Venoms and Toxins 2020 | Virtual Toxinology despite lock-down

7th International Toxinology Meeting 16 th - 17 th September 2020

@LPMHealthcare | #VenOx20V [email protected]

2 Hamish Ogston Foundation Our sole sponsor

We are grateful to The Hamish Ogston Foundation (HOF) for their continuing support of our Oxford toxinology meetings. HOF is a charitable foundation driven by a desire to contribute to lasting change, across three pillars: Health, Heritage and Music. Taking advantage of the commercial knowledge of its Founder, Hamish Ogston, and an experienced group of advisors, the Foundation introduces new disciplines and generates wider visions on impactful, fiscally prudent and longer-term giving.

The HOF is particularly committed to supporting and raising awareness of some lesser- known and preventable health issues, in particular snakebite. Having recognised the devastating impact snakebite can have, the Foundation aims to alleviate suffering by sponsoring high-quality antivenom research, and has recently committed to support research in three of the Asian countries worst affected by this neglected tropical disease. This will include hospital treatment of snakebite victims and first aid as well as support for studies into the effectiveness and safety of currently available and recently developed antivenoms.

Hamish Ogston CBE is an active philanthropist, supporting a range of initiatives related to health, heritage and music in and outside of the UK. Hamish was awarded his CBE in 2011 for services to business and the community in York.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 3 Contents

General Zoom instructions for participants 4 Information for speakers 6 Information for poster presenters 7 Timetable – Wednesday 16 th September 2020 8 Timetable – Thursday 17 th September 2020 10 Podium abstracts 13 Poster abstracts 33

DISCLAIMER

The information specified in oral and poster presentations, written abstracts and biographies come from diverse sources and it is not in the capacity of event organisers to validate it, and is provided on an ‘as-is’ basis. Responsibility for the literary and scientific content of the abstracts and the presentations, both oral and poster, remains with the authors and the presenters. Therefore, the event organisers accept no responsibility for literary or scientific correctness of this information, and shall accept no liability of any kind, should any of the information be incorrect. The event organisers and hosts make no representation or warranty of gain of business or profits as a result of use of services or information provided in connection with the even and shall not be liable for any direct or indirect damages, loss of business, employment, profits or anticipated savings resulting from the use of the services or information provided in connection with the event, in any country or court of law. Furthermore, the materials contained in the event handbook are provided on the understanding that speakers or presenters have the right to their presentation in this manner. Therefore, event organisers and hosts shall not be liable for infringement of third party rights by an event presenter, participant, sponsor, supporter or exhibitor. We cannot guarantee a minimum number of attendees.

This handbook is for use by the Venoms and Toxins 2020-Virtual (16-17 September 2020) participants only.

© 2020 Copyright Information: Textual and graphical contents of this handbook are copyright of presenters, sponsors, instructors and/or LibPubMedia Ltd, unless explicitly stated otherwise. No part of this handbook may be reproduced, distributed or transmitted in any form or by any means, electronic or mechanical, including but not limited to, photocopy, recording, or any other information storage or retrieval system, without the prior written permission of the legal copyright owners.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 4 General Zoom Instructions for Participants

Getting started

We will be using Zoom for our virtual meeting, which is a commonly used video conferencing platform. If you have not used Zoom previously, please see the following link, which has many useful short videos about this platform, including about joining a meeting : https://support.zoom.us/hc/en-us/articles/206618765-Zoom-video-tutorials https://support.zoom.us/hc/en-us/articles/360034967471-Quick-start-guide-for-new-users

To use Zoom, you will need the following:

• A computer (laptop/desktop) with a microphone and webcam (or a tablet or smartphone with speaker or headphones ) * • Speakers/headset/earbuds • High-speed internet *NB: Please log in from one device only. If you are logged in from two devices, one of them will be removed from the meeting. Thereafter, you may not be able to join the rest of meeting from this device, especially if this was your main meeting access device.

Logging in/joining the meeting and downloading Zoom client

You will receive an email notice from your meeting chair to join the meeting via Zoom on your registered day(s). The email will include information such as:

• A link to join the meeting • The meeting title and scheduled times • Phone numbers for a conference call option • The meeting ID (9 or 11 digits) and passcode, etc.

If you have registered to attend both days, you will receive separate email notifications for each day. For security reasons, do not share the links, user or conference IDs or passwords with anyone else – this may prevent you from accessing the meeting. You may also be required to register your email beforehand.

When you click on the link provided by your host, you will be asked to download or launch Zoom client on your device. We recommend you install the software before the start of the meeting. Zoom releases regular updates to their desktop program. It is important to ensure you are running the most recent version of Zoom so that you have the most up-to-date controls when you are joining the virtual conference.

You can either click on the link provided, or go to https://join.zoom.us/ and enter the 9- or 11-digit Meeting ID number (see the red box on next page).

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 5 Closing all programs that will not be required for the duration of the virtual conference will improve the connection quality.

Waiting for the virtual conference to begin

When you join the meeting on 16 th and/or 17 th September, for security reasons, you will be sent to the ‘ waiting room ’, and will be admitted to the meeting manually against the list of registered participants that we hold. Therefore, it is important that your Zoom joining name is the same as it appears in your registration form/invoice - DO NOT use your initials or nickname . Otherwise, you may not be admitted to the meeting.

Before joining, you will have the opportunity to test your audio by clicking on ‘Test Computer Audio’. Once you are satisfied that your audio works, click on ‘Join audio by computer’.

The virtual conference

The Zoom menu bar will appear at the bottom of the Zoom window once the meeting begins. If you do not see the menu bar, move your mouse slightly and the bar will appear ( the bar disappears after a few seconds when in full-screen mode).

