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Accepted posters (unedited)
(Presenters in Bold)
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.
Present condition of Green pit viper envenomation in Bangladesh
Twitter hashtags: #VenOx22, #RArefine
Rezoana Arefine1, Borhan Biswas Romon2
1Department of Zoology, National University, Gazipur, Bangladesh
2Molecular Biology lab, Department of Genetic Engineering and Bio technology. Rajshahi University, Bangladesh
Although the Trimeresurus erythrurus looks very beautiful, it is a deadly venomous snake in this world . The Trimeresurus erythrurus’s venom is a hemotoxin, like all snakes in the family Vipriidae. Trimeresurus erythrurus is found in 18 districts of four divisions of Bangladesh.Although the treatment of snake bites is neglected but the treatment of Trimeresurus erythrurus bites is even more neglected. Only 3 percent of patients in Bangladesh come to the hospital for treatment of Trimeresurus erythrurus’s snake bites.Although a large proportion of people in Bangladesh have been bitten by the Trimeresurus erythrurus, there is no antivenom to treat the bite of the Trimeresurus erythrurus in Bangladesh. It doesn’t even have proper medication guide lines, too. Nowhere else in Bangladesh, Chittagong Medical College Hospital is the only Center providing treatment for Trimeresurus erythrurus stings. A large proportion of the victims of Trimeresurus erythrurus stings (bitten individuals) live in remote area of mountains.Their lack of awareness and inadequacy of communication system is one of the reasons for not taking medical care.Through this direct research, we have been able to find out the overall current status, barriers and remedies of Trimeresurus erythrurus bite in Bangladesh.
Evaluating Antivenom efficacy on Human Induced Pluripotent Stem Cell (hiPSCs) using Machine-Learning Algorithm
Twitter hashtags: #VenOx22, #SBhatia
Siddharth Bhatia1, Vasanthi Dasari2, Darshan Patel1, Siddharth Daga1, Subhadra Dravida2
1VINS Bioproducts Pvt. Ltd., Thimmapur, Hyderabad, India
2Transcell Oncologics, Technology Business Incubator, Hyderabad Central University, Hyderabad, India
The efficacy of Polyvalent antivenom should be tested for all batches at their different in-process production stages as per the regulatory guidelines. Antivenom manufacturers have to rely on use of mice for estimating median effective dose (ED50). To release every batch of Indian Polyvalent Antivenom, nearly 250-300 mice are sacrificed. Considering the cruelty and high cost involved in maintaining the mice facility, there is an urgent need to replace the use of mice for routine effective dose measurements. VINS Bioproducts Pvt. Ltd. in collaboration with Transcell Oncologics has initiated project on Next-generation approach to measure Antivenom effective dose using human induced pluripotent cells (hiPSCs) complemented with AI/ML trained digital platform algorithm-NeuroSAFE. Different phenotypic responses of hiPSCs were recorded using inverted phase-contrast microscopy at 20X magnification. NeuroSAFE’s digital AI/ML platform was trained to detect cellular phenotypes including healthy cells, cells in shock, apoptosis and necrosis. Dose-dependent response of hiPSCs on exposure to different concentration of venoms and antivenom-venom mixtures were plotted to logistic curve and median IC50 and EC50 were estimated using Probit analysis. Based on our current findings and further investigation, NeuroSAFE platform could be seamlessly integrated in the Antivenom production process to report median lethal dose and antisera efficacy without the use of mice.
Interaction analysis of Naja ashei venom proteins with commercially available antivenoms
Twitter hashtags: #VenOx22, #ABocian
Aleksandra Bocian1, Justyna Buczkowicz1, Konrad K Hus1, Vladimir Petrílla2,3, Jaroslav Legáth1,4
1Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Powstańców Warszawy 6, 35-959 Rzeszów, Poland
2Department of Biology and Physiology, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041-81 Košice, Slovakia
3Zoological Department, Zoological Garden Košice, Široká 31, 040 06 Košice-Kavečany, Slovakia
4Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy, Komenského 73, 041-81 Košice, Slovakia
Naja ashei (giant spitting cobra) is one of the lesser-known representatives of African spitting cobras, inhabiting open and dry areas of eastern sub-Saharan Africa including southern Somalia, southeastern Ethiopia, and the northeastern regions of Uganda, Tanzania, and Kenya. In all of these areas, except Tanzania (category 2), the species is classified in the highest (first) category of snakes, which includes highly venomous species whose bites result in high numbers of deaths and severe medical cases. Despite the high threat posed by this species, at this moment there is no monospecific antivenom acting on the venom of this snake. Moreover, the venom of Naja ashei is not included in any of the immunizing mixtures used to produce polyspecific antivenins. The purpose of this study was to test whether components of Naja ashei venom interact with antibodies found in four commercially available antivenoms used in the treatment of cobras envenomation in Africa: Antivipmyn Africa, EchiTab-Plus-ICP, SAIMR Polyvalent Snake Antivenom, and African IHS. Crude Naja ashei venom was separated into six fractions differing in protein composition using size exclusion chromatography. Shotgun LC-MS/MS analysis revealed that fraction A contains a large amount of metalloproteinases (SVMPs) (about 40%) and L-amino acid oxidases (about 30%), fractions B and C contain high molecular weight proteins, of which more than 60% are SVMPs. Fractions E and F are predominantly composed of phospholipases A2 (PLA2s), whereas fraction G is 99% composed of 3FTx proteins. The crude venom and the obtained fractions were separated by SDS-PAGE technique and then transferred to membranes and incubated with each of the four antivenoms (acting as a primary antibody) and HRP-conjugated secondary antibody. Western blot analyses performed showed that all antivenoms tested react with the most abundant groups of proteins in venom: SVMP, PLA2, and 3FTx, but with varying effectiveness.
