venom is commonly seen as a potent means of immobilizing prey and facilitating the digestive process. However not only can venom be used for predation, but also defence, with several species evolving the ability to spit venom rapidly and accurately over a distance of several meters. Evidence of unambiguously defensive spitting can be found in some members of the Naja and Hemachatus genus of the Elapidae family, however whilst there have been several studies in the past describing morphological differences in the venom ejection mechanism of spitting and non-spitting cobras, our knowledge of the flow properties of their venom is surprisingly limited. To address this gap in our knowledge we studied thirteen spitting and non-spitting cobras (genus Naja), as well as the rinkhals (Hemachatus haemachatus) and one viper (Bitis arietans), with a hypothesis that the physical properties of venom have been selected for a specific venom delivery mode: spitting or biting. For all samples pH, protein concentration and viscosity were measured and integrated into calculations of pressure requirements to eject venom for certain fang morphologies. Despite noticeable differences in the modes of venom delivery between the variety of snake species studied here, we found no significant differences in the rheological and physical properties of the studied venoms between these functional groups, suggesting possible selection for conservation. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our model of fang pressure suggests that the pressure requirements to eject venom are lower in spitting than in non-spitting cobras. In summary, this is the first study correlating the physical properties of venom in spitting and non-spitting cobras and our findings suggest that spitting behaviour in cobras should probably not be seen as a binary trait, but may vary continuously in prevalence among the species of the genus Naja. Thus, understanding the evolution, or lack of evolution, of specialised spitting behaviours and associated physical adaptations would likely require studying the efficacy and prevalence of spitting behaviour as a defence against natural predators.
Clinical-Epidemiological aspects of snakebite accidents in the State of Goiás, Central Brazil: a temporal analysis (2003-2018)
(Poster)
Twitter: #VenOx20V & #VLBarros1
Verônica Lacerda de Barros1, Anita de Moura Pessoa2, Raimundo Nonato Leite Pinto3, Raphael Ladislau de Alcântara4, Rafael Stuani Floriano5, and Nelson Jorge da Silva Jr2,3,6
1Undergraduate Program in Medical Veterinary, School of Biological and Agrarian Sciences, Pontifical Catholic University of Goiás, Av. Engler, s/n, Jardim Marilizia, 74605-010, Goiânia, GO, Brazil
2Graduate Program in Biotechnology and Biodiversity, Institute of Tropical Pathology and Public Health, Rua 253, s/n, Setor Leste Universitário, 74001-970, Goiânia, GO, Brazil
3Undergraduate Program in Medicine, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goiás, Rua 235, 15, Setor Leste Universitário, 74605-050, Goiânia, GO, Brazil
4Undergraduate Program in Biomedicine, Universidade Paulista, Rodovia BR 153, km 503, Fazenda Botafogo, 74845-090, Goiânia, GO, Brazil
5Graduate Program in Health Sciences, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2-205, 19067-175, Presidente Prudente, SP, Brazil
6Graduate Program in Environmental Sciences and Health, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goiás, Rua 232, 128, 74605-140, Goiânia, GO, Brazil
In this study, we have described the profile of snakebite accidents occurred in the State of Goiás (Brazil) between 2003–2018, based on the notification forms from the Toxicopharmacological Information Center of the State of Goiás. There were 24,726 accidents with venomous animals, being 11,245 with snakes (45.5%) and 13,481 with terrestrial arthropods (54.5%). Among those accidents with snakes, 6,552 of them were caused by Bothrops (58.3%), 1,747 by Crotalus (15.5%), 70 by Micrurus (0.3%) and 152 reported as non-venomous (1.4%), with 2,724 cases (24.2%) without information about the causal agent. From this total, 5,682 (50.5%) occurred in rural areas, 1,270 (11.3%) in urban areas and 4,293 (38.2%) were not informed, affecting 8,541 (76%) of male patients, 2,557 (22.7%) female and 147 (1.3%) without information. Seasonality is relevant in the wettest months (January-April), with 5,802 cases (51.6%) notified in this period. Patients between 11–60 years old were most frequently affected (79.6%), with an expressive number of accidents occurring between 20 and 50 years old (53%); 81.5% of the cases were attended in hospitals between 0 and 6 hours after the accident. The cases were classified as mild (4,469; 39.7%), moderate (4,081; 36.3%), severe (1,328; 11.8%) or unreported (1,367; 12.2%). The most affected body parts were lower limbs (8,175; 72.7%), followed by upper limbs (2,493; 22.2%) and other regions of the body (53; 0.5%), with 524 (4.6%) not informed. In 10,381 cases (92.3%) some type of antivenom (specific or polyvalent) was used, with 8,819 cases (80.1%) using between 1 and 10 ampoules and 1,417 (12.9%) between 11 and 20 ampoules. The main notified symptoms were pain (319; 22.4%), pain + edema (951; 66.9%) and pain + edema + ecchymosis (152; 10.7%). The main notified systemic manifestations were changes in vagal activity (291; 50.5%), myalgia (85; 14.8%), neuroparalysis (76; 13.2%), renal injury (75; 13%), hemorrhages (23; 4.0%) and compartment syndrome (26; 4.5%). From the total cases, 5,870 (52.5%) evolved to cure, 50 (0.4%) evolved to cure with sequelae, 28 (0.2%) resulted in death and 5,296 (47.1%) were not informed.
Accidents with terrestrial arthropods in the State of Goiás, Central Brazil: a temporal analysis (2003-2018)
(Poster)
Twitter: #VenOx20V & #VLBarros2
Verônica Lacerda de Barros1, Anita de Moura Pessoa2, Raimundo Nonato Leite Pinto3, Raphael Ladislau de Alcântara4, Rafael Stuani Floriano5, and Nelson Jorge da Silva Jr2,3,6
1Undergraduate Program in Medical Veterinary, School of Biological and Agrarian Sciences, Pontifical Catholic University of Goiás, Av. Engler, s/n, Jardim Marilizia, 74605-010, Goiânia, GO, Brazil
2Graduate Program in Biotechnology and Biodiversity, Institute of Tropical Pathology and Public Health, Rua 253, s/n, Setor Leste Universitário, 74001-970, Goiânia, GO, Brazil
3Undergraduate Program in Medicine, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goiás, Rua 235, 15, Setor Leste Universitário, 74605-050, Goiânia, GO, Brazil
4Undergraduate Program in Biomedicine, Universidade Paulista, Rodovia BR 153, km 503, Fazenda Botafogo, 74845-090, Goiânia, GO, Brazil
5Graduate Program in Health Sciences, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2-205, 19067-175, Presidente Prudente, SP, Brazil
6Graduate Program in Environmental Sciences and Health, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goiás, Rua 232, 128, 74605-140, Goiânia, GO, Brazil
In this study, we describe the profile of accidents by terrestrial arthropods occurred in the State of Goiás (Brazil) between 2003–2018, based on the notification forms from the Toxicopharmacological Information Center of the State of Goiás, Goiás, Brazil, being 10,746 with scorpions (97.7%), followed by spiders (1,718; 12.7%), bees (414; 3.2%), centipedes (333; 2.5%), wasps (128; 0.9%), caterpillars (123; 0.9%) and ants (19; 0.1%). Of these, the most relevant are scorpions, spiders and bees (12,878; 95.5%). The most common causal agents in accidents with scorpions were Tityus serrulatus, Tityus bahiensis, and Rhopalurus iglesiasi (scorpions), Lycosa sp. (spider) and Apis mellifera (Africanized bee). From this total, 8,740 (64.8%) occurred urban areas, 3,168 (23.5%) in rural areas and 1,526 (11.3%) were not informed, affecting 7,108 (52.7%) of male patients, 6,282 (46.6%) female and 91 (0.7%) without information. No seasonality of accidents was evident for any of the groups of arthropods, with very similar numbers per month suggesting the domestic pattern of these occurrences. Patients between 1–60 years old were most frequently affected (87.1%), without any expressive differences among age classes (10 years each); 73.9% of the cases were attended between 0 and 3 hours after the accident. The cases were classified as mild (10,137; 75.2%), moderate (1,919; 14.2%), severe (493; 3.7%) or unreported (932; 6.9%). The most affected body parts were upper limbs (7,852; 58.2%), followed by lower limbs (4,958; 36.8%) and more than one region of the body (47; 0.4%), with 624 (4.6%) not informed. In 4,043 cases (29.9%) some type of antivenom (specific or polyvalent) was used, with 3,813 cases (94.3%) using between 1 and 5 ampoules and 210 (5.2%) between 6 and 20 ampoules. The main notified symptoms were local pain (2,223; 42%) and local pain + edema (3,065; 58%). The main notified systemic manifestations were changes in vagal activity (fever, sweating, dizziness, and headache) (11,152; 86.6%), myalgia (2,643; 20.5%), and neuroparalysis (2.764; 21.5%). From the total cases, 6,515 (48.3%) evolved to cure, 18 (0.1%) evolved to cure with sequelae, 24 (0.2%) resulted in death and 6,923 (51,4%) were not informed.