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 6

You can:

1) Mute/unmute your own microphone (by default during speaker sessions, all microphones will automatically be muted) 2) Turn on/off your camera (‘Start/Stop Video’) (by default during speaker sessions, videos sharing will automatically be turned off) 3) View the participant list – opens a pop-out screen that includes a ‘Raise Hand’ icon that you may use to raise a virtual hand to ask a question 4) Share your screen (only for speakers and for use in breakout rooms) 5) Chat – the chat function allows attendees to send messages to be seen by all attendees. We ask participants to be polite and respectful when asking questions, keeping discussions courteous and non-confrontational and based around the scientific content 6) Record (this feature will be disabled for participants) 7) Leave the meeting – please note: once you leave the meeting, you will no longer be able to join on the same device

On your Zoom screen (usually top right) you will also see a choice to toggle between ‘speaker’ and ‘gallery’ view. ‘Speaker view’ shows the active speaker. ‘Gallery view’ tiles all of the meeting participants.

Information for speakers

Your talk will continue as scheduled. As with a physical meeting, each session will proceed in the order identified and maintain the schedule.

• Please allow at least 5 minutes for questions and switching to the next speaker at the end of your talk (guide: ca. 6-7 slides for a 10 + 5 minute talk and 10-12 slides for a 15 + 5 minute talk). • Ensure video and audio are enabled for your presentation and are working prior to the virtual conference, and test the share screen option. Plan to join the meeting 15-20 minutes early in case there are any issues with your video/audio. • If you intend to share a third-party audio/video, please select ‘Share Computer Sound’ (bottom left corner). • Using the ‘Share Screen’ option ( 5), share your presentation screen with the participants. Once the screen has been shared, please start your presentation (please ensure you are not on ‘presenter view’, as participants will not be able to see your slides – you will still be visible to the participants while screen sharing in the top right-hand corner of the screen). •

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 7 • For those with Zoom experience, you may use the whiteboard screen if you wish to draw/write out anything during your presentation. • Once the presentation is finished, the chair will moderate questions from the participants.

Click on the ‘Stop Sharing’ button to stop screen sharing, and your video will automatically resume.

Information for poster presenters

http://lpmhealthcare.com/venoms-and-toxins-2020-virtual/posters

Prepare your poster as you would normally do for printing, and submit your final poster as both PDF and JPEG/JPG/PNG files online, no later than 07 th September 2020 . Late posters may not be included in the conference programme. Please DO NOT send your poster files by email . Posters will be made available via a secure page to the conference participants before the meeting.

Poster presentation: Posters will be made available via a secure page to the conference participants before the meeting.

The participants will be able to interact with the poster presenters in two ways:

• private interaction with the presenters: the participants will be able to ask questions via the Zoom chatbox during the mid-conference break each day; and/or • public interaction with the presenters: 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).

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. However, the presenters are not compelled to respond via Twitter.

We ask participants to be polite and respectful when asking questions, keeping discussions courteous and non-confrontational and based around the scientific content.

Any further information about the poster presentations at this meeting will be posted on the website.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 8 Provisional Agenda

(Subject to change) All times mentioned refer to British Standard/Summer (BST). World Time Converter: https://www.worldtimebuddy.com We have tried to group similar talks, but due to the wide geographical locations of our speakers, this may not always been possible.

Day 1: Wednesday 16 th September

1000: Welcome to Oxford on behalf of the conference co-chairs by Professor Anna Nekaris, Professor in Primate Conservation, Oxford Brookes University, Oxford, UK

Painful plants 1010: Professor Irina Vetter, Co-Director, IMB Centre for Pain Research and Principal Research Fellow, Institute for Molecular Bioscience, the University of Queensland, Queensland, Australia Neurotoxic venom peptides from the giant Australian stinging tree

1030: Symposium on snakebite in Africa, from West to East - Chair Professor David Warrell

1035: GHANA - Dr John Amuasi, Leader, Global Health and Infectious Diseases Research Group, Kumasi Collaborative Center for Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Reducing Snakebite Envenoming in Ghana: A low-hanging fruit

1055: NIGERIA - Professor Abdulrazak Habib and Dr Saidu Abubakar Ballah, Infectious Disease Unit, Aminu Kano Teaching Hospital, Bayero University, Kano, Nigeria Challenges and Prospects of Snake Antivenom Supply in Sub-Saharan Africa 1115: CAMEROON - Dr Frank Tianyi Tianyi, Chief Medical Officer, Mayo Darle Sub Divisional Hospital, Cameroon; and Oxford University Africa Society, UK Shortcomings in snakebite management in rural Cameroon 1135: KENYA - Dr Mitchel Otieno Okumu, Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya Management and cost of snakebite injuries at a teaching and referral hospital in Western Kenya 1155: Discussion and close of the symposium 1200: Break and Posters-I

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 9 1235: Venom therapeutics - Chair Dr Eddie Rowan

1240: Dr Igor Krizaj, Professor of Biochemistry, Scientific Counsellor, Group Leader and Head, Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia New viper venom proteins with high therapeutic potential for blood and cardiovascular disorders 1300: Professor Gisele Picolo, Scientific Researcher, Group Leader, Laboratory of Pain and Signaling, Butantan Institute, São Paulo, Brazil Potential use of compounds isolated from rattlesnake venom as central modulators of pain and inflammation

1320: Early Career Researchers Latin America Coordinator: Dr Rita de Cássia Collaço 1325: Miss Vanessa Costa de Oliveira, Universidad de Buenos Aires, Argentina Antineurotoxic arachnidic polyvalent antivenom experimental production in Argentina 1335: Miss Luz E. Romero, University of Antioquia, Colombia Recombinant immunogens type phospholipase A2 and three-finger toxin for the production of antivenoms of clinical interest in Colombia 1345: Mr Andrés Agurto, Universidad Nacional Mayor de San Marcos, Peru Design of primers for the identification of some poisonous Peruvian snakes by the multiplex loop- mediated isothermal amplification reaction 1355: Dr Karen Sarmiento, Pontificia Universidad Javeriana, Colombia Comparison of the specificity and neutralizing capacity of two antivenoms for snakebite challenged with Bothrops rhombeatus venom 1405: Dr Caroline B F Mourão, University of Brasília (UNB), Brazil ToPI1, from Tityus obscurus scorpion venom, and its head-to-tail cyclization after interaction with trypsin 1415: Dr Manoela Torres-Rêgo, Federal University of Rio Grande do Norte, Brazil Biodegradable cross-linked chitosan nanoparticles improve anti-Candida and anti-biofilm activity of TistH, a peptide identified in the venom gland of the Tityus stigmurus scorpion