Shared Landscapes: Preventing snakebites through outreach, data and policy
Twitter hashtags: #VenOx22, #GnaneswarCh
Gnaneswar Chandrasekharuni and Romulus Whitaker
Centre for Herpetology/Madras Crocodile Bank Trust, Chengalpattu, Tamil Nadu, India
The Centre for Herpetology/Madras Crocodile Bank Trust has been working to understand and mitigate snakebite in India for more than three decades. In the last 6 years, we have reached out to the far corners of India, where snakebite is an occupational and household hazard. The majority of snakebites happen in rural India which is over 70% of the country. The problem is proportionate to the population density of humans and snakes. With at least 58,000 deaths every year, India gets the bleak label of snakebite capital of the world. Snakes are an invaluable part of the ecosystem and killing snakes can lead to an increase in the rodent population. Several studies have proven that an uncontrolled rodent population is a major problem for grain storage and food security. Snakebites are preventable, and prevention has the potential to dramatically reduce the burden of the problem. However, to date in India, there have been few large-scale efforts to create awareness. Our project is working towards establishing prevention models that can be replicated across India with minimal implementation challenges. We have been working to reduce snakebite incidents by sensitizing rural communities through education and capacity-building programs. Through our programs, we have reached over 1.6 million people. We have conducted acceptability studies where we donated numerous kits of protective equipment gumboots, mosquito nets and torch lights. The findings of this study were very insightful and promising to replicate this work across. We are also partnering with herpetologists, educators, healthcare providers and other professionals from various regions to develop enhanced prevention programs. Our region-specific educational tools are designed to deliver effective results. We have prioritized data-driven measures by conducting epidemiological, implementation, and evaluation surveys with a notable achievement of establishing a specialized snakebite registry for the state of Tamil Nadu. Our mitigation work is recognised by the Indian government through the Ministry of Health and Family Welfare. The overarching goal of our efforts is to support a safe environment for people and snakes to coexist.
Anticancer potential of Androctonus mauritanicus scorpion neurotoxins: In silico modeling approach by virtual screening
Twitter hashtags: #VenOx22, #KDaoudi
Khadija Daoudi1, 2, Inès Hilal1,3, Samah Nassir1, Amal Safi3, Rachida Cadi2 and Naoual Oukkache1
1Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, 1 Place Louis Pasteur, Casablanca 20250, Morocco
2Laboratory of Molecular Genetics, Physiopathology and Biotechnology, Faculty of Sciences Ain Chock, Hassan II University of Casablanca, Morocco
3Laboratory of Biosciences, Integrated and Molecular Functional Exploration, Faculty of Sciences and Techniques of Mohammedia, Morocco
The Moroccan Androctonus mauritanicus scorpion (Buthidae) represents an important medical interest in the North African region. Its venom is highly toxic and encloses a large number of diverse neurotoxins, mainly targeting sodium (NaScTxs) and potassium channels (KScTxs). Since they are endowed with interesting biological and structural properties (specificity, stability and affinity), these toxins present interesting pharmacological activities modulating the ion channels often overexpressed in certain human pathologies such as cancer. Thus, as part of the evaluation of the anticancer effect of the Androctonus mauritanicus scorpion toxins, we carried out a molecular modeling study by virtual screening to explore the anticancer potential of three neurotoxins (alpha -insect toxin Lqq3, potassium channel toxin alpha-Ktx 8.1 and potassium channel toxin alpha-KTx 3.9) while targeting the ion channels Kv1.3 and TASK-3 overexpressed at the membrane level of certain cancer cells (Colon cancer, Glioblastoma, Neuroblastoma, Colon cancer, Melanoma…). The anticancer activity prediction results showed a very high anticancer potential for all neurotoxins with 81.31% for potassium channel toxin alpha-KTx 8.1; 89.13% for potassium channel toxin alpha-KTx 3.9 and 93.8% for alpha-toxin Lqq3. While the results of virtual screening of the neurotoxins against the ion channel receptors Kv1.3 and TASK-3 have shown that these toxins have a high binding affinity towards these ion channels. However, the strongest binding affinity is observed for the neurotoxin alpha-insect toxin Lqq3 for both ion channels. The in silico modeling approach by virtual screening opens the way to the development of drugs with anticancer effects from Moroccan scorpion toxins, which can later serve as a specific and non-aggressive treatment intended to target specific receptors overexpressed at the level of the cell membrane of cancer cells.
An artificial intelligence model to identify snakes from across the world: Opportunities and challenges for global health and herpetology
Twitter hashtags: #VenOx22, #IBolon
Isabelle Bolon1, Lukáš Picek2,3, Andrew M. Durso4, Gabriel Alcoba1,5,6, François Chappuis5,7, Rafael Ruiz de Castañeda1,5
1Institute of Global Health, Department of Community Health and Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland
2Department of Cybernetics, FAV, University of West Bohemia, Pilsen, Czechia
3PiVa AI s.r.o, Plzeň, Czechia
4Department of Biological Sciences, Florida Gulf Coast University, Fort Myers, Florida, USA
5Division of Tropical and Humanitarian Medicine, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
6Médecins Sans Frontières – Doctors Without Borders, Geneva, Switzerland
7Department of Community Health and Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Snakebite envenoming is a neglected tropical disease that kills an estimated 81,000 to 138,000 people and disables another 400,000 globally every year. WHO (World Health Organization) aims to halve this burden by 2030. To achieve this ambitious goal, we need to close the data gap in snake ecology and snakebite envenoming epidemiology and give healthcare providers up-to-date knowledge and access to better diagnostic tools. An essential first step is to improve the capacity to identify biting snakes taxonomically. The existence of Artificial Intelligence (AI)-based identification tools for other animals offers an innovative opportunity to apply machine learning to snake identification. We developed an AI model using Vision Transformer, a recent neural network architecture, and a comprehensive snake photo dataset of 386,006 training photos covering 772 snake species from 188 countries. We gathered photos from online biodiversity platforms (iNaturalist, HerpMapper) and photo-sharing sites (Flickr). We open-sourced the code and publicly shared the model weights and the snake photo dataset. The model macro-averaged F1 score, calculated as the mean of all species F1 scores, is 92.2%, with a species and genus level accuracy of 96.0% and 99.0%, respectively. The average model accuracy per country is 94.2%. The model accurately classifies selected venomous and non-venomous lookalike species from Southeast Asia and sub-Saharan Africa. To our knowledge, this model’s taxonomic and geographic coverage and performance are unprecedented. This model could provide high-speed and low-cost snake identification to support snakebite victims and healthcare providers in low-resource settings, as well as zoologists, conservationists, and nature lovers from across the world.