Comparison of antinociceptive activity of topical formulations containing extracts of plants of the genus Jatropha against the venom of the scorpion Tityus stigmurus
(Poster)
Twitter: #VenOx20V & #BKBatista
Beatriz K Da C Batista; Jacinthia B Xavier-Santos; Júlia G R Passos; João F O Silva; Arnóbio A Silva-Júnior; Matheus De F Fernandes-Pedrosa
Department of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil
Scorpion envenomings are common in tropical and subtropical regions, with the genus Tityus the most relevant in Brazil. In the Brazilian Northeast region the most significant species is Tityus stigmurus, which is responsible for triggering greater toxicity, when compared to other species of the same genus. The main symptoms caused by its venom are pain, edema, erythema, paraesthesia, headaches and vomiting, the pain being present in 94.4% of cases. The treatments performed against envenoming are supportive therapy and serotherapy, in more severe cases. However, serotherapy is not able to reduce the intense inflammatory response caused by the presence of toxins, does not effectively combat the local effects generated by the venom and has difficult access in some regions of the country. Therefore, the use of medicinal plants is seen as an alternative or complement to serotherapy and has been investigated in recent years, among them are the species of the genus Jatropha, with great anti-inflammatory and analgesic potential, being able to act in the local effects caused by venom, mainly in the hyperalgesia characteristic of the species. Therefore, this study seeks to evaluate semi-solid formulations based on the leaves extract of J. molissima and J. gossypiifolia regarding its antinociceptive activity against the venom of the scorpion T. stigmurus, with a comparison of activity between the species. In addition, this approach aims to contribute to the development of herbal products, resulting in a lower cost and easier access when compared to serotherapy, which contributes to the easing of the pain caused in the affected individuals and an alternative for the portion of the population that does not have access to anti-scorpion serum.
Taxonomic identity analysis of Bothrops atrox snake from Peru and Brazil using enzymatic markers
(Poster)
Twitter: #VenOx20V & #CCayo
Carmen Cayo, Dan Vivas, Jorge Electo, Edith Rodríguez, Walter Silva, Armando Yarlequé
Laboratory of Molecular Biology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima – Perú
Bothrops atrox is the specie with the greatest geographic distribution and clinical importance in wide region of South America. It is distribution, could suggest differences at the taxonomic level between the Peruvian and Brazilian populations, according with varied biological effect even is considered as a single specie. Therefore, the objective of this study was to carry out an analysis of the possible differences between the populations of B. atrox from Peru and Brazil, based on molecular characteristics. Taking as a starting point the amino acid and nucleotide sequences deposited in Genbank and generated in this work, the proteins with the greatest relevance in poisoning were analyzed: metalloprotease type III, L-amino acid oxidase and hyaluronidase, determining the variable positions, preference of use codon and phylogenetic analysis, thus obtaining the degree of genetic distance between both populations. The results indicate that the metalloprotease type III and L-amino acid oxidase enzymes show marked different positions, while the hyaluronidase enzyme showed less variability. Likewise, regarding variability in codon use preference, metalloprotease type III presented the following results: Proline (CAA, CCA – Peru / CCA – Brazil); L-amino acid oxidase: Alanine (GCT, GCC – Peru / GCT, GCC, GCA – Brazil), Glycine (GGA – Peru / GGA, GGT – Brazil), Isoleucine (ATT – Peru / ATC – Brazil) and Valine (GTG– Peru / GTG, GTC – Brazil) and for hyaluronidase: Alanine (GCT, GCA – Peru / GCT – Brazil), Proline (CCT – Peru / CCT, CCA – Brazil) and Glutamine (CAA, CAG – Peru / CAA – Brazil ). On the other hand, the phylogenetic analysis of these markers showed variations in the grouping of the Peruvian and Brazilian populations. These results would indicate that there is a marked difference between both populations, which, added to the analysis of mitochondrial and nuclear markers, would be the basis for their taxonomic revision. Financial support: Contrato N° 101-2018-FONDECYT-BM-IADT-AV (FONDECYT-Perú) and Vicerrectorado de Investigación (UNMSM-Perú).
Venomics approach reveals a high proportion of Lactrodectus-like toxins in Steatoda nobilis venom – First link to post-bite symptomology
(Poster)
Twitter: #VenOx20V & #JPDunbar
John P. Dunbar1, Antoine Fort2, Damien Redureau3, Aiste Vitkauskaite1, Ronan Sulpice2, Michel M. Dugon1 & Loïc Quinton3
1 Venom Systems & Proteomics Lab, School of Natural Sciences, Ryan Institute, National University of Ireland Galway, Galway, Ireland 2 Plant Systems Biology Lab, Plant and AgriBiosciences Research Centre, School of Natural Sciences, Ryan Institute, National University of Ireland Galway, Galway, Ireland3 Mass Spectrometry Laboratory, MolSYS RU, University of Liège In recent years, the Noble false widow spider Steatoda nobilis has expanded its range globally and may represent a potential threat to native ecosystems and public health. Increasing numbers in synanthropic habitats has led to an increase in human encounters resulting in envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). A combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins with unknown function. Among the toxins, the most represented in numbers are α-latrotoxins (61), 𝛿-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), 𝛿-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. We present symptomology from 23 cases (15 new) of S. nobilis envenomations confirming necrosis and Latrodectus-like symptoms including debilitating pain, tremors, fatigue, nausea and hypotension. The continued rising numbers of S. nobilis will undoubtedly result in further bites and this study will help provide the medical community with a better understanding of the potential medical outcomes from bites by this species and alert them to the possibility of medically important outcomes.
Topical herbal gel in combination with commercial antivenom reduces local toxicity of Bothrops venom: a new approach for snake envenomation treatment?
(Poster)
Twitter: #VenOx20V & #JFSilva
Juliana Felix-Silva, Jacinthia B X Santos, Julia G R Passos, Jacyra A S Gomes, Arnóbio A Silva-Junior, Matheus F Fernandes-Pedrosa
Laboratory of Pharmaceutical Technology and Biotechnology (TecBioFar), Faculty of Pharmacy, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
Bothropic envenomation, clinically characterized by prominent local tissue damage, comprises about 90% of snakebites in Latin America. Antivenom immunoglobulins, in general, has limited efficacy against these effects. Thus, the search for complementary alternatives to treat snakebites is of great interest. Bothrops erythromelas is a snake of medical importance in Northeastern Brazil, however, it is not included in the pool of venoms used in production of snake antivenoms distributed in whole country. Jatropha gossypiifolia L. (Euphorbiaceae), popularly known as “bellyache bush”, is a medicinal plant widely used in folk medicine as antidote for snakebites. Previous studies have demonstrated the antiophidic potential of its aqueous leaf extract against Bothrops venom. Here, the efficacy of a polyvalent bothropic-crotalic antivenom, alone or in association with an herbal gel containing J. gossypiifolia extract, was evaluated against local toxicity induced by B. erythromelas venom in mice. Both antivenom (intraperitoneally) and herbal gel (topically) were administered after venom injection. Antivenom was able to significantly reduce haemorrhage (58.9 ± 6.3 of inhibition, P<0.05), but not edema (P>0.05). When antivenom was associated with herbal gel, its antiedematogenic activity was significantly increased (70.6 ± 10.3% of inhibition, P<0.05), being effective as fast as 30 min after envenomation. The association inhibited haemorrhage in 74.5 ± 10.0% (P>0.05 related to antivenom alone). Myotoxicity was not inhibited. Together, these results indicate the limited efficacy of bothropic-crotalic antivenom against B. erythromelas local effects, and bring evidence of the antiophidic activity of J. gossypiifolia topical gel as an adjunct in the treatment of bothropic local effects, suggesting the potential application of this product as a prototype for the future development of genuinely Brazilian herbal products in the complementary treatment of local toxicity of snakebites.
Composition and characterization of venom from the aquatic bug Ilyocoris cimicoides
(Poster)
Twitter: #VenOx20V & #MLFischer
Maike L Fischer1, Natalie Wielsch2, Sol A Yepes Vivas1, David G Heckel1, Andreas Vilcinskas3, Heiko Vogel1
1Department of Entomology, Max-Planck Institute for Chemical Ecology, Jena, Germany
2Research Group Mass Spectrometry/Proteomics, Max-Planck Institute for Chemical Ecology, Jena, Germany
3Institute for Insect Biotechnology, Justus Liebig University, Giessen, Germany
Predatory Heteroptera secrete complex venom mixtures that fulfil various functions including prey paralysis, extra-oral digestion and defense against vertebrates and invertebrates. The origin of these venoms is the salivary gland complex that comprises an accessory gland (AG), anterior main gland (AMG) and posterior main gland (PMG). With an integrated transcriptomics and proteomics approach, we analyzed and compared the composition of venom from the AMG and the PMG of the nepomorphan aquatic bug Ilyocoris cimicoides. Both glands secreted distinct, complex protein mixtures. The majority of identified compounds included digestion-associated proteins and numerous uncharacterized proteins. Putative neurotoxins were only present in the PMG secretions, while putative hemolysins were specifically expressed and secreted in the AMG. The differences in the protein compositions of AMG and PMG were also reflected in their bioactivity. Enzymatic and hemolytic activity was considerably different between the two glands, with PMG venom conferring strong activity and AMG venom exhibiting only low or no activity. Proteomic analysis of venom samples revealed an exclusive use of PMG venom for prey overwhelming, extra-oral digestion and defense, thus leaving the role of the AMG unclear. A fractionation of AMG and PMG venom proteins, with subsequent functional characterization of individual fractions will help us to further characterize venom components and to resolve their putative functions. Our study contributes to a better understanding of venom composition and function in Heteroptera, a species-rich but so far largely neglected group of venomous arthropods. It forms a basis for future research aimed at identifying compounds that may be relevant for drug or pesticide development.
Application of Top-Down and Bottom-Up Proteomics in Iranian Saw-Scaled Viper, Echis carinatus sochureki, Venom
(Poster)
Twitter: #VenOx20V & #PGhezellou
Parviz Ghezellou,1 Wendell Albuquerque,2 Vannuruswamy Garikapati,1 Nicholas R. Casewell,3 Seyed M. Kazemi,4 Alireza Ghassempour,4 Bernhard Spengler1
1Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany
2Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Germany
3Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, United Kingdom
4Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran
The proteinaceous components of snake venoms have been shown to vary among species and to exhibit a wide range of biological functions. Among all venomous snakes, the saw-scaled or carpet vipers (family Viperidae; genus Echis) are thought to be responsible for a higher global snakebite mortality than any other snake genus. Echis carinatus sochureki (ECS) is a widely distributed snake species, including being found across the thirteen provinces of Iran, where it is assumed to be responsible for the majority of snakebite envenomings. Here, we collected specimens of ECS from three different Iranian populations. Subsequently, we used a combination of bottom-up and top-down proteomics approaches to identify and characterize in detail venom protein compositions and to give an overview of conspecific venom variation of Iranian ECS. The combined proteomics data revealed great complexity in the protein composition of all ECS venoms which was not reported before.