1425: Recovery from envenoming and treatment - Chair Professor David Warrell

1430: Dr Naira Ayvazyan, Orbeli Institute of Physiology of the National Academy of Sciences, Yerevan, Armenia Pros and cons of paraspecificity and cross-reactivity of different monospecific anti-Vipera antivenom products towards the Middle East vipers’ venoms

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 10 1450: Professor Cesare Montecucco (and Mr Marco Stazi) , Emeritus Professor of Neurosciences, CNR Neuroscience Institute, Emeritus Professor of General Pathology, Department of Biomedical Sciences, University of Padova, Padova – Italy NUCC-39 activates the CXCR4 receptor and accelerates the recovery from the peripheral neuroparalysis induced by Taipan snake envenomation 1510: Professor Andreas Laustsen, Associate Professor, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark Progress towards next-generation antivenoms 1530: Professor José María Gutiérrez, Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica Why is skeletal muscle regeneration impaired after necrosis induced by viperid snake venoms?

1550: Presentation of Lifetime Achievement Award to Dr. Charlotte L Ownby , Professor Emeritus, Physiological Sciences, OSU Director Emeritus, OSU Microscopy Laboratory Stillwater, OK, USA (citation by Professor José-María Gutiérrez)

1610: Close of Day 1

Day 2: Thursday 17 th September

(Subject to change) All times mentioned are British Standard/Summer Time (BST). World Time Converter: https://www.worldtimebuddy.com We have tried to group similar talks, but due to the wide geographical locations of our speakers, this may not always been possible.

0955: Welcome by Dr Eddie Rowan

1000: Professor Glenn King, Professorial Research Fellow, Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia A spider-venom peptide for treatment of chronic visceral pain

1020: Toxin Themes from SE Asia - Chair Professor David Warrell

MYANMAR 1025: Professor Yi Yi Khine, Myanmar, Senior Nephrologist and Head of National Snakebite Control Programme, Myanmar

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 11 The snakebite control programme in Myanmar 1045: Dr Bethany Moos, General Practitioner, Hedena Health, Oxford National Health Services, UK A retrospective descriptive study of snakebite patients admitted to Yangon General Hospital in Myanmar, in 2018 THAILAND 1105: Dr Lawan Chanhome, Head of Snake Farm, Queen Saovabha Memorial Institute, The Thai Red Cross Society, Bangkok, Thailand Updates in Venomous Snakes of Thailand: An Issue of Protobothrops kelomohy venom and its envenomation MALAYSIA 1125: Dr Michelle Khai Khun Yap, School of Science, Monash University Malaysia, Bandar Sunway, Malaysia The myth of venom cytotoxin – more than just cytolytic actions

1145: Venomous taxa and venom evolution - Chair Professor Anna Nekaris

1150: Dr Ronald Jenner, Department of Life Sciences, The Natural History Museum, London, UK Horizontal gene transfer is a key mechanism of centipede venom evolution 1210: Miss Mathilde Wells, University of Mons, Laboratory of Pharmaceutical Analysis, Hainaut, Belgium Discovery strategies for novel antiplasmodial compounds from the venom of Bufo toads 1230: Mr Agneesh Barua, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan Toxin expression in snake venom evolves rapidly with constant shifts in evolutionary rates 1250: Professor Juan Calvete, Laboratorio de Venómica Evolutiva y Traslacional, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain What's in a mass?

1310: Break and Posters-II

1345: Toxin Themes from the Western hemisphere - Chair Professor José María Gutiérrez

1350: Professor Denise Tambourgi, Principal Investigator and Lead Researcher, Laboratório de Imunoquímica, Instituto Butantan, Brazil Complement inhibition: a possible therapeutic strategy for controlling cobra envenomation

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 12 1410: Professor Elda Sánchez, Director, National Natural Toxins Research Center, Texas A&M University-Kingsville, TX, USA Paraspecific neutralization of venoms 1430: Professor Fan Hui Wen, Project Manager, Strategic Nucleus for Poison and Antivenom (NEVAS), Instituto Butantan, São Paulo, Brazil Revisiting the big antivenom crisis in Brazil 1450: Dr Dabor Resiere, Head of Clinical Toxicology Resuscitation Department, Critical Care Unit, University Hospital of Martinique, Fort-de-France, Martinique Bothrops lanceolatus update

1510: Poster Prizes – announcement

1530: Discussion and close

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 13 Podium Abstracts (Unedited) Neurotoxic venom peptides from the giant Australian stinging tree

Edward K. Gilding, 1† Sina Jami, 1† Jennifer R. Deuis, 1† Mathilde R. Israel, 1,2 Peta J. Harvey, 1 Aaron G. Poth, 1 Fabian B.H. Rehm, 1 Jennifer L. Stow, 1 Samuel D. Robinson, 1 Kuok Yap, 1 Darren L. Brown, 1 Brett R. Hamilton, 3 David Andersson, 2 David J. Craik, 1 Irina Vetter ,1,4 * Thomas Durek 1*

1Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia 2Wolfson Centre for Age-Related Diseases, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, SE5 8AF, United Kingdom 3Centre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia 4School of Pharmacy, The University of Queensland, Brisbane, QLD 4102, Australia

Stinging Trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours and intermittent painful flares can persist for days and weeks. Our recent work shows that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate sensory neurons and delay inactivation of voltage- gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure and pharmacology. Reducing Snakebite Envenoming in Ghana: A low-hanging fruit