Construction and cloning of an expression vector containing the pictobin gene, a snake venom thrombin-like enzyme from Bothrops pictus
Twitter hashtags: #VenOx22, #JEspinoza
Jordano Espinoza1, Dan E Vivas-Ruiz1,2, Daniel Torrejón1, Félix A Urra2,3, Alex Proleón1, Frey Romero4, Fanny Lazo1, Edith Rodríguez1,2, Armando Yarlequé1,2
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos (Perú)
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com/
3Clinical and Molecular Pharmacology Program, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Santiago – Chile. 4Facultad de Ciencias Biológicas, Universidad Nacional San Agustín, Arequipa, Peru
Snake venom thrombin-like enzymes (SV-TLEs) are proteins present in the vipers’ venom that participate during the snakebite envenomation affecting some components of the hemostatic system. Pictobin is an SV-TLE isolated from Bothrops pictus venom, an endemic species of Perú. Previously, this protein was identified and characterized by our investigation group. Notably, pictobin inhibit of fibronectin-dependent migration of breast and lung cancer cells in vitro assays and it was described as the first SV-TLE that induces mitochondrial fragmentation and dysfunction. Therefore, to continue with the studies on the mechanism of action of this protein, construction an expression vector is a preliminary step to achieve this objective. In this work, it was performed the construction and cloning of an expression vector containing the pictobin gene. For these purpose, the pictobin gene was synthesized by a commercial house. Then, the ligation between the pictobin gene and the pPICZα-C expression vector was performed by a T4 DNA Ligase. Afterward, the vector containing the pictobin cDNA was transformed into E. coli competent cells and these transformed cells were cultivated in plates containing LB agar supplemented with zeocin as a selection marker. PCR colony was performed for the colony selection. Finally, the selected colonies were cultivated in tubes containing medium LB supplemented with zeocin and the cloned vectors containing pictobin gene were extract from the cells. The results in the bacterial cultures were approximately 28 colonies per plate. Likewise, the results in the PCR colony amplification were a bright band of approximately 800 pb that is like the size reported for the pictobin gene (760pb). In conclusion, we report the construction and cloning of the expression vector containing the pictobin gene. Financial support: Grant: 079-2021-Fondecyt (PROCIENCIA-Perú), Grant: PINTERDIS B2110007i (VRIP-UNMSM), FONDECYT 11201322 (ANID-Chile) and Redbio0027 (ANID-Chile).
A novel model in snake bite management from a rural community health center of Assam, North East India: Impact on morbidity and mortality
Twitter hashtags: #VenOx22, #SGiri
Surajit Giri, Simanta Jyoti Taye, Deepak Agarwalla, Hridoy Baruah, Gaurav Choudhary, Bijit Borah
Demow Model Hospital cum community Health Centre, Sivasagar, Assam,785662, India
Snakebite is a public health disease which severely affects the tropical and sub-tropical countries of Asia and Africa. The major constraints in the bettermemnt of situation is the lack of public awareness due to which the victims succumb to traditional faith healers, thereby, not being able to seek medical treatment on time. To overcome this problem, a snakebite management protocol was designed from Demow model community hospital, Assam, India. The local rural population was sensitized with community awareness and eduction program in 2018 and as a part of this which, a Venom Response Team (VRT) comprising a group of trained volunteers was made. The function of the VRT was to notify the nearest clinician and help the victim reach the nearest health center with snakebite treatment facility. Another group of health worker volunteers, the fast response team (FRT) were trained to identify the early symptoms of venomous bite and administer antisnake venom serum (ASV), neostigmine or airway management of bite patients in snake bite treatment room. The snakebite room was a two bedded dedicated ward in the health centre which contained all necessary medicines, ASV and equipment necessary for the snakebite treatment. From 2018-2022, 820 snake bite victims were admitted in the health center and all were successfully treated except for one patient who died due to late arrival at hospital. Because of extensive community awareness, in 2022, most bite victims attended this hospital approximately 15 minutes after a snake bite, in contrast to arrival time of 3-4 hours post bite in 2018. In 2021, this rural health center registered 464 snake bite victims with 128 numbers of venomous bites and zero mortality. A non-breakable chain from community to hospital in the form of VRT, FRT, and snake bite room was successfully established at the community health centre preventing many snakebite deaths.
Intraspecific differences in the immunochemical pattern of Naja mossambica venom from different regions of Africa
Twitter hashtags: #VenOx22, #KKHus
Konrad K Hus1, Justyna Buczkowicz1, Vladimir Petrilla2,3, Mirosław Tyrka1, Jaroslav Legáth1,4, Aleksandra Bocian1
1Department of Biotechnology and Bioinformatics, Rzeszow University of Technology, Powstancow Warszawy 6, 35-959 Rzeszow, Poland
2Department of Biology and Physiology, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041-81 Košice, Slovakia
3Zoological Department, Zoological Garden Košice, Široká 31, 040 06 Košice-Kavecany, Slovakia
4Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy,Komenského 73, 041 81 Košice, Slovakia
Recent clinical experiences with the treatment of Naja mossambica envenomations in Southern Africa have been shown geographically-based differences in the neutralizing potential of the available antivenoms. The limited neutralizing capacity of antivenins observed in Eswatini and Mozambique could indicate intraspecies differences in protein composition of venoms from that region in relation to venoms collected from
N. mossambica specimens residing in a different part of the continent. Therefore, the study aimed to analyze the immunochemical profiles of N. mossambica venoms acquired from the Eswatini and compare them with the patterns obtained for the samples from two provinces of South Africa (Limpopo and KwaZulu-Natal), where patients are effectively treated with the use of the same antivenins. The analysis of 52 venom samples was performed with the Western Blot technique against three commercially available antivenins: EchiTab-Plus-ICP, Antivipmyn Africa, and SAIMR. Obtained results showed that immunoglobulins in all tested antivenoms were able to bind to the toxins from Naja mossambica, however, the extent of the interaction depended on both the specific antivenom used and the geographical origin of venom.
Snakebite in South Africa: a retrospective study between May 2015 and June 2020
Twitter hashtags: #VenOx22, #TKellermann
Anné Lermer, Carine J Marks, Tracy A Kellermann
Division of Clinical Pharmacology, Department of Medicine, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
Snakebite envenomation in Sub-Saharan Africa presents a significant public health problem. To establish the severity of this problem from a South African context, snakebite related Telelog call records reported to the Poison Information Helpline of the Western Cape (PIHWC) were reported. The aim was to reveal insights into the origin and incidence of snakebite and report on trends observed in snakebites caused by venomous snake species that may add predictive value in diagnosis and management of victims. An investigation into how often species responsible for envenomation were correctly identified, as well as which venomous species caused the most frequent and problematic envenomation symptomatology was performed. A retrospective cross-sectional study of AfriTox Telelog data consisting of calls received by the PIHWC from 1 June 2015 – 31 May 2020 was conducted. Descriptive statistics were used to quantitatively describe the variables in the study dataset. Over this period 1411 snakebite related calls were received. The calls were delineated according to the toxidrome caused by the venom as neurotoxic, cytotoxic, and haemotoxic. In 44% of all snakebite calls the bite was inflicted by an unidentified snake species. The most snakebites occurred during the summer months from December – March. The most bites occurred in males (20 – 39 years). The incidence of snakebite in South Africa was 2.39 per 100 000 population, with the highest incidence of snakebite in North-West province of South Africa. In sub-Saharan Africa there is a major underestimation of the incidence of snakebite due to under reporting and absence of physical attendance to health care facilities. The PIHWC provides an invaluable service in assisting and informing medical personnel and the public on the management of snakebites. Data collected by centers provides a useful source of information on the prevalence of snakebites and medically important species towards which research should directed.