A small octopus peptide targets BRAF melanoma via blockage of PI3K/AKT/mTOR and other metabolic pathways
(Poster)
Twitter: #VenOx20V & #MIkonomopoulou
Manuel A. Fernandez-Rojo1,2, Javier Moral-Sanz1, Jeremy Potriquet2, Joshua Daley2, Yaiza A. López1, Pamela Mukhopadhyay2, Andreas Brust3, Patrick Wilhelm3, Taylor Smallwood3, Richard Clark3, Nic Waddell2, Bryan Fry3, Paul Alewood3, Jason Mulvenna2, John Miles2,4 & Maria P. Ikonomopoulou1,2
1Madrid Institute for Advanced Studies in Food, Madrid, E28049, Spain
2QIMR Berghofer MRI, Queensland, 4006, Australia
3The University of Queensland, Queensland, 4072, Australia
4James Cook University, Centre for Biodiscovery and Molecular Development of Therapeutics and Centre for Biosecurity in Tropical Infectious Diseases, Cairns, 4811, Australia
The incidence of skin cancer is high and continuously growing, causing a huge economic burden to the health systems worldwide. Melanoma is a lethal form of skin cancer that affects around a tenth of all skin cancer patients. Even though at early stages it can be treated by surgical resection, when it reaches the metastatic stage the percentage of successful therapies is reduced. Common practices involve targeted therapies and/or immune check-point inhibitors, which show unprecedented responses in approximately 60% of patients. However, it remains an approximate 40 % of metastatic patients that develop drug resistance, show adverse effects or are unresponsive to the current regimes. Hence, there is still an unmet medical need for new melanoma drugs to target metastasis. This prompted us to study the antiproliferative profile of a small linear peptide (Octpep-I) derived from Octopus Kaurna in BRAF-mutated human patient-derived melanoma cells. Octpep-I exhibited melanoma-specific cytotoxic activities. To elucidate its mechanism of action, we used “omic” approaches. Proteomics SWATH time-course analysis at 5 minutes and up to 24h revealed various affected pathways, including granzyme A signalling, oxidative phosphorylation, and PI3K/AKT/mTOR among others. The role of PI3K/AKT/mTOR was validated by western blots and complemented further by RNAseq at 1h, 3h & 6 h, which showcased various additional metabolic and immune-related pathways in melanoma cells. Accordingly, seahorse experiments validated that numerous metabolic parameters including ATP production, maximal respiration and non-mitochondrial respiration were consistently downregulated in melanoma-treated with Octpep-1 cells. Altogether, highlight that Octpep-1 alters metabolism, acting mainly via PI3K/AKT/mTOR pathway to exert its antiproliferative properties in melanoma BRAF mutated cells. We show the potential of OctPep-1 to be developed further as a targeted melanoma drug candidate. Future steps entail selecting the best therapeutic approach (alone or in combination with other pathway inhibitors) as well as its study in in vivo experimental models of melanoma tumors and metastasis.
Ecology and conservation of Europe’s largest viper (Macrovipera lebetina) on Cyprus
(Poster)
Twitter: #VenOx20V & #DJestrzemski1
Daniel H Jestrzemski1,2, Irina Kuzyakova3, Ulrich Kuch1
1Institute of Occupational Medicine, Social Medicine and Environmental Medicine, Goethe University, Frankfurt am Main, Germany
2Faculty of Forest Sciences and Forest Ecology, Department of Forest Zoology and Forest Conservation, University of Göttingen, Göttingen, Germany
3Faculty of Forest Sciences and Forest Ecology, Department of Ecoinformatics, Biometrics and Forest Growth, University of Göttingen, Büsgenweg 4, 37077 Göttingen, Germany
The medically important blunt-nosed viper Macrovipera lebetina (Linnaeus, 1758) is Europe’s largest viper species. Although its nominate subspecies M. l. lebetina is endemic to Cyprus, there is a lack of data on its ecology, morphology and conservation status. In 2014, 2015 and 2017, the first author investigated the morphometric characteristics, proximity to water and conservation status of M. l. lebetina in Paphos district (Republic of Cyprus). Additionally, local people were interviewed about the species’ conservation situation. Morphological data were collected from 10 adult males, 16 adult females and eight unsexed juveniles. Rounded total length (ToL) ranged from 23.5 cm to 133.0 cm and weight between 10 g and 1456 g. Adult males significantly exceeded adult females in tail length (TaL), ToL and head length (HL). No significant sex-specific differences were found in snout-vent length (SVL), head width (HW), weight and body condition index (BCI). Adult females from late summer (2015) had a significantly lower mean BCI than those from spring (2014). Distances of 38 blunt-nosed vipers to the nearest water bodies did not differ significantly between spring (2014) and late summer (2015). There was also no significant difference between the distances of vipers to natural and to artificial water bodies in spring (and late summer). Key threats to M. lebetina in Cyprus are persecution, road traffic and habitat destruction due to real-estate development and wild fires. People involved in outdoor activities encounter vipers more often. Widespread public aversion against this species is an obstacle to effective legal protection. Adult females are likely in a more vulnerable body condition in late summer. Periodic drying out of water bodies in summer probably does not affect the species’ occurrence. Educational workshops and habitat conservation are recommended for reducing human-viper conflict.
A 7-year review of snakebite envenoming in the Republic of Cyprus (2013-2019)
(Poster)
Twitter: #VenOx20V & #DJestrzemski2
Daniel H Jestrzemski1,2, Maria Athanasiadou3, Vasos Scoutellas3, Parviz Ghezellou4, Bernhard Spengler4, Frank Gessler5,6, Ulrich Kuch1
1Institute of Occupational Medicine, Social Medicine and Environmental Medicine, Goethe University, Frankfurt am Main, Germany
2Faculty of Forest Sciences and Forest Ecology, Department of Forest Zoology and Forest Conservation, University of Göttingen, Göttingen, Germany
3Health Monitoring Unit, Ministry of Health, Nicosia, Republic of Cyprus
4Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Giessen, Germany
5miprolab Mikrobiologische Diagnostik GmbH, Göttingen, Germany
6Institut für angewandte Biotechnologie der Tropen e.V., University of Göttingen, Göttingen, Germany
The Mediterranean island of Cyprus is inhabited by three venomous snake species, of which the blunt-nosed viper (Macrovipera lebetina) is the only medically important, front-fanged species. Reviewing the period from 2013-2019, we present first-time epidemiological data of snakebite in the Republic of Cyprus. Data on hospital admissions with a history of snakebite envenoming were obtained by the Ministry of Health, data on population growth and rainfall from the Statistical Service and Department of Meteorology. Further data were obtained from 10 interviews with representatives of Cypriot institutions. From 2013-2019, 288 patients with snakebite envenoming were recorded by hospitals. Annually, 41 persons on average were admitted, ranging from 29 in 2017 to 58 in 2015. The number of hospitalized snakebite patients increased from 4.55 per 100,000 population in 2013 to 6.84 in 2015 but declined until 2018 (4.22). One death related to snakebite occurred in July 2004, and another, indirectly related, in April 2015. Of all cases, 266 (92%) happened between April and October, with a peak in September (88 cases or 31%). Most patients were male (70%). The most affected group was 60-69 years old (20%). Most patients were admitted to Paphos General Hospital (51%), Limassol (30%) and Nicosia (11%) hospitals. Of all patients, 242 (84%) were discharged within four days; the mean duration of hospitalization was 2.65 (0-13) days. Snakebite-related deaths are very rare on Cyprus. Most cases of envenoming happen in late summer. The hospital admission data suggest that snakebite risk may be highest in Paphos district. Males, middle- to older-aged people and those engaged in outdoor occupations are at highest risk. The hospital data indicate that most cases of M. lebetina envenoming in this period were not severe. Prospective studies of M. lebetina envenoming and its clinical management are needed to ascertain this and to assess the safety and efficacy of currently applied treatments.
Biochemical and functional characterization of the first member of the new P-IIIe subclass of snake venom metalloproteinases
(Poster)
Twitter: #VenOx20V & #ALeonardi
Adrijana Leonardi1, Kity Požek1,2, Igor Križaj1
1 Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia
2 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia
Snake venoms are a rich source of bioactive proteins. These include snake venom metalloproteinases (SVMPs), which are closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif), but differ in domain structure and size. SVMPs have diversified structurally and functionally through domain loss and post-translational processing. Depending on their structure, they are organized in three classes (P-I, P- II, P- III). The P-III SVMPs consist of a catalytic metalloproteinase (MP) and non-catalytic disintegrin-like (D) and cysteine-rich (C) domains. The other two classes are the result of a non-catalytic domain loss. Recently, in the Vipera a. ammodytes (Vaa) venom, we have discovered an SVMP called VaaMPIII-3, which on the contrary is the result of the loss of the MP domain. It consists only of a truncated D-domain and a C-domain. For this and similar proteins consisting of partial or complete D and C domains, the new subclass P-IIIe of SVMPs has been introduced (Leonardi A et al., 2019, J Proteome Res 18, 2287-2309). We have purified VaaMPIII-3 from the Vaa venom using several chromatographic steps. The isolated protein of 21 kDa is acidic and highly glycosylated. N-terminal Edman sequencing and the use of covalent thiol affinity chromatography confirmed the existence of a free cysteine residue in the molecule. Although the protein is usually present in a monomeric form, it can also form a covalent dimer. The pharmacological effect of VaaMPIII-3 has been studied in platelet-rich plasma and it has been found to inhibit platelet aggregation stimulated by either collagen, ADP or arachidonic acid. Platelet aggregation is an important part of the blood clotting process as it enables the formation of a platelet plug (thrombus) that temporarily seals the wound and stops bleeding. VaaMPIII-3 therefore has an antithrombotic effect and could be a suitable basis for the development of a new anticoagulant.