John H. Amuasi 1,2 , Leslie Aglanu 2, Ibrahim Kwaku Duah 2, Louis Adu-Amoah 2, Nana Kena Frempong 3, Anthony Afum-Adjei Awuah 2, Franklin Asiedu-Bekoe 4, Yaw Badu-Sarkodie 4

1Department of Global and International Health, School of Public Health, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 2Global Health and Infectious Diseases Research Group, Kumasi Center for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 3Department of Statistics and Actuarial Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana 4Ghana Health Service, Accra, Ghana

In Ghana, reducing the burden of snakebite envenoming (SBE) can be described as a “low-hanging fruit which is yet to be plucked” i.e. the negative impact of snakebites can be prevented with relative ease. However, the major factor slowing down Ghana’s progress towards reducing the incidence and negative effects of SBE is the unavailability of reliable data on the types and distribution of snakes and the burden of snakebites to inform effective and efficient intervention. The research conducted made use of data from Ghana’s District Health Information Management System and other sources, and reveals the incidence and distribution of snakebites in Ghana over the past five years. Findings show that in Ghana for 2015 – 2019, there were on the average 9,600 snakebites per year, although it is not known how many of these bites resulted in deaths. The Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 14 months of May and June have consistently accounted for the highest number of bites, while those aged between 20 and 34 years suffer the most bites. The average number of bites per year (2015 – 2019) show that the Upper West (1,425), Ashanti (1,161), Eastern (987), Central (897) and Northern (781) regions account for the top five. However, in 2019 for the first time in five years, the Ashanti region recorded the highest number of bites (1,535), followed by the Eastern (1,242), Upper West (961), Central (886) and Northern (799) regions. More importantly, when the population size of each region is taken into consideration, even in 2019 we calculated 114 bites for every 100,000 persons in the Upper West region followed by the Savannah (103 for every 100,000 persons) and the North East and Western North regions (tied at 64 for every 100,000 persons). While on the average, males account for 56% of bites, this is not a reflection of the impact of the snakebites based on gender, which will require further research to determine. We further model the rate of SBE in each region using a count time series approach and forecast the number of snakebites expected in 2020. The data available is unlikely to provide the full picture of snakebite in the country, because they rely on only those figures recorded and reported by health facilities. Nonetheless they are by far the best available evidence to-date of the burden of the disease in Ghana, and can be used to inform immediate targeted interventions to address snakebite envenoming across the country. Challenges and Prospects of Snake Antivenom Supply in Sub-Saharan Africa

Abdulrazaq G Habib 1,2,3,4* , Baba M Musa 1,4 , Garba Iliyasu 1,2,3,4 , Muhammad Hamza 2,3 , Andreas Kuznik 5, Jean-Philippe Chippaux 6

1Health Economics & Outcomes Research (H-CORE) Group, 2Venom and Antivenom Research Project (VASP), 3African Snakebite Research Group (ASRG) Project, 4Africa Center of Excellence in Population Health and Policy Bayero University Kano, Nigeria 5Regeneron Pharmaceuticals, Tarrytown, NY, USA 6Université de Paris, MERIT, IRD, F-75006, France; Institut Pasteur, CRT, F-75015, France

Snakebite envenoming (SBE) is a major public health concern that causes substantial morbidity and mortality in Sub-Saharan Africa (SSA). Despite highly favourable antivenom cost-effectiveness and an annual burden estimated at over 1 million DALYs, resource allocation and consequent antivenom supply, access, affordability and availability have been grossly inadequate. The recently launched Roadmap strategy for prevention and control of SBE by WHO seeks to halve the burden by 2030. It utilizes multifaceted approaches involving phased-in antivenom stockpiling, hoping to make available 3 million treatments annually at full roll-out by 2030. However, in SSA an estimated annual amount of $51 to 66 million will be needed to halve the burden based on antivenom therapy prices of $100 to 153 with 54 to 65% of the amount required for antivenom procurement. Furthermore as a non-communicable disease antivenom stockpiling for SBE may pose peculiar challenges to sustain in the long-term. Innovative approaches such as sub-regional joint pooled antivenom procurements, reduced antivenom acquisition costs through volume procurements and economies of scale, and inclusion of antivenom supply in subsidy-oriented sustainable revolving funding programmes facilitated by international and regional health agencies are recommended. It is imperative for countries to increase budgetary allocation as well as establish sub-regional local antivenom production facilities through public-private-partnerships using either north-south or Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 15 south-south collaborations. Logistics of procurement, distribution and monitoring, as well as forecasting and training on rational utilization of AV should be strengthened in SSA. Shortcomings in snakebite management in rural Cameroon

Frank Tianyi 1,2 , Armand Nkwescheu 2

1Nuffield Department of Population Health, University of Oxford, Old Road Campus, Oxford, UK 2Cameroon Society of Epidemiology, Yaoundé, PB 1411, Cameroon

Snake bites represent a major public health problem, disproportionately affecting poor rural communities. A high incidence has been associated with rural agricultural activities with farmers and children representing the most vulnerable groups. Prompt administration of potent anti- venom remains the mainstay of management. However, in Cameroon, the use of anti-venoms is limited by non-availability, high cost (where available) and poor mastery of treatment guidelines. Furthermore, treatment for snakebite envenoming is time-sensitive, with early access to appropriate medical care significantly decreasing the risk of death and disability. We present three case-studies of snakebite victims in Cameroon, and how an interplay of the biting snake, antivenom availability and health seeking behaviour affected their outcomes. We will highlight the shortcomings of snakebite management in Cameroon, and steps being taken by the Cameroonian Ministry of Public Health and the Cameroon Society of Epidemiology in reducing the burden of snakebites in Cameroon. Management and cost of snakebite injuries at a teaching and referral hospital in Western Kenya

Mitchel O Okumu 1, 2 , Minal N Patel 1, Foram R Bhogayata 1, Francis O Ochola 3, Irene A Olweny 1, Joshua O Onono 2, Joseph K Gikunju 4