Intravenous anti-V. berus Fab fragments and intramuscular anti-V. ammodytes F(ab’)2 fragments in V. ammodytes-envenomed patients
Twitter hashtags: #VenOx22, #TKurtovic
Tihana Kurtović1, Svjetlana Karabuva2, Damjan Grenc3, Mojca Dobaja Borak3, Igor Križaj4, Boris Lukšić2, Beata Halassy1, Miran Brvar3
1Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, 10000 Zagreb, Croatia
2Clinical Department of Infectious Diseases, University Hospital of Split, Šoltanska 1, Split, Croatia
3Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana, Zaloška cesta 7, 1000 Ljubljana, Slovenia
4Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
Before 2015, snakebites caused by Vipera ammodytes were successfully treated with intravenous Viperfav (France), a formulation containing polyvalent equine F(ab’)2 fragments against the venoms of V. aspis, V. berus and V. ammodytes, or intramuscular “Zagreb” antivenom (Croatia) composed of monospecific equine F(ab’)2 fragments against V. ammodytes venom. During the period of their shortage, in Slovenia V. ammodytes bites were managed with ViperaTAb (UK), a formulation containing monospecific ovine Fab fragments against the venom of V. berus, whose eligibility for use in the clinical setting hasn’t been previously assessed despite its different specificity and type of bioactive compound. “Zagreb” antivenom was continuously used in Croatia. Newly established treatment approach opened up the possibility for a comparative study of V. ammodytes-envenomed patients that were treated intravenously with ViperaTAb or intramuscularly with “Zagreb” antivenom. The aim was to gain insight into pharmacokinetic profiles of two antivenoms and to resolve their influence on venom level decrement as well on resolution of envenomation pathology, providing findings that might serve as useful guidance to clinicians. The kinetics of Fab fragments after one or more i.v. applications was characterized by immediate rise in systemic circulation and matched better with the venom concentration in the early envenomation phase, enabling their use according to the clinical progress. Multiple doses were needed for efficient therapy of edema and thrombocytopenia due to venom recurrence, while the progression of rhabdomyolysis and neurotoxic effects could not be prevented. The intramuscular use of F(ab’)2 fragments resulted in slower increase of their level in the bloodstream. They had 25- and 14-fold longer clearance and elimination half-time, respectively, compared to Fab fragments given i.v. Such pharmacokinetic behavior demands for early administration but without need for additional doses.
Intra-specific variation in the venom composition of the Cape cobra (Naja nivea) from two regionally distinct areas in South Africa
Twitter hashtags: #VenOx22, #ALermer
Anné Lermer1, Nicolaas M Vlok2, Tracy A Kellermann1
1Division of Clinical Pharmacology, Department of Medicine, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
2Proteomics Unit, Central Analytical Facilities, Stellenbosch University, Cape Town, South Africa
The African non-spitting cobra Naja nivea (Cape cobra) is endemic to South Africa and one of the most medically significant snake species in the country due to the severe neurotoxicity caused by the venom. Variation in the proteomic profile of snake venoms on inter- and intrapopulation levels is a well-known phenomenon. Medical implications of the variability in venom composition include pathophysiological effects following snakebite envenomation, efficacy of antivenom therapy and the development of diagnostic tools. This study investigates the intra-specific variation in venom composition in pooled N. nivea venom from two regionally distinct areas in South Africa by proteomic analysis. The proteomic composition of crude N. nivea venoms from two regionally distinct areas in South Africa, the Western Cape (WC) and the Northern Cape (NC) were compared by high resolution LC-MS/MS following a bottom-up proteomic approach. Combined, a total of 56 proteins were identified in the venoms, 72% of the N. nivea venom proteome comprised three-finger toxins. Upregulation of the toxins long neurotoxin 1, long neurotoxin OH-55, short neurotoxin 2, Zn-dependent-metalloproteanase-disintegrin-like-atrase-B and Zn-dependent-metalloproteanase-disintegrin-like-kaouthiagin-like was observed in N. nivea venoms from the Western Cape. Proteomic analysis of N. nivea venom provides insight into the distribution and diversification of N. nivea venom components from regionally different populations. This information is of high value in the choice of target toxins during the development phase of point of care diagnostic assays for snakebite envenomation in South Africa. The outcome of this study has shown that further research into the neutralisation capabilities of the SAVP polyvalent antivenom towards N. nivea envenomation may contribute to effective management strategies of envenomed victims in South Africa to provide the best outcome.
P-III metalloproteinases from Bothrops pictus and B. leucurus snake venoms reduce cell proliferation, migration, and mitochondrial metabolism in breast cancer cells
Twitter hashtags: #VenOx22, #CLTorres
Camila López-Torres1,2, Alex Proleon2,3, Daniel Torrejón1,2,3, Ana B Tenorio1,2, Cristopher Almarza1,2, Eladio Sánchez2,4, Dan Vivas-Ruiz2,3, Armando Yarlequé2,3 Félix A Urra1,2
1Laboratorio de Plasticidad Metabólica y Bioenergética, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile. 2 Network for Snake Venom Research and Drug Discovery, http://snake-research.com/ 3Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 01, Lima, Peru. 4Laboratory of Biochemistry of Proteins from Animal Venoms, Research and Development Center, Ezequiel Dias Foundation, Belo Horizonte, Brazil. Snake venom metalloproteinases P-III (SVMP-III) are components relevant of the Bothrops species venom, exhibiting different biological effects. Interestingly, this toxin class exhibits metalloproteinase (M), disintegrin or disintegrin-like (D), and cysteine-rich (C) domains that can interact with extracellular matrix-cell cues impairing proliferation and cell migration. Since breast cancer cells exhibit high migratory abilities and proliferation compared to non-malignant cells, SVMP-III may exhibit selectively anti-cancer effects. In work, we evaluate the anti-cancer effect of two SVMP-III, LeucoB and Pictolysin III isolated from B. leucurus and B. pictus snake venom, respectively, on proliferation, migration, and cellular metabolism. In breast cancer cell lines (MCF7 and MDA-MB-231), SVMP-III toxins reduced the viability in a concentration-dependent manner, which had less effect on non-malignant cells HEK293. Additionally, the effect on mitochondrial ROS (mtROS) production and migration were evaluated by flow cytometry and Transwell assays, respectively. The real-time changes in the cellular metabolism induced by SVMP-III were determined using XFe96 Analyzer in MDA-MB-231 cells. Our results indicate that SVMP-IIIs increased the mtROS production, altering the extracellular acidification and oxygen consumption rates, and reducing the migration of MDA-MB-231 cells. Collectively, the SVMP-III toxins from B. pictus and B. leucurus exhibit relevant anti-cancer effects, highlighting the need for further mechanistic studies. Acknowledgment/Funding: This work is funded by Anillo Grant ACT210097 (ANID-Chile), Redbio0027 (ANID-Chile), UI-024/20 (VID-UChile), PhD fellowship (21221998). FONDECYT 11201322 (ANID-Chile), and PROCIENCIA-Peru (079-2021-Fondecyt).