Use of venom blocked with chelating agent for anti-bothropic serum production
(Poster)
Twitter: #VenOx20V & #GLLopez
Gisela L Lopez1, Andrea Van de Velde1, David Hernandez2, Laura C Leiva1, Luciano S Fusco1
1Laboratorio de Investigación en Proteínas (LabInPro), IQUIBA-NEA CONICET, FaCENA, Universidad Nacional del Nordeste, Av. Libertad 5470, Corrientes, Argentina
2Cátedra de Histología y Embriología, Facultad de Ciencias Veterinarias –UNNE, Sargento Cabral N° 2139, Corrientes, Argentina
Snakes belonging to genus Bothrops are responsible for more than 85% of the bites occurring in South America. Bothrops alternatus is a pitviper widespread in this area and it is one of the most important species associated to snakebites not only in Argentina but also Brazil. Antivenoms are the only specific treatment for envenoming by snakebites. They are made by animals (e.g. horses or sheep) immunizations with sublethal doses of venom. Snake Venom Metalloproteinases (SVMPs) play an important role in envenomation, causing relevant local effects such as hemorrhage, edema and myotoxicity as well as systemic bleeding. They represent around ̴43.1% of the protein composition from B. alternatus venom and causing lesions during the immunization of animals. In this work, an alternative immunization protocol was developed in mice where SVMPs activity was previously blocked by Na2EDTA as chelating agent. For this proposal, the B. alternatus venom (BaV, 10 mg/mL) was treated with 50 mM Na2EDTA (1 h, 37°C) and excess chelator was removed by Sephadex G-25 chromatography. Proteolytic activity was assayed to control the block processing. Groups of 5 BALB/c mice were immunized s.v. on 0-15-30 days with BaV (15-30-45μg) and BaV/Na2EDTA (45-90-135 μg). Blood samples were collected on days 14-29-41 for antibody analysis. Sera from BaV/Na2EDTA protocol have a titer (5.1×104) higher than those treated with BaV (1.3×104). The neutralizing ability of both antivenoms was tested against proteolytic, coagulant and PLA2 activity, resulting that it was significantly higher (p <0.05) for sera from mice bowing to blocked venom. Histological analysis of mice lungs showed that pulmonary parenchyma from BaV immunized mice was significantly affected in relation to those BaV/Na2EDTA treated. In conclusion, BaV/Na2EDTA showed to be an appropriate immunogen, not only proved that serum has a high neutralizing capacity but also has a lower organic impact in animals to antivenom production.
A new generation of F(ab’)2 antivenoms made of highly purified and specific immunoglobulin fragments
(Poster)
Twitter: #VenOx20V & #InosanBiopharma
Henri Mathé, Raul Soria
Inosan Biopharma, S.A. Arbea Campus Empresarial, Edificio 2, Planta 2. Carretera Fuencarral a Alcobendas. Madrid. Spain
After years of research and collaboration with experts, Inosan biopharma has developed a unique manufacturing platform using the latest biotechnology equipment and operating in compliance with the highest international quality standards. This newly designed production platform exceeds by far the performance of previous manufacturing processes especially regarding the purity level and the neutralizing capacity of the antivenoms. A series of analyses have been performed to compare 3 antivenoms: Inosan Biopharma polyvalent F(ab’)2 immunoglobulin fragments antivenom dedicated for sub-Saharan Africa (Inoserp PAN-AFRICATM) and 2 other F(ab’)2 antivenom also dedicated for sub-Saharan Africa and currently available in the region (called Antivenom B and Antivenom C to not disclose the commercial names). According to the product specifications, each of the 3 antivenoms considered is able to neutralize the venom of Echis ocellatus with a potency of at least 250 LD50. HPLC have been conducted to analyze the composition of each antivenom and especially the level of F(ab’)2. Bradford method has been used to quantify proteins concentration per vial. Neutralization potencies have been measured for Echis ocellatus through standard mouse model testing. Based on these results, the neutralizing effectiveness of F(ab’)2 has been estimated by calculating the neutralization per mg of proteins for each antivenom. F(ab’)2 level reaches 95.6 % for Inoserp PAN-AFRICATM whereas both HPLC profiles of Antivenom B and Antivenom C show several pics revealing different types of impurities and a rather low F(ab’)2 level of 86.5% and 74.3% respectively. According to Bradford method, the protein concentration has been estimated to 9.8 mg/ml for Inoserp PAN-AFRICA, 11.2 mg/ml for Antivenom B and 11.7 mg/ml for Antivenom C. According to the mouse model, 1 vial of Inoserp PAN-AFRICA is able to neutralize 678.6 LD50 of venom of Echis ocellatus whereas the neutralization potency has been evaluated to 279.9 LD50 and 109.8 LD50 for Antivenom B and Antivenom C respectively. When reported to the quantity of proteins per vial, the neutralization potency for Inoserp PAN-AFRICATM has been estimated to be almost 3 times greater than Antivenom B and more than 7 times greater than Antivenom C. In conclusion, the results of these analyses illustrate the very high performance of antivenoms manufactured by Inosan Biopharma. Each operation unit of the production process has been optimized, especially the fractionation and filtration process, resulting in very pure F(ab’)2 antivenoms. In addition, the modernization of immunization strategies and the use of last-generation adjuvants have led to a significant increase of the neutralization capacity of the antivenoms. This high-yield production process enables the inclusion of antibodies of additional species in the same vial for a broader coverage. Inosan Biopharma antivenoms are polyvalent and cover most species that are medically important (WHO category 1) for a given region. Inosan Biopharma antivenoms offer very safe and effective options to patients suffering from animal envenomation worldwide.
Protease characterization from Mexican rattlesnake (Crotalus sp.) venoms as a potential source for chronic wound therapeutics
(Poster)
Twitter: #VenOx20V & #DMMartinez
David Meléndez-Martínez1, José Manuel Aguilar-Yáñez1,2 and Cuauhtémoc Licona-Cassani1
1Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Av. Eugenio Garza Sada 2501 sur, Monterrey, N.L. 64849 and México
2Scicore Medical SAPI de CV, Av. Alfonso Reyes 2612-13, Del Paseo Residencial. Monterrey, N.L. 64920, México
Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drives the normal wound healing process. However, the use of these proteases has demonstrated low reproducibility results. Therefore, alternative sources of proteases such as snake venom have been proposed to develop new chronic wound treatments. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. proteases we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases such as the universal protease assay and zymography, using several substrates as gelatin, casein, hemoglobin, L-TAME, fibrinogen and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen and fibrin, but not hemoglobin. C. ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight metalloproteases (P-I subtype), whereas C. m. nigrescens possessed mostly high molecular weight metalloproteases (P-III subtype). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots,whereas serine proteases were capable of degrading fibrinogen generating fibrin clots, mimicking the activity of several human proteases: matrix metalloproteases, plasmin and thrombin. Moreover, we demonstrate that Crotalus spp. are a valuable source of proteases that can aid chronic wound healing treatments.
Identification of a novel octopus-derived peptide with antiproliferative properties and its therapeutic potential against BRAF-mutated melanoma
(Poster)
Twitter: #VenOx20V & #JMSanz
Javier Moral-Sanz1, Yaiza A. López1, Andreas Brust2, Patrick Wilhelm2, Glen Boyle3, Bryan Fry2, Paul Alewood2, John Miles3,4, Manuel A. Fernandez-Rojo1,3 & Maria P. Ikonomopoulou1,3
1 Madrid Institute for Advanced Studies in Food, Madrid, E28049, Spain
2 Institute for Molecular Bioscience, The University of Queensland, Queensland, 4072, Australia
3 QIMR Berghofer MRI, Queensland, 4006, Australia
4 James Cook University, Centre for Biodiscovery and Molecular Development of Therapeutics and Centre for Biosecurity in Tropical Infectious Diseases, Cairns, 4811, Australia
Melanoma is an aggressive form of skin cancer in which BRAF mutations account for 40–60% of all cutaneous melanomas and with BRAFV600E as the most common variant. BRAF mutations cause a constitutive activation of the mitogen-activated protein kinases (MAPK) signalling, leading to uncontrolled cell proliferation in the absence of mitogens. Unfortunately, current therapies targeting MAPK are limited by the appearance of drug resistance and relapse. In that regard, metabolic rewiring and Akt activation are key elements linked to drug resistance, cell survival and tumorigenesis. The aim of this study was to investigate the pharmacological properties of the Octopus Kaurna-derived peptide Octpep-I and its therapeutic potential in human BRAFV600E -mutated melanoma cells. Octpep-I was tested in human non-transformed Neonatal Foreskin Fibroblast (NFF) and in several lines of melanoma cells carrying the BRAFV600E mutation (i.e., MM96L, A2058, HTT144, JA, SKMEL28, A02). The MTT colorimetric assay revealed a dose-dependent cytotoxicity for Octpep-I. Most importantly, 48h treatment with 200 µg/mL Octpep-I selectively reduced the cellular viability in BRAFV600E mutants, but not in NFF cells. Evaluation of mitochondrial respiration and glycolysis using the Seahorse technology revealed that MM96L cells rely on a limited glycolytic reserve and a forced dependence on their spare mitochondrial respiratory capacity, where the later was impaired by Octpep-I but not by its inactive analogue. Moreover, Octpep-I virtually abolished Akt phosphorylation in Serine473 in response to insulin stimulation. Finally, and as a proof of concept, Octpep-I significantly slowed the progression of melanoma in xenograft mouse models. Octpep-I slows the progression of BRAFV600E melanoma, showcasing its potential as a melanoma candidate to prevent drug resistance and relapse in combinatorial treatments with MAPK inhibitors.