1Department of Pharmacy, Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, Kenya 2Department of Public Health, Pharmacology, and Toxicology, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya 3Department of Pharmacology and Toxicology, Moi University, Eldoret, Kenya 4Department of Medical Laboratory Science, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

Data on the cost of snakebite injuries may inform key pillars of universal health coverage including proper planning, allocation, and utility of resources. This study evaluated the injuries, management, and costs resulting from snakebites at the Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Western Kenya. In total, medical records of 127 snakebite victims attending the hospital between January 2011 and December 2016 were purposively selected. Data on the age, gender, type of residence (urban or rural),part of the body bitten, time of bite, injuries, pre-hospital first aid, time to hospital, length of stay, treatment, and direct medical costs were collected from these records. Regression analysis was used to predict the direct medical cost of snakebite injuries and p≤ 0.05 was considered significant. Mortality and loss of income of hospitalized victims were considered as indirect medical costs. It was established that 33.9 % of victims were 13-24 years of age, 50.4 % were female, and 74.0 % were from rural areas. 72.4 % were bitten on the lower limbs, 38.6 % were bitten between 6.00 pm and midnight, 33.9 % attempted pre-hospital first aid, and the

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 16 median time to hospital was 4.5 hours. 54.3 % of the victims presented with neurotoxic symptoms while 18.9 % presented with both neurotoxic and hemotoxic symptoms. Antivenom, supportive fluid therapy, antibiotics, antihistamines, corticosteroids, analgesics, and non-steroidal anti- inflammatory drugs were used in snakebite management. Cellulitis, compartment syndrome, gangrenous foot, psychiatric disorder, and death were the main complications. Only 21.3 % and 29.2 % of victims who exhibited neurotoxic symptoms or both neurotoxic and hemotoxic symptoms respectively received antivenom. Most victims spent 1-5 days in the hospital and the median direct cost of treating snakebite injuries was 2652 KES (~$26); range 1100-41399 KES (~$11-414). Drugs, ward charges, and nursing procedures were the highest contributors to the direct medical cost of snakebite injuries. Victims hospitalized for 6-10 days and >10 days incurred 32 % and 62 % more costs, respectively, compared to those hospitalized for 1-5 days. The longer snakebite victims are hospitalized, the higher the direct medical cost incurred. Despite the low utility of antivenom in the study area, snakebite outcomes were generally good. However, in view of the extended length of stay and high costs incurred by some snakebite victims, we recommend continuous medical education on the correct management of snakebite to minimize complications that may increase hospital stays and costs incurred. New viper venom proteins with high therapeutic potential for blood and cardiovascular disorders

Igor Križaj

Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia

Recently, we isolated two haemostasis-affecting glycoproteins from the venom the nose-horned viper ( Vipera a. ammodytes ; Vaa ), VaaSPH-1 and VaaSP-VX. The former is strongly anticoagulant, while the latter acts oppositely on the blood coagulation. Both molecules are serine proteases by structure, however, only VaaSP-VX expresses proteolytic activity. Description of the molecular mechanism behind anticoagulant action of VaaSPH-1 exposed this molecule as an ideal natural template molecule to design original anticoagulants to attenuate thrombus formation and propagation without increasing the risk of bleeding. This endows VaaSPH-1 with a promising medical potential concerning therapy of venous thromboembolism (VTE), the pathological process behind two very serious cardiovascular diseases (CVD), deep vein thrombosis and pulmonary embolism. These CVDs represent the third leading cause of CVD-related mortality, in the first place because no adequate therapy is available. The procoagulant VaaSP-VX also turned to be unique in its way of action. As we demonstrated, its blood coagulation-inducing effect relies on its ability to activate blood coagulation factors V and X simultaneously. Following the activation, these two coagulation factors combine to form prothrombinase complex, the key element of the enzymatic cascade leading to blood coagulation. VaaSP-VX is the first protease known to possess such dual activity, which makes it very efficient procoagulant. Since it is also resistant to inhibition by serpins in the blood, its potential to treat patients suffering from haemophilia is evident.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 17 Potential use of compounds isolated from rattlesnake venom as central modulators of pain and inflammation

Sant’Anna MB; Giardini AC; Teixeira NB; Vieira LFK; Zambelli VO; Picolo G

Butantan Institute, Laboratory of Pain and Signaling, Sa ̃o Paulo, Brazil

The management of pain is still a challenge, since many patients suffer with incomplete relief or undesirable side effects, especially in chronic conditions. Then, the search for new analgesic compounds is continuous. Animal venoms and toxins are rich sources of bioactive molecules. Studies performed in the last years by our group have demonstrated the analgesic effect of crotalphine and crotoxin, two molecules obtained from crotalid venom. Crotalphine is a 14-aa synthetic peptide that induces analgesic activity by endogenous opioid release and TRPA1 channels desensitization. Crotoxin is the main neurotoxin from crotalid venom that has prolonged anti-inflammatory, immunomodulatory and antinociceptive activities already described. We have investigated the central effect of these compounds as modulators of pain and inflammation. Crotalphine effect was investigated in the experimental autoimmune encephalomyelitis (EAE) model, an animal model of multiple sclerosis. It caused partial reversion of EAE-induced hyperalgesia and decreased the severity of the clinical signs. Crotalphine reduced, in the spinal cord, the immunoreactivity of the Egr-1 (marker of neuronal activity), CD11-b (microglia/macrophages marker) and GFAP (astrocytes markers) induced by EAE. In addition, it increased IL-6 and reduced TNF-α expression and inflammatory infiltrate. Crotoxin was investigated in the same EAE model and in the neuropathic pain induced by partial sciatic nerve ligation model. CTX induced a long-lasting analgesic effect in both models. CTX inhibited IFN-γ-producing T cells, GM-CSF-producing T cells, reduced the frequency of activated microglia/macrophages within the CNS and decreased the number of migrating cell to spinal cord and cerebellum induced by EAE. In PSNL model, CTX inhibited the expression of Iba-1 (microglia/macrophages marker) and GFAP (astrocytes marker) induced by the surgery. Taken together, these results point out the potential of both compound in attenuate the neuroinflammation, conferring them as promising agents for the control of chronic pain. Finantial support: Fapesp and CNPq. Antineurotoxic arachnidic polyvalent antivenom: experimental production in Argentina