Does the purification methods’ roughness affect the stability of IgG-based snake antivenoms?
Twitter hashtags: #VenOx22, #SMLukacevic
Sanja Mateljak Lukačević1,2, Tihana Kurtović1,2, Juraj Borić1,2 and Beata Halassy1,2
1University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova 10, HR-10000 Zagreb, Croatia
2Center of Excellence for Virus Immunology and Vaccines, CERVirVac, Rockefellerova 10, HR-10000 Zagreb, Croatia
Antivenoms contain either pure animal IgGs or their fragments as an active substance and are the only specific therapeutics against envenomation arising from snakebites. Although they are highly needed, low sustainability of such preparations’ manufacture is causing constant global shortages. One reason for this is the stability of the product, which contributes not only to the manufacture sustainability but the product safety as well. It has been hypothesized that roughness of conditions to which IgGs are exposed during downstream purification disturbs their conformation, making them prone to aggregation, particularly after exposure to secondary stress. The aim of this research was to investigate how the roughness of the downstream purification conditions influences the stability properties of purified IgGs. For this purpose, equine IgGs were extracted from unique hyperimmune plasma by two mild condition-based operational procedures (anion-exchange chromatography and caprylic acid precipitation) and three rougher ones (ammonium sulphate precipitation, cation-exchange chromatography and protein A affinity chromatography). The stability of refined preparations was studied under non-optimal storage conditions (37 °C, 42 °C, transient lowering of pH) by monitoring the changes in their aggregate content and thermal stability of pure IgGs. Mild purification protocols generated IgG samples with a lower aggregate share in comparison to the rougher ones. Their tendency for further aggregation was significantly associated with the initial aggregate share. The thermal stability of IgG molecules and the aggregate content in refined samples was inversely correlated. Since the initial proportion of aggregates in the samples was influenced by the operating conditions, we have shown a strong indication that each of them also indirectly affected the stability of the final preparations. This suggests that mild condition-based refinement protocols indeed generate more stable IgGs.
Defining Antivenom Design Guidelines Using Computational Pharmacokinetic Modelling
Twitter hashtags: #VenOx22, #NMorris
Natalie M Morris, Johanna A Blee, Sabine Hauert
Department of Engineering Mathematics, Ada Lovelace Building, University of Bristol, University Walk, Bristol, BS8 1TW, UK
In recent years, the design space of snakebite antivenoms has greatly expanded. In vitro antibody selection techniques and recombinant protein expression has enabled the production of toxin-neutralising antibodies in a wide range of formats. Computational pharmacokinetic simulations of venom and antivenom dynamics can be used to quantitatively explore and compare the function of different antivenom scaffolds in different treatment contexts. These simulations can facilitate the methodical testing of a wider area of parameter space than would be possible in vivo. We have built a two-compartment pharmacokinetic model of systemic snakebite envenomation and treatment in rabbits, which tracks the movement of toxins through blood and tissue. The model was parameterised with existing experimental data to allow simulation of antivenom scaffolds ranging in size from 15 to 150 kDa. The model additionally allows control of other parameters including antivenom dosing, affinity, treatment time, and venom type. We are performing a range of simulations to better understand the most important features in antivenom design, comparing outcomes for elapid and viper envenomation. We are identifying the optimal combinations of antivenom molecular size, dosing ratio, and affinity parameters within these studies. Thus far, global sensitivity analysis has indicated the primary importance of the antivenom-to-venom dosing ratio and the on-binding affinity rate on the treatment of low molecular weight venoms. Global parameter optimisation has indicated that the most effective antivenoms generally constitute low molecular weight scaffolds, at high dosing ratios and with high kon-binding affinities. The benefits of low molecular weight antivenoms become more apparent with delayed treatment. This in silico modelling approach can be used to explore the dynamics of envenomation-treatment systems, and help inform the development of low-cost, high coverage antivenoms for snakebite.
A cell-based fluorescence assay of nAChR function for investigating venom neurotoxin activity and detecting toxin inhibitory molecules
Twitter hashtags: #VenOx22, #RPatel
Rohit N Patel, Rachel H Clare, Mark C Wilkinson, Nicholas R Casewell
Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Liverpool, L3 5QA
Venoms from many medically important elapid species result in neurotoxicity via blocking of the neuromuscular junction. Neurotoxic three-finger toxins (3FTxs) are abundant in many of these venoms and act by inhibiting the postsynaptically located muscle-type nicotinic acetylcholine receptor (nAChR). This can result in muscle paralysis, including of the respiratory muscles, which presents a serious medical emergency requiring immediate treatment. Traditional methods of investigating this interaction and the ability of venom neutralisers to disrupt it can be expensive, low-throughput and labour intensive. Here, we outline an alternative assay investigating muscle-type nAChR function using the immortalised TE671 cell line which expresses the muscle-type nAChR and a fluorescent dye that reports changes in membrane potential resulting from nAChR activation. The ability of this assay to detect inhibitory activity of elapid venoms abundant in neurotoxic 3FTxs from cobras (Naja spp.) and mambas (Dendroaspis spp.) and isolated neurotoxic 3FTxs was assessed. Thereafter, this approach was used to profile elapid venom toxin fractions for nAChR activity. Finally, we assessed the suitability of the assay for quantifying the neutralising capability of toxin-inhibitory molecules. We demonstrated that we can detect venom/toxin inhibition by specific commercial antivenoms, monoclonal antibodies, small molecules and receptor-mimicking peptides, thereby demonstrating the usefulness of this approach as an in vitro screening tool to identify toxin-inhibitory molecules, as well as to define muscle-type nAChR-targeting toxins.