Death from naja nigricollis poisoning and use of traditional therapy in Benguela – Angola
(Poster)
Twitter: #VenOx20V & #POliveira
Paula Oliveira1, Guilherme Kilembeketa2, Mercedes Bardaji2, Audreys Rosário2, Signey Coimbra3, Eduardo Kedisobua2
1 Faculty of Medicine, Katyavala Bwila University, Angola
2 Benguela General Hospital, Angola
3 National Center for Scientific Research, Angola
Poisoning by snakebites is an important public health problem and a frequent medical-surgical emergency that affects rural communities in Africa. It is responsible for a huge number of victims that can evolve to death, mainly in rural areas, where these communities are the ones that suffer the most because they live far from health services and end up undergoing traditional treatments, preventing early handling and adequate care in the hospital environment. This poster aims to describe the first fatal clinical case that occurred in a child in Benguela-Angola Province that has been poisoned by naja nigricollis; the species that is considered the second most dangerous in Africa after dendroaspis polylepsis. It became a particularly feared snake in the countryside because it is nocturnal and mostly found inside homes in search of small rodents, its accident take place mostly at night while the victims sleep. Although rarely fatal, the poison is highly cytotoxic with no observed neurotoxic symptoms: The two-year-old girl has been bitten at her neck at night while sleeping, having the same snake bitten on her right foot the next day. For five days after the accident, the parents underwent traditional herbal treatment with a healer from the Dombe Grande region, who went to the hospital with a septic condition of necrotizing fasciitis, severe anemia, having performed the necrectomy of the affected regions and adequate treatment of anemia. The child’s death after 4 days in intensive care at the General Hospital of Benguela. The fatality of this clinical case demonstrates that despite of adequate late surgical and clinical handling, the spectrum of severity of poisoning by naja nigricollis still constitutes a reality that could be avoided if we educated our populations regarding the belief in the use of traditional therapy in snake accidents.
Development of an enzyme linked immunosorbent assay to detect Russell’s viper (Daboia russelii and Daboia siamensis) venoms in South and Southeast Asia
(Poster)
Twitter: #VenOx20V & #YADOO
Yadanar Oo1, Katrin Hampe-Krone2, Sibylle Pagel-Wieder2, Ulrich Kuch3, Frank Gessler2,4
1Department of Medicine, University of Veterinary Science, Yezin, 15013, Myanmar
2miprolab Mikrobiologische Diagnostik GmbH, Göttingen, Germany;
3Institute of Occupational Medicine, Social Medicine and Environmental Medicine, Goethe University, Frankfurt am Main, Germany;
4Institut für angewandte Biotechnologie der Tropen e.V., University of Göttingen, Göttingen, Germany
Diagnosing the species of snake that bit a patient has been difficult not only in tropical developing countries. However, obtaining this information is of crucial importance for clinical research on antivenoms and other therapeutic interventions, and in many settings also for making treatment decisions. The use of sandwich enzyme linked immunosorbent assays (ELISA) for snake venom detection has been one of the earliest and most successful approaches but is not widely applied in clinical practice. Russell’s vipers (Daboia russelii and Daboia siamensis) belong to the medically most important snake species, causing a tremendous burden of disease, death and disability as well as economic loss in the regions of South and Southeast Asia where they occur. Early clinical signs of Russell’s viper envenoming may be confused with envenoming by certain species of pitviper which often occur in the same habitats. In Myanmar, monovalent antivenom against Daboia siamensis venom is manufactured but no pitviper antivenom. Hence, laboratory confirmation of Russell’s viper envenoming may help avoid the use of this monovalent antivenom where it is not indicated. We developed an ELISA for the detection of D. russelii and D. siamensis venoms using rabbit IgG raised against D. russelii venom from Pakistan and horse immunoglobulins raised against D. siamensis venom (Russell’s viper antivenom from Queen Saovabha Memorial Institute, Thailand). The ELISA detected Russell’s viper venoms with a limit of detection of 1 ng/ml in buffer solution but did not cross-react with venoms from other vipers and pitvipers such as Echis carinatus, Trimeresurus spp. or Protobothrops mucrosquamatus. Although other immunodiagnostic test formats are better suited for use in the field or low-resource rural clinic settings, this test is useful where healthcare staff are already familiar with performing ELISA, and where large numbers of samples need to be processed for epidemiological or clinical research.
Structure analysis of analog peptides of the antimicrobial peptide Stigmurin from the Tityus stigmurus venom
(Poster)
Twitter: #VenOx20V & #AMSParente
Adriana M S Parente1, Bruno A Carmo1, Jarbas M Resende2, Renata M Araujo1, Matheus F F Pedrosa1
1Federal University of Rio Grande do Norte, Natal, Brazil
2Federal University of Minas Gerais, Belo Horizonte, Brazil Stigmurin is an antimicrobial peptide that present activity against Gram-positive bacteria identified by transcriptomic analysis of the venom gland of the scorpion Tityus stigmurus. From this molecule, StigA6, StigA16, StigA25 and StigA31 analog peptides with higher net charge and hydrophobic moment were designed aiming an enhanced antimicrobial activity. The analog peptides showed lower minimum inhibitory concentration and broader activity spectrum against bacteria, and Candida and Trypanosoma species. Thus, with the purpose of evaluating the structure of these peptides, thereby giving insights on their action mechanism, nuclear magnetic resonance (NMR) as well as circular dichroism (CD) and molecular dynamics (MD) were performed. Regarding the CD analysis, the peptides exhibited mostly α-helix content in hydrophobic solvents and random structure in hydrophilic mediums. However, when studied their interaction with lipid vesicles, it was observed that when in contact with negatively charged vesicles the molecules presented as more structured than when interacting with zwitterionic vesicles, indicating that the peptides show a higher interaction with negatively charged lipids. In the MD simulations with a membrane model, StigA25 and StigA31 showed a larger contact area than Stigmurin, suggesting a higher interaction between the analog peptides and the membrane. For better understanding their tridimensional structure, StigA6 and StigA16 were analyzed by NMR assays; 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC and 1H-15N HMQC were executed. A general analysis of the obtained spectra demonstrates that both peptides show a high percentage of α-helix, indicating that they are tightly arranged peptides. A more profound evaluation of these NMR spectra is needed to construct their tridimensional structure, and thus, together with other assays, shed light on their action mechanism, and increase their pharmacological and biotechnological potential.
Inhibitory potential of rosmarinic and chlorogenic acids against the effects of envenomation with the Bothrops leucurus snake venom
(Poster)
Twitter: #VenOx20V & #DPSilva
Diana Pontes-da Silva1, Sarah de Sousa Ferreira1, Manoela Torres do Rêgo1, Allanny Alves Furtado1, Fabiana de Oliveira Yamashita1, Juliana Felix da Silva2, Karla Patricia de Oliveira Luna3, Matheus de Freitas Fernandes Pedrosa1
1Health Sciences Center, Federal University of Rio Grande do Norte, Natal / RN, Brazil.
2Pharmacy departament, Unifacex, Natal / RN, Brazil.
3Center of Biological and Health Sciences, State University of Paraíba, Campina Grande / PB, Brazil
Snake venoms are complex mixtures of components that act in the disruption of different physiological mechanisms. Several classes of toxins act, alone or synergistically, on specific molecular targets to trigger the observed toxic effects. Antivenom sorotherapy is the treatment to the symptoms, but it has limitations to the effectiveness of neutralization and distribution logistics. In this context, the study of phenolic compounds such as rosmarinic acid and chlorogenic acid has indicated its ability to interact in silico with animal toxins of different classes, which promises an analysis of its inhibitory effect against the effects of envenomation. Thus, this project aims to evaluate the inhibitory potential of chlorogenic and rosmarinic acids against the effects of the Bothrops leucurus snake venom. For inhibition tests with phenolic compounds, analyzes of inhibition of edematogenic and hemorrhagic activities were carried out, as well as analysis of enzymatic activities in vitro and evaluation of the inhibitory potential of phenolic compounds on systemic toxicity. The results obtained so far indicate that both phenolic compounds were effective in inhibiting the effects of envenomation, both being more effective than antibiotic serum for some of the tested effects. However, comparing the efficiency of chlorogenic acid with rosmarinic acid, it was noted that rosmarinic acid had the greatest protective action in most tests. With the data obtained, it is expected to provide subsidies for the development of complementary therapeutic alternatives to antivenom sorotherapy currently available.
Functional characterization of a phospholipase A2 from Bothrops atrox snake venom and its neutralization by three types of polyclonal antibodies
(Poster)
Twitter: #VenOx20V & #AProleon
Alex Proleón, Fanny Lazo, Edith Rodríguez, Daniel Torrejón, Gustavo Sandoval, Edwin Quispe, Armando Yarlequé
Laboratory of Molecular Biology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima – Perú
Snake venoms often contain toxins that cause a rapid muscle necrosis, which are classified as myotoxins. The most common are phospholipases A2 (PLA2s), enzymes characterized by triggering many pharmacological effects independent of their enzymatic activity. The snake Bothrops atrox, the most responsible of the ophidic accidents in Peru, causes envenoming characterized in great measure by myotoxicity, which is attributed to PLA2s. Thus, in the present work, some pharmacological effects of a PLA2, such as enzyme activity, myotoxicity, edema formation and anticoagulation were experimentally evaluated. For this purpose, the protein was isolated from crude venom of specimens kept in captivity in the Serpentario “Oswaldo Meneses” of UNMSM. Likewise, polyclonal antibodies anti-PLA2 and anti-B. atrox venom were produced in rabbits following standard immunization protocols. These antibodies were used for neutralization test against the myotoxicity triggered in mice by the PLA2. The commercial antibothropic serum produced by INS (Peru) was also used for neutralization tests. The isolated PLA2 showed poor enzymatic activity using phosphatidylcholine as substrate and anticoagulant activity on human citrated plasma. The minimum myotoxic dose was 12.3 ± 0.95 µg, while that the minimum edematic dose was 26 ± 1.15 µg. On the other hand, using 0.5 mL of serum per mg of myotoxic PLA2, the anti-PLA2 serum was able of neutralize a 50.28 ± 10.16 % of myotoxicity, which was superior to total antivenom serum of B. atrox (27.83 ± 6.52 %), but less that antibothropic serum of INS (75.84 ± 11.56 %). It is concluded that myotoxic PLA2 also has edematic and anticoagulant activity and its neutralization is greater using the INS antibothropic serum. Financial support: Contrato N° 168-FONDECYT-2017 (FONDECYT-Perú) and Vicerrectorado de Investigación (UNMSM-Perú).