Vanessa C. de Oliveira 1,2 ; Laura C. Lanari 2; Jantine H. van Grootheest 2; Carlos F. Damin 1;

Adolfo R. de Roodt 1,2

1Primera Cátedra de Toxicología, Facultad de Medicina, Universidad de Buenos Aires, Argentina 2Área Investigación y Desarrollo – Venenos, INPB-ANLIS “Dr. Carlos G. Malbran”, Ministerio de Salud y Desarrollo Social, Argentina

Human neurotoxic arachnid envenomation can lead to death. In Argentina is caused by Latrodectus and Phoneutria spider genera and the scorpion genus Tityus. In many regions, these three species overlap in geographic distributions and, unfortunately, in accidents caused. Venoms are composed by a variety of toxins and physiopathology of envenomation occurs differently, although, clinical development of symptoms may be very similar. In severe cases may lead to pulmonary edema, respiratory distress and cardiac failure leading to death mostly in scorpion envenomation. In many Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 18 attended cases anamnestic data are not always reliable or directly absent, especially in pediatric cases. This poses a serious problem for therapeutic success, as the fast instauration of specific treatment is vital, and demands the rapid application of antivenom. In those cases of overlapped species regions, lacking proper identification of the arachnid, and similar clinical manifestations, the use of polyvalent antivenom avoid binding of different toxins to their target sites and thus enhance the possibility of success of the treatment. An equine anti Phoneutria-Latrodectus-Tityus antivenom was produced, applying the classical procedures. This hyperimmune serum or its purified IgG were able to neutralize the different venoms. Mice (CF-1, 18-22g) were fully protected against a challenge dose of 3.0LD 50 s of Latrodectus spp. venom incubated with 100 μl of crude serum. One ml of crude serum protected guinea pigs (Hartley) against 1.5MMD of Phoneutria nigriventer envenomation. The purified IgG of the antivenom protected 100% of mice challenged with 3.0LD 50 s of Tityus trivittatus venom, in a dose of 250 μl. Considering the neutralizing potency of the specific antivenom currently used in Argentina, the arachnidic polyvalent produced is a valuable therapeutic option, since a 5.0 -10.0 ml vial has a similar or higher neutralizing potency than the ones required by the sanitary authorities. Recombinant immunogens type phospholipase A2 and three-finger toxin for the production of antivenoms of clinical interest in Colombia

Luz E. Romero 1* , Vitelbina Nuñez 1, Paola Rey 1, Sergio Pulido 2, Mónica Saldarriaga 3, Jaime A. Pereañez 1

1Research Group in Toxinology, Pharmaceutical, and Food Alternatives. * Financial support COLCIENCIAS Project 111577757474. University of Antioquia. Medellín, Colombia 2Tropical Disease study and Control Program – PECET. University of Antioquia. Medellín, Colombia 3The University Bernardo O’Higgins. Santiago de Chile, Chile

Snakebite envenoming is a neglected tropical disease of high impact in the rural areas of Asia, Africa, Latin America, and Papua-New Guinea. A wide distribution of snakes throughout the national territory, especially in regions densely populated, constitutes a risk in morbimortality in Colombia. The Crotalus and Micrurus genera are snakes of clinical interest that cause between 1% and 2% of neurotoxin envenomings from snakes in Colombia, respectively. The neurotoxicity of the genus Micrurus venom is due to β-neurotoxins that inhibit the release of acetylcholine at the pre- synaptic level such as phospholipase A2 (PLA2), and α-neurotoxins such as three-finger toxins (3FTx) that block the nicotinic receptor of acetylcholine. For its part, the presynaptic neurotoxicity produced by the venom of the genus Crotalus lies in β-neurotoxins such as enzymatically active PLA2. In this context, antivenom is the most effective immunotherapy for the treatment of victims of envenomings from snakes. However, obtaining and availability of the venom necessary to produce antigens is difficult, this is due to factors such as lack of individuals, the captivity condition, and in the case of the Micrurus genus the anatomical characteristics underlying the venomous system. In other words, the real problem is the difficulty to obtain the venom necessary to produce antibodies. In this order of ideas, the objective of this project is to develop recombinant immunogens type phospholipase A2 and three-finger toxin to produce antivenoms of clinical interest in Colombia. The first part of methodology was based on the synthesis and cloning of neurotoxins type PLA2 and 3FTX, selected based on their major abundance, toxicity and immunological cross-reactivity in the venom of three species of venomous snakes with neurotoxic Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 19 effects: Micrurus mipartitus , Micrurus dumerilii and Crotalus durissus terrificus . Amino acid sequences both of α and β-neurotoxins from these snake venoms were collected from the UniProt Protein database (http://www.uniprot.org). The neurotoxins primary structure was used as a template to generate a DNA sequence, by reverse translation and then optimized with preferential codon usage in Leishmania tarentolae and Spodoptera frugiperda according to database to http://www.kazusa.or.jp/codon. Furthermore, the restriction sequences for NcoI, NotI and XbaI were introduced in the genetic construct for every neurotoxin optimized for L. tarentolae , and BamHI and NotI in the constructs optimized for S. frugiperda , respectively. Additionally, a polyhistidine tag, a Glicin-Serin linker, and the coding sequence for the recognition site for TEV- protease was introduced downstream of the restriction sites of N-terminal. For the synthesis of the genetic constructs, we hired with General Biosystems which the commercial cloning vector pUC57_BsaI_Free. Then, different expression vectors: pET28a, pLEXSY and pFastBac, and heterologous hosts: BL21 (DE3)-ArticExpress, BL21 (DE3)-RIPL y BL21 (DE3) pLysS of E. coli , L. tarentolae cells and Sf9 insect cells, that allows the expression of the recombinant proteins will be evaluated, and the best expression model will be standardized. Once the recombinant neurotoxins are obtained, their functionality is evaluated in vivo for their ability to be lethal and in vitro for their phospholipase A2 and myotoxic activity. Subsequently, recombinant anti-neurotoxin hyperimmune sera will be produced from the generation of antibodies in an immunization scheme in rabbits, with increasing doses of immunogens and at intervals of 20 days after the first inoculation for five months. Antibody titers and recognition will be analyzed using the double antibody enzyme immunoassay, which will detect the antibody levels of each antivenom. Finally, for the neutralization of the recombinant neurotoxins by the specific hyperimmune serum, the lethality neutralization test will be used. As preliminary results we selected four neurotoxins identified with Uniprot code: B3EWF8, C0HKB9, C0HKB8, and P0CG56. These sequences were the base for the design of eight genetic constructs: four optimized for L. tarentolae and other four for S. frugiperda . Due to the similarity of the codonic use between L. tarentolae and E. coli , the genetic constructs included sequences for restriction sites of E. coli too. Thus, we successfully cloned in pET28a. BL21 (DE3)-ArticExpress and BL21 (DE3)-RIPL colonies, transfected with every construct, were able to express the four neurotoxins. This was determined by expression analysis using SDS-PAGE 14% Tris-Trycin, confirming the presence of the expressed protein with an apparent molecular weight at the 10–17 kDa region. At the end of this project, it is expected to obtain: 1) Recombinant neurotoxins. 2) Specific antibodies for each recombinant neurotoxin. In this way, the development of recombinant DNA technology will be used to innovate in the production of proteins of clinical relevance and in the generation of antibodies with new and better properties, contributing to the decrease in morbidity and mortality rates in the country. Design of primers for the identification of some poisonous Peruvian snakes by the multiplex loop-mediated isothermal amplification reaction