Recombinant expression of a basic phospholipase A2 from Peruvian Bothrops pictus snake venom (BpMtx-I) in Pichia pastoris
Twitter hashtags: #VenOx22, #AProleon
Alex Proleón1, Daniel Torrejón1, Dan Vivas-Ruiz1,a, Fanny Lazo1, Angie Regalado1 Edith Rodríguez1, Felix A. Urra2,a, Javier Cárdenas3, Armando Yarlequé1,a
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, , Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 01, Peru.
2Laboratorio Laboratorio de Plasticidad Metabólica y Bioenergética, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Santiago, Chile, Av. Independencia 1027.
3Laboratorio de Bioquímica, Facultad de Ciencias de la Salud. Universidad Nacional del del Callao. Av. Juan Pablo Ⅱ 306, Bellavista 07011, Perú
aNetwork for Snake Venom Research and Drug Discovery, http://snake-research.com/
Recombinant expression of snake toxins allows obtaining the protein for more detailed studies. In the present work, the recombinant version of BpMtx-I, a basic phospholipase A2 from Peruvian Bothrops pictus snake venom, was obtained. For this, BpMtx-I gene was amplified with restriction sites in the 5’ (SnaBI) and 3’ (AvrII) regions. The amplified gene and pPIC9K vector were digested with restriction enzymes and ligated to construct the recombinant vector. Then, the vector was linearized with the enzyme StuI to carry out the transformation via electroporation to Pichia pastoris strain KM71 cells. These cells were seeded in culture media without histidine, only cells containing the pPIC9K (His4) vector grew. In transformed cells, the BpMtx-I was amplified by colony PCR using AOX primers. The sequencing of the amplicons showed the correct open reading frame of the gene in the recombinant vector. However, 5 nucleotide differences were found that generated 4 amino acid changes (Ser1->Asn1, Val3-> Trp3, Glu4->Gln4 and Gln35->Arg35). Therefore, structural modeling of the expressed PLA2 (rBpMtx) was performed to predict the differences at the structural level with the native protein. The P. pastoris colonies that contained the recombinant vector were cultivated in a BMGY culture medium until reaching an OD600: 2.0 (approximately 24 h). After that, the P. pastoris cells were transferred to a BMMY culture medium for seven days. Every 24 h, 1 mL of culture medium was collected to analyze expression level via SDS-PAGE. In the electrophoresis gel, a 23 kDa band was observed at day 4, similar in size to the native protein BpMtx-I. In this way, the preliminary results of the expression of rBpMtx are presented. Financial support: Grant PCONFIGI B22100141 and B21101991 (VRIP-UNMSM), Grant PINTERDIS B2110007i (VRIP-UNMSM), Grant ACT210097 (ANID-Chile) and FONDECYT 11201322 (ANID-Chile). FONDECYT 079-2021.
Electrophoretic Separation & variation of Daboia Russelii venom In Bangladesh
Twitter hashtags: #VenOx22, #BBRomon
Borhan Biswas Romon1, Rezoana Arefine2
1Molecular Biology lab, Department of genetic Engineering and Bio technology. Rajshahi University, Bangladesh
2Department of Zoology, National University, Gazipur, Bangladesh
Daboia Russelii is one of the reason for one-third of snake bite death in Bangladesh. Daboia russelii was once thought to be extinct from Bangladesh, but Daboia russelii survived in limited numbers in Barendra region. Later in 2010/2011, Daboia russelii came out in flood through Padma River and they increased alarmingly in the pastures and banks of Padma.Currently, Daboia russelii is spread in 28 districts of Bangladesh and increasing day by day. In the treatment of Daboia russelii’s bite, it has been observed that there is a difference in antivenom dose in envenomation and post-treatment disability in the stings of Daboia russelii floating in Padma and Daboia russelii surviving in Barendra region.By looking at the difference in dose and post-treatment disability in envenomation, we were able to show the quantitative variables of molecules and proteins with kda difference through SDS profiling with the venom lab of Daboia russelii from Padma river and surviving Daboia russelii from Barendra region of Bangladesh.
Purification and molecular characterization of Pictolysin-III, a Metalloproteinase from the venom of Peruvian Pit Viper Snake Bothrops pictus (Jergón de la Costa)
Twitter hashtags: #VenOx22, #PRRamos
Paola Rosas-Ramos1, Nadia F. Vera-Munárriz1, Dan E. Vivas-Ruiz1,a, Felix Urra2,a, Fanny Lazo1, Armando Yarleque1,a
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 01, Peru
2Laboratorio de Plasticidad Metabólica y Bioenergética, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile
aNetwork for Snake Venom Research and Drug Discovery, http://snake-research.com/
In the present work, the purification and molecular characterization of Pictolysin-III, a P-3 Metalloproteinase (SVMP-III) from the venom of the Bothrops pictus snake was carried out. First, venom was obtained from 2 specimens kept in the Oswaldo Meneses serpentarium – Natural History Museum – UNMSM. Pictolysin-III purification was performed by size exclusion chromatography on a Sephacryl S-200 column (1.15× 57 cm). The proteolytically active fractions were grouped together and separated using gel filtration chromatography DEAE A-50. The fractions containing the enzyme of interest were grouped together and separated by using G-100 column. Their purity was validated using MPLC. The enzyme was purified 15.90 times with a yield of 2.48%. SDS-PAGE analysis revealed a single protein band of 56.5 kDa under non-reducing conditions. Molecular assays showed that the Pictolysin-III cDNA sequence has 2295 bp and encodes 610 amino acid residues. The analysis of the amino acid sequence reveals the presence of a prodomain and three structural domains: Metalloprotease region with an active site of conserved sequence, a disintegrin-like region with the presence of ECD instead of RGD sequence, and a final a cysteine-rich domain, which identifies it as a P-3 Metalloproteinase. Its identity percentage with others SVMP-III from Bothrops species. is higher than 94% (97.9% with Bothropasin, from B. jararaca; 97.5% with BITM06A, from B. insularis; 96.7% with Batroxrhagin, from B. atrox; 96.5% with Jararaghin, from B. jararaca and 94.3% with Atroxlysin -II, from B. atrox). Phylogenetic analysis placed Pictolysin-III in the same group as P-3 Metalloproteinases, differentiating it from type I and II. The structural prediction of Pictolysin-III shows 97.85% identity to the protein structure of Bothropasin. Financial support: Contrato N° 079-2021-FONDECYT (PROCIENCIA Perú), Proyecto N° B22100151 VRIP-UNMSM (Peru) and Anillo Grant ACT210097 (ANID-Chile).