ANTI-EDEMATOGENIC POTENTIAL OF A GEL CONTAINING THE LEAVES EXTRACT FROM Jatropha mollissima (Pohl) Baill. FRONT OF THE INFLAMMATION INDUCED BY THE ENVENOMING OF Bothrops jararaca
(Poster)
Twitter: #VenOx20V & #JPassos
Júlia G R Passos1, Jacinthia B X Santos1, Jacyra A S Gomes1, Fabiana O Yamashita1, Juliana F Silva1,2, Arnóbio A S Júnior1, Matheus F F Pedrosa1
1Department of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil
2Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, RN, Brazil
Snakebites are considered a major public health problem in several countries, mainly in tropical and subtropical countries. In this context, the Bothrops genus is responsible for about 90% of accidents caused by snakes, the main local effects of envenoming being: edema, myotoxicity, hemorrhage and tissue necrosis, leading to irreversible consequences for the patient. Currently, the main treatment available for snake envenoming consists of bothropic antivenom, but it has some limitations, such as: low efficacy due to local effects and difficulty in access in some regions. Medicinal plants are used for thousands of years for various purposes. Jatropha mollissima (Pohl) Baill. has several popular uses, among them: anti-inflammatory and anti-ophidic action. This study evaluated the antiophidic potential of a topical formulation containing the hydroethanolic extract of J. mollissima against the inflammation produced by Bothrops jararaca envenoming using the in vivo paw edema model in mice, also carried out a treatment associated with the developed formulation and the bothropic antivenom, and evaluation of the effectiveness of bothropic antivenom. The main results showed that the topical formulation significantly reduced the edema during the four hours analyzed. The animal groups that received treatment with the formulation associated with bothropic antivenom, and the isolated bothropic antivenom significantly inhibited the edematogenic activity from one hour after the edema induction. However, it is noteworthy that the inhibition generated by the topical formulation associated with bothropic antivenom obtained an almost total inhibition of edema after four hours, showing a percentage of inhibition around 90% in this period. These data demonstrate the effectiveness of a topical formulation as a complementary alternative to the treatment of local effects induced by botropic envenoming.
Acute systemic toxicity induced by Lachesis muta muta (South American bushmaster) venom in rats and neutralization by N-acetyl-L-cysteine and antivenom
(Poster)
Twitter: #VenOx20V & #CVPires
Carina V Pires1, Aline G Leão-Torres1, Amanda C Ribelato1, Maria C Zerbinatti1, Rogério Giuffrida2, Rosa MB Nogueira2, Inês C Giometti2, Nelson J Silva Jr3, Rafael S Floriano1
1 Graduate Program in Health Sciences, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2-205, 19.067-175, Presidente Prudente, SP, Brazil
2 Graduate Program in Animal Science, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2-205, 19.067-175, Presidente Prudente, SP, Brazil
3 Graduate Program in Environmental Sciences and Health, School of Medical, Pharmaceutical and Biomedical Sciences, Pontifical Catholic University of Goiás, Rua 232, 128, 74.605-140, Goiânia, GO, Brazil
Envenomation by Lachesis muta muta, found in Amazon river basin, is characterized by local/systemic myotoxicity, acute renal failure, hemorrhage, coagulopathy and hypotension, with the treatment being conditioned to a nonspecific antivenom. L. m. muta causes ≤ 4% of venomous snakebites reported annually in Brazil, although potentially severe. N-acetyl-L-cysteine (NAC), an antioxidant agent, has been singled out as a potential supporting agent to antivenom for Viperidae snakebites. We investigated the neutralizing action of NAC associated or not to antivenom on the systemic toxicity induced by L. m. muta venom in rats. Male Wistar rats (300–350 g) were grouped in: (1) control saline, (2) venom, (3) venom+antivenom, (4) NAC, (5) venom+NAC and (6) venom+NAC+antivenom (n=6 each) [administration via: venom 1.5 mg/kg (IM), NAC (Sigma-Aldrich) 150 mg/kg and anti-Bothrops/Lachesis (Vencofarma) at antivenom:venom ratio of 1:1.5 (v/w) (IP)]; NAC and antivenom were administered subsequently to venom for groups (3), (5) and (6). The animals were clinically monitored for 120 min and then euthanized for collecting of blood samples, which were biochemically analysed for determination of liver, kidney and muscle injury plasmatic markers, i.e., alanine aminotransferase (ALT), creatinine (Cr) and creatine kinase (CK and CK-MB), respectively. All results were expressed as mean±SEM, with p<0.05 indicating significance. Venom produced systemic toxicity characterized by significant increase of ALT, Cr and CK release [53±1 vs. 151±17*, 0.2±0.04 vs. 0.5±0.04* and 901±62 vs. 5592±389* for groups (1) and (2), respectively; *p<0.05 compared to (1)]; venom produced no alteration in CK-MB levels. The nonspecific antivenom alone showed to be efficient to attenuate the hepatic, renal and muscle injuries produced by L. m. muta venom [81±6*, 0.4±0.07 and 1391±256* for (3); *p<0.05 compared to (2)], whereas NAC alone failed to attenuate the hepatotoxicity and nephrotoxicity, but neutralized significantly the CK release [3040±307* for (5); *p<0.05 compared to (2)]. The combined action of NAC and antivenom did not result in greater protection compared to antivenom alone. In conclusion, the nonspecific antivenom is efficient to neutralize the systemic toxicity by L. m. muta venom and NAC contributes to prevent systemic myotoxicity.
Potential application from Harpalyce brasiliana roots extract for auxiliary treatment of snakebites
(Poster)
Twitter: #VenOx20V & #EERocha
Enos Emanuel Azevedo Rocha1, Manoela Torres-Rêgo1,2, Felipe França Cavalcanti1, Fabiana de Oliveira Yamashita2, Diana Pontes da Silva2, Sarah de Sousa Ferreira2, Ana Karoline Silva de Aquino1, Renata Mendonça Araújo1, Matheus de Freitas Fernandes-Pedrosa2
1Chemistry Institute, Federal University of Rio Grande do Norte, Avenue Senador Salgado Filho 3000 Lagoa Nova, Natal BR
2Laboratory of Technology and Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, Federal University of Rio Grande do Norte, Street General Gustavo Cordeiro de Farias, Petrópolis, Natal BR
Snakes of the Bothrops genus is responsible for 87% of cases of envenomation in Brazil and the only treatment is antivenom therapy, however, it is ineffective in most cases of local tissue damage. New alternative therapeutics for treat snakebites using plant extracts have revealed species for complement the conventional therapy. Harpalyce brasiliana is popularly known in Northeast of Brazil as “raiz-de-cobra” (Port. Lit.: snake’s root) due the antiofidic medicinal use of the hydroalcoholic extract from its roots. This study evaluated the ability of hydroalcohol extract from H. brasiliana roots inhibits the local effects induced by Bothrops leucurus and B. brazili venoms, not previously reported in the literature. For this, the effect of the extract against proteolytic, phospholipase A2 and hyaluronidase enzymatic activities of venoms was evaluated in vitro. The in vivo inhibition of bothropic venoms-induced edematogenic and local hemorrhagic effects was assayed in mice, by oral route treatment with extract (400 mg/kg). The extract demonstrated promising inhibitory effects for proteolytic and hyaluronidase enzymatic activities assays. In edematogenic model, the extract exhibited an expressive reduction of paw edema followed by a decrease of myeloperoxidase activity against both snake venoms. In bothropic venoms-induced local hemorrhagic model was observed a decrease of hemoglobin concentration, as well as, reduction of the hemorrhagic halos in the dorsal region of the animal, after treatment with extract. These results suggest that the hydroalcohol extract from H. brasiliana roots decrease edematogenic and hemorrhagic effects induced by B. leucurus and B. brazili, demonstrating a potential adjuvant treatment of bothropic envenomation local effects, and these effects can be associated, in part, to presence of antiophidic pterocarpans (Cabenegrins A-I and A-II) on this extract.
Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test
(Poster)
Twitter: #VenOx20V & #ARummel
Jasmin Weisemann1, Nadja Krez1, Uwe Fiebig2, Sylvia Worbs2, Martin Skiba2, Tanja Endermann2, Martin B. Dorner2, Tomas Bergström3, Amalia Muñoz4, Ingrid Zegers4, Youssef Fikri5, Christian Müller6, Stephen P. Jenkinson6, Marc-Andre Avondet6, Laurence Delbrassinne5, Sarah Denayer5, Reinhard Zeleny4, Heinz Schimmel4, Crister Åstot3, Brigitte G. Dorner2, Andreas Rummel1
1toxogen GmbH, Hannover, Germany;
2Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany
3Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden; 4Joint Research Centre, Institute for Reference Materials and Measurements, European Commission, Geel, Belgium
5Scientific Service of Food-Borne Pathogens, Operational Directorate of Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium
6Federal Department of Defence, Civil Protection and Sport—Spiez Laboratory, Spiez, Switzerland
The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.