Andres Agurto Arteaga , Dan Vivas Ruíz, Armando Yarlequé Chocas

Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú

In Peru, snake bite envenomation is a public health major concern that affects an average of 2150 individuals per year, and may result in death or invalidating injury to their victims, due to the lack of Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 20 effective treatment available in areas with the highest incidence of ophidic accidents, which coincide with impoverished human populations, as many of the snakes responsible for accidents are not identifiable by health personnel, which makes it difficult to choose treatment using antivenom serums. Based on this, we focused on the development of a method of snake’s identification that is fast, sensitive, specific, easy to handle and cost- effective, by applying the multiplex loop-mediated isothermal amplification technique. To this end, the nucleic acid sequences of Peru's major venomous snakes were extracted from the GenBank database, of the mitochondrial genes COI, CYTB and 12S rRNA, and an alignment was performed using the CLUSTAL W tool, to subsequently design degenerate primers for the PCR reaction and obtain those sequences that were not available, and also to confirming those that were. In addition to this, genomic DNA was extracted from the main snakes of the genus Bothrops (Bothrops atrox , Bothrops pictus , Bothrops brazill i, Bothrops barnetti and Bothrops Chloromelas ), of the snake Crotalus durissus, and the snake Lachesis muta , from the moulting of the skins of specimens held in captivity in the serpentine Oswaldo Meneses of the UNMSM, using the commercial kit GeneJet, according to the recommendations of the manufacturer. PCR reactions for the 3 genes under study were standardized and PCR products were sequenced using the Sequence service of Macrogen (Korea), and the chromatograms thus obtained were analyzed for the respective sequences, which were used for the design of mLAMP reaction primers using the online software PrimerExplorer V5.0 obtaining the best result in sequence divergence using the COI gene for Crotalus durissus, the CYTB gene for Lachesis muta, and the 12S rRNA gene for snakes of the genus Bothrops. It is concluded that the mitochondrial genes used are appropriate for the design of isothermal reaction primers in this preliminary study, and it is expected that, in conjunction with different fluorescent probes for each genus of snakes, the identification of the snake responsible for the ophidic accident will be achieved in order to administer appropriate treatment in favour of patients. Comparison of the specificity and neutralizing capacity of two antivenoms for snakebite challenged with Bothrops rhombeatus venom

Karen Sarmiento 1, Alejandro Alagón 2 Ana L Castiblanco 3

1Department of Physiological Sciences, Faculty of Medicine, Pontificia Universidad Javeriana, Bogotá, Colombia 2Toxins Analysis Laboratory, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México 3Instrumental Analysis Laboratory, Instituto de Biotecnología, Universidad Nacional de Colombia, Bogotá, Colombia

The aim of this research was to evaluate the specificity and affinity of INS (Colombia) and AVP-T (Mexico) antivenoms with Bothrops rhombeatus , Bothrops asper and Bothrops atrox venoms. The test was in Mus musculus CD1 of 18-20 g. SDS-PAGE was performed and RP-HPLC to analyze the protein profile. The mean lethal dose (LD 50 ) of the venom and the mean effective dose (ED 50 ) of the antivenoms were calculate. The immunochemical specificity and affinity of the antivenoms for the venom were evaluated with affinity chromatography, Western Blot, and enzyme-linked immunosorbent assay (ELISA). The LD 50 of B. rhombeatus was 6,6 mg/kg; B. asper was 6,4 mg/kg and B. atrox was 6,7 mg/kg. The ED 50 of INS was 119,7 mg/3LD 50 and AVP-T was 270 mg/3LD 50 with

B. rhombeatus venom. Challenged with B. asper venom ED 50 of INS was 62,74 mg/3LD 50 and AVP-T

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 21 was 143,8 mg/3LD 50 . Challenged with B atrox venom ED 50 of INS was 75,15 mg/3LD 50 and AVP-T was 190,2 mg/3LD 50. We found phospholipases A2 (PLA 2), metalloproteinases (svMP) type I, II, III and serinoproteases (SP) in B. rhombeatus venom. Similarities of high, medium and low molecular weight protein were found between the profile of the three venoms with bands between 50-70