General characterization of the most dangerous venom in Morocco Cerastes cerastes
Twitter hashtags: #VenOx22, #KSoukaina
Khourcha Soukaina1,2, Ennaji Hanane3, Hilal Ines4, Chablaoui Mohamed1, Ibenmoussa Samir3, Safi Amal4, Hmyene Abdelaziz2, Oukkache Naoual1
1Laboratory of Venoms and Toxins, Institut Pasteur of Morocco, Casablanca, Morocco.
2Laboratory of Biochemistry, Environment and Food Technology, Faculty of Sciences and Technologies of Mohammedia, Hassan II University, Morocco
3Laboratory of Chemistry-Biochemistry, Environment, Nutrition and Health Faculty of Medicine and Pharmacy, Hassan II University, Morocco
4Laboratory of Biosciences, Functional Integrated and Molecular Exploration, Faculty of Sciences and Technologies of Mohammedia, Hassan II University, Morocco
The varied landscapes and geomorphology of Morocco make it one of the richest Mediterranean countries in terms of biodiversity, especially with regard to its ophidian fauna. In total, they are eight species of venomous snakes exist, seven of which belong to the Viperidae family, causing about 80% of envenomation rates. According to the Anti Poison and Pharmacovigilance Center of Morocco, Cerastes cerastes (Cc) (Saharan horned viper) is classified as one of the most potentially dangerous venoms in the cases of envenomation accidents reported in the Kingdom. These vipers cause varying degrees of local and systemic symptoms, such as local edema, blistering, dermonecrosis, myotoxicity, and bleeding diathesis (associated with defibrinogenation and thrombocytopenia). However, for an effective treatment of viper envenomations, it is important to develop a comprehensive characterization of this. This prompted us to study the lethality, hemorrhagic, edematous and myotoxic activities and physiophatological alterations induced by Cc viper. In addition, we studied the immune cross-reactivity of Cc venom against that of another dangerous Moroccan viper, Bitis arietans (Ba) and Daboia mauritanica (Dm), to identify the best candidates (venom or mixture of venoms) to produce the more effective and protective of anti-venom. Our results indicated that the venom of Cc represents a toxic, hemorrhagic power by inducing myonecrosis of skeletal muscles that decreases serum creatine phosphokinase (CPK) levels. For the study of the histopathological aspect, it can be concluded that the Cc venom causes the formation of hemorrhagic foci and lesions on the liver, kidneys and heart. As expected, Cc venom is immunogenic and with highly protective anti-venom against Dm and Ba venom antigens. These results can be used to guide the development of an antivenom against the bites of dangerous Moroccan snakes.
Recombinant expression of the catalytic domain of a haemorrhagic P-III metalloproteinase from Bothrops atrox snake venom in Pichia pastoris: Preliminary results
Twitter hashtags: #VenOx22, #DTorrejon
Daniel A Torrejón1, Alex Proleón1, Felix Urra2a, Fanny Lazo1, Armando Yarleque1a, Eladio Flores3a, Luciana S. Oliveira3a, Dan E Vivas-Ruiz1a
1Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima, Peru.
2 Laboratorio de Plasticidad Metabólica y Bioenergética, Facultad de Medicina, Universidad de Chile, Santiago, Chile, Av. Independencia 1027.
3Research and Development Center, Ezequiel Dias Foundation, 30510-010 Belo Horizonte, MG, Brazil
a Network for Snake Venom Research and Drug Discovery, http://snake-research.com/
Snake venom metalloproteinases (SVMP) are known to trigger bleeding and are structurally classified into three groups (P-I to P-III) based on the organization of their domains: metalloproteinase (M), disintegrin or disintegrin-like (D), and cysteine-rich (C). Great attention has been directed toward the M domain, not only because it is the catalytic region; but also due to the conformational dynamism, certain post-translational modifications and other properties, which in synergy with the rest of the domains extend and determine the functional diversity. Thus, this work reports the heterologous expression of the catalytic domain of a type III metalloprotease identified from Bothrops atrox snake venom in Pichia pastoris cells. Technically, our work consisted of isolation of total RNA (1.0 to 6.0 ng of total RNA per mg of lyophilized venom), synthesis cDNA (0.5 to 1.2 ng of cDNA was obtained per 10 ng of total RNA), amplification and sequencing transcripts encoding-M domain (~600 bp), cloning into Escherichia coli TOP10 cells (transformation efficiency: 2.5 x 108 CFU/µg plasmid), construction of an expression vector (M domain into pPIC9K plasmid), inducible expression of Pichia pastoris strain GS115 and KM71, and confirmation of expression by Western blotting (25.8 kDa) and ELISA. Financial support: Grant: 079-2021-Fondecyt (PROCIENCIA-Perú), Grant: PCONFIGI B20100101 (VRIP-UNMSM) and FONDECYT 11201322 (ANID-Chile)
Reloadable magic bullets: Discovery of recycling monoclonal antibodies against snake venom toxins
Twitter hashtags: #VenOx22, #TTulika
Tulika Tulika1, Shirin Ahmadi1, Fulgencio Ruso-Julve2,3, Jack Wade1 , Christoffer V. Sørensen1, Rasmus W. Pedersen1, Markus-Frederik Bohn1 , Anne Ljungars1, Charlotte Rimbault1, Line Ledsgaard1, Sara P. Bjørn1, Karen K. Brøndum1, Sanne Schoffelen1, Bruno Lomonte4, Jan Terje Andersen2,3, Andreas H. Laustsen1
1Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark
2Centre for Immune Regulation and Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway
3Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway
4Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Jose, Costa Rica
Snakebite envenoming is a neglected tropical disease causing 100-150,000 deaths each year, mostly affecting people from impoverished rural areas. The current snakebite antivenoms consist of polyclonal antibodies extracted from the plasma of immunized animals. Though these antivenoms have saved countless lives, their animal origin is associated with major drawbacks, including substantial cost, high dosage, and a propensity to cause adverse reactions. However, recent research has focused on the development of next-generation snakebite antivenoms based on monoclonal antibodies. For such products, low cost is essential, and a promising strategy to achieve this is by engineering (i) the Fc region for enhanced binding to the neonatal Fc receptor (FcRn) and (ii) the antibody-toxin interaction to be pH-dependent. By altering the binding properties of both antibody paratope and Fc, antibodies can be made to work as shuttles, binding toxins in bloodstream at pH 7.4 and releasing them for degradation in the endosomes at pH 5.4, while the antibodies themselves are rescued back to bloodstream via the FcRn-mediated recycling mechanism. Thus, such a single antibody can possibly neutralize multiple toxin molecules in its lifetime, resulting in lowered dosage, and consequently lower treatment cost. In this project, we used phage display technology to discover pH-dependent antibodies against snake venom toxins. Using a binding immunoassay and Bilayer interferometry, we characterized 12 antibodies showing varying binding affinity and pH-dependency towards the toxins. Furthermore, we are currently employing a human endothelial cell-based recycling assay (HERA) to investigate the recycling properties of these antibodies inside cells. If successful, this project may not only contribute in making recombinant snakebite antivenoms more cost-effective with lowered dosage, but also lead to new strategies for developing recyclable antibodies using phage display technology and Fc engineering.