Naja nigricincta nigricincta – (Zebra snake) bites in children – a complex clinical picture with high mortality, a retrospective case study review – Namibia
(Poster)
Twitter: #VenOx20V & #ELSaaiman
E L Saaiman 1, P J C Buys 2
1 Anaesthetist, Private practice, Windhoek, Namibia; Member of the Namibian Snakebite Interest Group
2 Ear, Nose and Throat Surgeon, Private practice, Windhoek, Namibia; Namibian snakebite management expert; Founding member and head of the Namibian Snakebite Interest Group, Namibia
Naja nigricincta nigricincta (Zebra snake) is a spitting cobra, endemic to Namibia and southern Angola. Specific antivenom is unavailable. Considered to be mainly cytotoxic, children, especially those younger than 36 months, often present with confusing and fatal clinical pictures of systemic and local envenomation after being bitten by this snake. In order to develop targeted management, 15 case reports of Zebra snake bites in children younger than 8 years were reviewed. Four patients died, all within 48 hours post-bite and all younger than 3 years. Raised CK, AST, ALT, LD, INR, CRP values and hypoalbuminaemia were invariably present. Nine patients exhibited thrombocytopaenia. In five of these patients, fragmented red cells were noted, as in one patient with a normal platelet count. D-dimer values were done on five patients, one normal and four raised. Six patients developed a transient hyponatraemia, two patients suffered severe pre-mortem hypoglycaemia and two displayed decreased TSH, T3 and T4 hormone levels. Decreasing kidney function was noted in six patients, the four who died and two others. The latter resolved with supportive non-invasive treatment. Wound swabs were done on eleven patients. In all cases mainly gram-negative organisms resistant to the penicillin group, 1rst and 2nd generation cephalosporins, were cultured. The following envenomation pathologies need to be considered: rhabdomyolysis, coagulopathy, systemic inflammatory response and capillary leak. Haemolysis and/or thrombotic microangiopathy may occur in a subset of patients. Pituitary injury, resulting in hypoglycaemia, hyponatraemia and abnormal T3, T4 and TSH, should be excluded. Kidney injury may be linked to envenomation severity. Bacterial wound infections often occur and empiric ceftriaxone is recommended. Anticipating envenomation pathologies, and their interactions, are crucial for life saving strategies in treating paediatric zebra snakebite victims.
A basic phospholipase A2 from Bothrops diporus venom inhibits cancer cell adhesion and migration
(Poster)
Twitter: #VenOx20V & #DJSasovsky
Daniela J. Sasovsky1, Laura D. Galarza1, Laura C. Leiva1, Elisa D. Bal de Kier Joffé2, Soledad Bustillo1
1 Grupo de Investigaciones Biológicas y Moleculares (GIByM) IQUIBA-NEA, CONICET. Northeast National University, Corrientes, Argentina
2 Universidad de Buenos Aires. Instituto de Oncología “Ángel H. Roffo”. Área Investigación. Ciudad Autónoma de Buenos Aires, Argentina.
The vast majority of snakebite envenomings in northeastern Argentina are caused by Bothrops diporus. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, we isolated a basic PLA2 from B. diporus venom, and examined its potential adhesion and migration inhibition effects on a tumoral cell line. Purification was made by a two-step procedure: ion exchange (HiTrapSP XL-AKTAprime) and gel filtration chromatography (Sephadex G-75). Enzyme non-cytotoxic concentrations on a murine tumoral epithelial cell line (LM3) were selected for adhesion and migration inhibition assays. In order to evaluate cell adhesion, LM3 cells (3×104/well) were preincubated for 30 min at 37°C with PLA2 (1.25-10 μg/mL) or culture medium (DMEM 5% FBS-100% adhesion) and then added to 96-well plates. After 1.5 h, adherent cells were fixed and stained with crystal violet and the percentage of cell adhesion was determined. Cell migration was measured by wound-healing assay. Cells were grown in a 6-well plate and a wound was created by a sterile pipette tip. Culture medium alone or PLA2 (0.125-0.25 μg/mL) were added to the cells and incubated for 24 h. Wound widths were measured and percentage of cell migration was calculated. Results indicate that the isolated enzyme PLA2 induces a dose-dependent inhibition of cell adhesion and migration. The lowest dose assayed (1.25 μg/mL) evidenced a 20% of adhesion inhibition effect and with 10 μg/mL, 60% of cells didn´t adhere to the substrate. On the other hand, the wound scratch in control cells was completely closed after 24 h of incubation. However, treatment with 0.125 and 0.25 μg/mL of PLA2 resulted in the suppression of cell migration in a concentration-dependent manner, decreasing respect to controls by 8 and 55% respectively. Although more studies are needed, these findings demonstrate the therapeutic potential of this basic PLA2 isolated from B. diporus venom.
Evaluation of the multifunctional therapeutic potential of chitosan nanoparticles containing Crotalus durissus cascavella snake venom
(Poster)
Twitter: #VenOx20V & #FGSilva
Fiamma G Silva1, Karla S R Soares2, Arnóbio A S Júnior1,2, Matheus F F Pedrosa1, 2
1Department of Pharmaceutical Sciences, Federal University of Rio Grande do Norte, Natal, Brazil
2Department of Development and Technological Innovation in Medicaments, Federal University of Rio Grande do Norte, Natal, Brazil
The substantial increase on cases of infectious diseases caused by resistant bacteria, yellow fever and dengue viruses, and diseases such as leishmaniases and Chagas disease, with high mortality rate, evidences the necessity of implementation of new therapeutic alternatives. Active compounds with great therapeutic potential have been identified in snake venoms, with testified antimicrobial and antiviral activities. The venom of the South American rattlesnake (Crotalus durissus) contains various proteins biologically active with important and verified therapeutic action, being profitable and valuable as pharmacological tools in the search of new potential drugs. Nanosystems have being studied as release systems for therapeutic macromolecules. Chitosan, a biopolymer obtained by the partial deacetylation of chitin, due to its properties, have been extensively investigated on the formulation of nanocarriers, specially of genes and proteins. The present work aims to seek a release system from chitosan nanoparticles for the Crotalus durissus cascavela’s venom peptides/proteins, capable of providing a new pharmacological model for modified release of these bioactive proteins, with the intention of enlarging its therapeutic potential, which can be used as prototypes for obtaining new drugs with biotechnological and therapeutic applications.
Biochemical characterization of Trimeresurus albolabris (White-lipped Pit Viper) venom from Bangladesh
(Poster)
Twitter: #VenOx20V & #SRSohan1
Ibrahim Khalil Al Haidar1, Mohammed Noman1, Sohanur Rahman Sohan 2, Mahedi Hasan2, M. Monirul Islam3, Mohammad Abdul Wahed Chowdhury3, Aniruddha Ghose1, Abdullah Abu Sayeed,1 Md. Abu Reza2*
1Venom Research Centre, Chittagong Medical College, Chattogram, Bangladesh
2Molecular Biology and Protein Science Laboratory, Department of Genetic Engineering & Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh
3Department of Biochemistry and Molecular Biology, University of Chittagong, Chattogram
Snakebite is a potentially life threatening occurrence in rural areas of many tropical countries including Bangladesh. It is one of the neglected health problems in our country. There are approximately 100 species of snake in Bangladesh, 35 of which are venomous. White-lipped pitviper (Trimeresurus albolabris) bite is one of the most common causes of snakebite envenoming in Bangladesh (57-62%). There is no antivenom against the venom of T. albolabris in Bangladesh. Our doctors/health professionals provide symptomatic treatment to the envenomed patients. However, for effective treatment of snakebite it is important to know the characteristics of the venom composition of the snakes involved. Therefore, for the first time in Bangladesh we have initiated the characterization of T. albolabris venom. We determined the LD50 of T. albolabris venom which was found to be 1.2 mg/kg in mice by i.p. injection, significantly lower than the reported LD50 from other countries which implies that the venom composition is different, measured the PLA2 activity of the venom, carried out SDS, gel filtration profile, and RP-HPLC profiling, and for the first time studied the effects of this venom on the liver, cardiac and renal tissues of mice using histopathology. Huge histological changes were observed in different tissues including haemorrhage and tissue damage. Thus, our current study clearly supports the clinical experience that an antivenom to neutralize the venoms of Trimeresurus pitvipers is needed in our country. Further studies on the characterization of pitviper venoms in Bangladesh and neighbouring countries will be required to inform the process of developing the best antidote for treating this very common syndrome of snakebite envenoming.
Characterization of Naja kaouthia snake venom from Bangladesh: study reveals the necessity of specific antivenom for snake bite treatment in Bangladesh
(Poster)
Twitter: #VenOx20V & #SRSohan2
Md. Jahangir Alam1, Sohanur Rahman Sohan1, Md. Mahmudul Hasan Maruf1, Khaled Mahmud Sujon1, Masum Miah2, Archana Deka2, Robin Doley2 and Md Abu Reza1*
1Molecular Biology and Protein Science Laboratory, Department of Genetic Engineering & Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh
2Department of Molecular Biology and Biochemistry, Tezpur University, Assam, India
Snake bite particularly in rural Bangladesh is a major cause of mortality and morbidity. A suitable medical treatment for snake bite depends on the better understanding of the site and mode of action of its venom components. LD50 value was found to be 0.12mg/kg which is significantly lower than the reported LD50 value of Naja kaouthia from other countries. The comparative RP-HPLC profile of N. kaouthia from Bangladesh also shows significant difference to that from North East India. Neutralization studies also show that the polyvalent antivenoms (either from Haffkine, India or from VINS, India) used in Bangladesh does not properly neutralize the PLA2 activity, which is one of the major lethal protein components of N. kaouthia venom. We also for the first time have studied the effects of venom on liver, cardiac and renal tissues of mice using microtomy and histopathology. Profound histological changes were observed in both the tissues including hemorrhage and tissue damage. Thus, our current study clearly indicates the need of specific antivenom to be raised against the N. kaouthia species of our country.
Profiling of signaling pathway in human dermal blood and lymphatic endothelial cells induced by snake venom cysteine-rich secretory protein (svCRiSP) from Mohave rattlesnake venom
(Poster)
Twitter: #VenOx20V & #MSuntravat
Montamas Suntravat1, 2, Oscar Sanchez1, Armando Reyes1, Abcde Cirilo1, Emelyn Salazar1, Peter Davies3, Elda E Sanchez1, 2
1 National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA
2 Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA
3 Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA
Snake venom Cysteine-Rich Secretory Proteins (svCRiSPs) are important components in the venom of many species of snakes including Viperidae and Elapidae. Although the widespread distribution of svCRiSPs in snake venoms, little is known of the contribution that they make to the local pathophysiology of snakebite. This work aimed to investigate the role of svCRiSPs from the most medically significant species of North American vipers (Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus, and Crotalus horridus), focusing on the cellular and molecular mechanisms underlying vascular biology in snakebite. We evaluated the biological activity of svCRiSPs (Css-CRiSP, Catrox-CRiSP, Cada-CRiSP, and Chor-CRiSP) by using both in vitro assays on human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs) permeability and in vivo Miles assay of vascular permeability. Css-CRiSP is the most potent cause of acutely increase vascular permeability compared to other crotaline CRiSPs. To elucidate the main pathway underlying the endothelial permeability induced by svCRiSP, we initially screened the changes in protein expression and phosphorylation in HDLECs and HDBECs 30 min after treatment with Css-CRiSP using reverse phase protein arrays (RPPA). We observed that protease activating receptor (PAR) and several signaling molecules involved in cell mobility, immune response, and MAPK pathway, were elevated to varying degrees in HDBECs and HDLECs. These preliminary observations suggest that Css-CRiSP induce the increased endothelial monolayer permeability in HDBECs and HDLECs via different mechanisms. Knowledge gained from these studies provides insights into the molecular mechanisms that underlie the effects of svCRiSPs on vascular function and contributes to a new level of understanding of the pathophysiology of snakebite.
Identification and molecular comparative study of basic phospholipase A2 of three snake venoms of the genus Bothrops from Perú
(Poster)
Twitter: #VenOx20V & #DTorrejon
Daniel Torrejón, Dan Vivas, Gustavo Sandoval, Andrés Agurto, Lorgio Bautista, Armando Yarlequé
Laboratory of Molecular Biology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima – Perú
The complex pathogenesis of ophidian poisoning is driven by individual and synergistic biological actions of the venom components. In Peru, approximately 2100 ophidian accidents are registered per year and the majority of these cases are inflicted by species of the genus Bothrops (Lancehead Pit-Vipers, Family Viperidae). Phospholipases are among the most toxic proteins in bothropic venom and are characterized by having edema-forming, myotoxic, haemolytic and anticoagulant effects. Previously, we isolated and characterized a Lys49 myotoxic PLA2 homolog from Peruvian Bothrops atrox (“Jergon”) venom. In this study, we report a new Lys49 basic PLA2-enconding cDNA sequence from the venom of Peruvian B. atrox, as well as their counterparts in the venoms from Bothrops pictus (“Jergón de la costa”) and Bothrops brazili (“Jergón shushupe”). This work consisted of venom RNA isolation from specimens kept under captivity in the Serpentario “Oswaldo Meneses” (UNMSM) followed by cDNA synthesis and sequencing, estimation of physicochemical parameters, structural modeling, phylogenetic analysis and prediction of epitopes. At sequence level analysis, the three phospholipases maintain the molecular determinants for membrane interaction and permeabilization proposed in other studies, such as canonical “protein-membrane docking” (MDiS) and “cationic membrane-docking” (MDoS) sites. Estimates of isoelectric point (strongly basic) and molecular weights (~14 kDa) do not differ significantly. Regarding structure-based analysis, we highlight differences at the level of the C-terminal region which is related to the variability in myotoxic capacity (“myotoxic bothropic cluster”). Bayesian analysis determined a well-defined cluster conformed for these PLA2 and other myotoxic PLA2s from Bothrops genus. Finally, the identified epitopes candidates are highly conserved in certain regions of the backbone of the three PLA2s. Financial support: Contrato N° 168-FONDECYT-2017 (FONDECYT-Perú) and Vicerrectorado de Investigación (UNMSM-Perú).
Preliminary Evidence for Mimicry of the Spectacled Cobra (Naja naja) in the Javan Slow Loris (Nycticebus javanicus)
(Poster)
Twitter: #VenOx20V & #ARWatkins
Anna R Watkins, Thais Q Morcatty, K Anne-Isola Nekaris
Nocturnal Primate Research Group, Faculty of Humanities and Social Sciences, Oxford Brookes University, Oxford OX3 0BP, UK
The slow lorises (Nycticebus spp.) are a group of nocturnal strepsirrhine primates native to Southeast Asia and are notable as one of only seven groups of mammals that possess the ability to produce venom. Previous research has suggested that several serpentine features of slow loris behaviour and morphology may have evolved to mimic that of the spectacled cobra (Naja naja) in addition to the venom itself. These include extra vertebra in the spine that result in a serpentine movement, defensive displays that include hissing and spitting vocalisations, a facial mask that resembles the anterior eye spots of the spectacled cobra, and a long dark dorsal stripe marking. In this study, we review the evidence for Müllerian mimicry of the spectacled cobra by the slow loris and present initial findings of dorsal stripe image analysis in the light of this evidence. From 2012-2020, we captured and photographed 56 wild Javan slow lorises multiple times in Java, Indonesia. Using ImageJ software, we extracted RGB values from 8 regions of the dorsal stripe. Using the gamlss R-package in R, we ran a Generalised Linear Mixed Model. We found that the dorsal stripes of Javan slow lorises changed according to seasonality, being longer and darker in the dry season than in the wet season. As during the dry season there is less ground vegetation cover, an evident dorsal stripe may protect lorises when the animal is forced to move terrestrially. A more snake-like resemblance during these risky periods could deter aerial predators in particular. Our study provides some of the first ecological evidence for the rare occurrence of Müllerian mimicry in a mammal.
The effect of disulfide bond mimetics on the structure-activity relationship for conotoxins, specifically αGI and µKIIIA
(Poster)
Twitter: #VenOx20V & #CWhitmore
Charlotte Whitmore1, Astrid Knuhtsen2, Rachel Whiting1, Thomas Piggot1, Brian O. Smith3, Christopher M. Timperley1 A. Christopher Green1, & Andrew G. Jamieson2
1 Chemical, Biological and Radiological Division, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ
2 School of Chemistry, University of Glasgow, Joseph Black Building, Glasgow, G12 8QQ UK
3 Institute of Molecular, Cell & Systems Biology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Joseph Black Building, Glasgow, G12 8QQ UK
Originally isolated from Cone snail venom, conotoxins are peptides that bind selectively to their targets, generally receptors and ion channels. As a consequence of their specificity and potency, conotoxins are being investigated as new therapeutic compounds, with ziconitide already available clinically to treat chronic pain. However, as a peptide, conotoxins have properties which limit their potential as therapeutics, including poor oral bioavailability and plasma instability. These can be overcome by using a suitable route of exposure; this approach is taken for ziconitide which is administered intrathecally. A second option is structural modification to increase plasma stability or enhance oral bioavailability whilst maintaining activity at the target. The disulfide bond network of most conotoxins is considered crucial for their structure and consequently activity. We investigated whether synthetic staples could replace disulfide bonds in two conotoxins (αGI and µKIIIA), chosen because they have different complexities and secondary structures. Here, we describe the assays developed to assess activities of the native conotoxin and synthesised compounds at both the human nicotinic acetylcholine receptor (αGI) and voltage gated sodium channels (µKIIIA). For αGI, we identified that, depending on which disulfide bond is replaced, compounds with similar activity and structure to the native could be developed. For µKIIIA, we showed that compounds with a disulfide bond mimetic are active at voltage gated sodium channels, albeit with reduced activity to the native, and link this to changes in the structural conformation. Taken together, this improves our understanding of the structure-activity relationship of αGI and µKIIA and shows the potential of these staples as disulfide bond surrogates for development of improved therapeutics.
Crown Copyright © [2020] under the Open Government Licence (OGL) (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/).
Antivenom activity of a gel containing Ipomoea pes-caprae extract against local hemorrhage induced by Bothrops erythromelas venom
(Poster)
Twitter: #VenOx20V & #JXSantos
Jacinthia B Xavier-Santos, Júlia G R Passos, Beatriz K C Batista, João F O Silva, Arnóbio A Silva-Jr, Matheus F Fernandes-Pedrosa
Department of Farmacy, Federal University of Rio Grande do Norte, Natal, Brazil
Snakebites is of great importance in public health, being included in the list of Neglected Tropical Diseases by the World Health Organization. About 90% of accidents are induced by Bothrops snakes that can cause between 81 and 138 thousand deaths annually. The local effects caused by the bite can evolve, bringing drastic consequences to the affected patient, who often do not have easy access to serotherapy, which is the only effective treatment, however not efficient in terms of local effects, especially when the time sting- treatment is over. The relevance of snakebite accidents arouses interest in the search for new therapies, among them, the use of medicinal plants is widespread throughout the world, and among plant species, Ipomoea pes-caprae is used in folk medicine for the most diverse pharmacological activities, including the use in snake bites. In the study developed by our research group, obtaining a gel for topical use incorporated with extract of the plant species I. pes-caprae implies activity in the in vivo model of local hemorrhage induced by B. erythromelas venom. The treatment with the isolated antiophidic serum was not able to reduce the hemorrhagic halo, despite the treatment carried out with the 5% Ipc gel showed reduced inhibition (p< 0.01), which probably did not result in any beneficial effect when treated with an association of gel with serum, results that corroborate with the reduction of the hemoglobin content in the hemorrhagic halos, except for the group that indicated only serum. Promising data, which demonstrate the potential of a topical basis, which may contribute to the development of a complementary alternative to serotherapy in the treatment of local effects of the venom of B. erythromelas with lower cost, greater ease of access, bringing improvements to the prognosis of the injured.