Effects of Lys49 and Asp49-phospholipases A2 from Bothrops pictus venom on mitochondrial bioenergetics and migration of breast cancer cells
Twitter hashtags: #VenOx22, #FUrra
Félix A. Urra1,2, Alex Proleon2,3, Daniel A Torrejón2,3, Ana Belén Tenorio1,2, Camila López-Torres1,2, Cristopher Almarza1,2, Víctor Andrades1,2, Armando Yarleque2,3, Dan Vivas-Ruiz2,3
1Laboratorio de Plasticidad Metabólica y Bioenergética, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile
2Network for Snake Venom Research and Drug Discovery, http://snake-research.com
3Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 01, Lima, Peru.
Snake venom phospholipase A2 (svPLA2) group IIA is subdivided into two types according to their evolutionary pathway: classical PLA2s and PLA2 homologs (or PLA2-like proteins). The latter group exhibits substitutions in the catalytic domain (Asp49Lys) and the calcium-binding loop (Tyr28Asn), lacking phospholipase activity. Emergent evidence suggests that some svPLA2 act on the mitochondrial metabolism of cancer cells, exhibiting unique and novel mechanisms that are not yet fully understood. In this work, we evaluate the effect of both Lys49- and Asp49-PLA2s isolated from Bothrops pictus venom, an endemic species of Peru, on proliferation, migration, and mitochondrial bioenergetics of breast cancer cell line MDA-MB-231. At 48 h of treatment, Lys49-PLA2 exhibited a greater inhibitory effect on proliferation compared to Asp49-PLA2. At non-cytotoxic concentrations, both toxins decreased the migration, evaluated by Transwell assays. On the other hand, the mitochondrial ROS (mtROS) and oxygen consumption rate (OCR) were evaluated using flow cytometry and XFe96 Analyzer. Both Lys49- and Asp49-PLA2s produced an increase in mtROS and a decrease in OCR, suggesting that these toxins induce mitochondrial dysfunction in MDA-MB-231 cells. Taken together, our results suggest that Lys49/Asp49-PLA2s from B. pictus venom may exhibit anti-migratory and anti-proliferative effects by induction of mitochondrial dysfunction in breast cancer cells. Funding: Anillo Grant ACT210097 (ANID-Chile), Redbio0027 (ANID-Chile), UI-024/20 (VID-UChile), PhD fellowship (21221998), FONDECYT 11201322 (ANID-Chile), Grant 079-2021-Fondecyt (PROCIENCIA, Peru) and PCONFIGI B22100141 (VRIP-UNMSM).
High-throughput screening of repurposed drug libraries to identify small molecule snakebite therapeutics
Twitter hashtags: #VenOx22, #AWesthorpe
Adam Westhorpe, Rachel Clare, Laura-Oana Albulescu, Charlotte Dawson, Rohit Patel, Steven Hall, Amy Marriott, Nicholas Casewell
The Centre for Snakebite Research & Interventions, Tropical Disease Biology Department, Liverpool School of Tropical Medicine, Liverpool, UK
Antibody-based antivenom therapies have existed for more than 100 years and are known to have several significant shortcomings, especially in the countries most affected by snakebite. The requirement of cold chain, intravenous administration by a trained specialist, cost, and snake species specificity all impede effective response and treatment of snake envenomation. Small molecule compounds have previously been demonstrated as promising alternative routes of snakebite therapy; however, there are only a handful of compounds that are currently progressing into clinical trials. The Small Molecules group at the Centre for Snakebite Research and Interventions have established a high-throughput workflow for screening thousands of novel and repurposed compounds against specific snake venom toxins per day, with hits progressing into dose-finding and secondary screening assays. Through these validated platforms, we have screened over 15,000 compounds and identified numerous small molecules with previously unknown activity against venom toxins, several of which demonstrate activity close to or matching our current gold standard. Work is ongoing to chemically modify the structures of these hits to improve their activity, bioavailability, and stability, further highlighting the potential for small molecules as a snakebite therapy.
Histological analysis of the effects of the BaMtx: A myotoxic Lys49-PLA2 homologue from Bothrops atrox snake venom
Twitter hashtags: #VenOx22, #JZRomero
Jacquelyne Zarria-Romero1, Alex Proleon1, Daniel Torrejón1, Fanny Lazo1,Armando Yarleque1, Betty Shiga1, Jose Pino1, Felix Urra2, Dan Vivas-Ruiz1
1Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela Cdra 34 S/N, Ciudad Universitaria, Lima 15081, Perú
2Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile
Acute muscle tissue damage (myonecrosis) is a typical consequence of snakebite envenomation by the Viperidae family. Lys49 phospholipases A2 homologues (Lys49-PLA2s) are abundant myotoxic components in viperid venoms that trigger plasma membrane damage by a mechanism independent of phospholipids hydrolysis. However, the exact mechanism of action remains unsolved. In this work, mice were used to assess the action of a Bothrops atrox myotoxin (named as BaMtx),x a Lys49-PLA2. The muscle tissue exposed to the myotoxin was processed for scanning electron microscopy (SEM), previously fixed with glutaraldehyde, dehydrated by an alcohol battery, desiccated at a critical point and metallized with plasmatic gold, in the same way the tissue was fixed, it was embedded in paraffin to subsequently make histological sections by conventional light microscopy with hematoxylin eosin (HE) staining. As a result of the scanning and white light histology, we obtained that the tissue suffers a severe rupture from the M line of the muscle tissue sarcomeres, disorganizing these structures from their center, mainly attacking myomesin/myosin, evidencing the myotoxic damage generated in the tissue. Financial support: Grant PCONFIGI B22100141 and B21101991 (VRIP-UNMSM), Grant N° 079-2017-FONDECYT (PROCIENCIA-Perú), and Anillo Grant ACT210097 (ANID-Chile)