KDa corresponding to svMP III, band around 20-25 KDa of svMP I and between 11-15 KDa of PLA 2. Both antivenoms showed immunochemical recognition towards PLA2 and svMP. INS showed recognition to svMP III fractions of B. atrox venom, same as towards svMP I and III of B. rhombeatus venom and svMP I, II and III fractions of the B. asper venom. AVP-T showed immunochemical recognition to SP of B. rhombeatus venom and almost all the svMP fractions of B. asper venom, with higher intensity to svMP III and PLA 2 of B. atrox venom. The quantification of antibodies coupled was for INS to B. rhombeatus was 94,2% and to B. asper venom was 92,7%; while AVP-T was 90,4% to B rhombeatus and 96,6% to B. asper venom. Both antivenoms were effective by cross-reactivity towards PLA 2 and svMP. ToPI1, from Tityus obscurus scorpion venom, and its head-to-tail cyclization after interaction with trypsin

Caroline B F Mourão 1,2 , João Paulo C Fernandes 3, João Alexandre R G Barbosa 3, Guilherme D Brand 4, Maura V Prates 5, Carlos Bloch Jr 5, Elisabeth F Schwartz 1

1Neuropharma Lab, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília-DF, 70910-900, Brazil 2Instituto Federal de Brasília, Campus Ceilândia, Brasília-DF, 72220-260, Brazil 3Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brazil 4Laboratório de Biofísica Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brazil 5Laboratório de Espectrometria de Massa, EMBRAPA Recursos Gene ́ticos e Biotecnologia, Brasília-DF, Brazil

Due to the wide role of peptidases (proteases) in biological processes of living beings, peptidase inhibitors (PIs) have been extensively studied, aiming at understanding these processes, the development of new therapeutic treatments for related diseases, among others. In this study, we isolated and characterized a potent trypsin inhibitor, named ToPI1, from Tityus obscurus scorpion venom. This species is found in the Amazonian region, and is responsible for many envenomation cases in the northern part of Brazil. The peptide was initially isolated by RP-HPLC from the crude venom through three consecutive fractionation steps, being tested against trypsin after each step. With a molecular mass of 3806.89 Da, its amino acid sequence was partially determined by MALDI in-source decay (ISD) method, and by the cDNA library. The mature toxin has 33 amino acid residues and 3 disulfide bonds, with a C-terminal amidation. Since ToPI1 amount in the venom was insufficient, it was chemically synthesized by means of the Merrifield technique, and oxidized to form the 3 disulfide bonds. This synthetic peptide, named ToPI1 s, also inhibits trypsin, with a 1:1 stoichiometric ratio, with no activity against chymotrypsin. The X-ray structure of ToPI1 s:trypsin complex shows Lys 32 as the P1 residue of ToPI1 at the S1 site of trypsin, in an interaction similar to canonical inhibitors. The head-to-tail cyclization happens through a sequential set of events. Initially, the complex is formed, with Lys 32 in the trypsin S1 pocket. Then, the inhibitor C-terminal residue Ser 33 is cleaved and the cyclization of ToPI1 occurs via a peptide bond between residues

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 22 Ile 1 and Lys 32 . ToPI1 shares no sequence similarity with any other PIs characterized to date. However, in its cyclic form, it shares structural similarities with plant cyclotides, such as MCoTI-I and II, which also inhibit trypsin. ToPI1 is the first PI with CS-α/β motif described from animal venoms. Biodegradable cross-linked chitosan nanoparticles improve anti-Candida and anti-biofilm activity of TistH, a peptide identified in the venom gland of the Tityus stigmurus scorpion

Manoela Torres-Rêgo 1, Fiamma Gláucia-Silva 1, Karla SR Soares 1, Luanda BFC Souza 2, Igor Z Damasceno 3, Emanuell Santos-Silva 1, Ariane F Lacerda 1, Guilherme M Chaves 2, Arnóbio A Silva- Júnior 1, Matheus F Fernandes-Pedrosa 1

1Laboratory 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 2Laboratory of Medical and Molecular Micology, Department of Pharmaceutical Sciences, Federal University of Rio Grande do Norte, Street General Gustavo Cordeiro de Farias, Petrópolis, Natal BR 3Department of Materials Engineering, Federal University of Rio Grande do Norte, Avenue Senador Salgado Filho 3000 Lagoa Nova, Natal BR

The TistH (T. stigmurus Hypotensin) is one bioactive peptide identified from the venom glands of the Tityus stigmurus scorpion with hypotensive action, however demonstrated have multifunctional and biotechnological applications. The maximum efficacy of this molecules can be achieved by immobilizing in specific and suitable biomaterials or carriers. In this approach, different entrapment methodologies of TistH in chitosan nanoparticles was evaluated using its incorporation (CN-TistH- Inc) or adsorption (CN-TistH-Ads) methods by ionotropic gelification. Physico-chemical characteristic as well as cytotoxic and antifungal effect were assessed for different samples. Atomic force microscopy and scanning electronic microscopy images associated with particle size measurements demonstrated that for both methods induced cationic spherical, small (< 160 nm), and narrow-sized (PdI about 0.3) nanoparticles, with peptide loading greater than 96.5%, which was confirmed using Fourier transform infrared spectroscopy. The suspensions evidenced to be stable for 8 weeks and were able to induce the desired slow in vitro peptide release. Cytotoxicity assays performed in normal cells originated from murine macrophages (RAW 264.7) and kidneys of African green monkeys (Vero E6) suggested biocompatibility and safety of samples. The CN-TistH- Inc and CN-TistH-Ads showed a minimal inhibitory concentration of 89.2 μg/mL against Candida albicans, 11.1 μg/mL for C. parapsilosis and C. tropicalis, confirmed by minimum fungicidal concentrations assay. Moreover, the TistH-loaded cross-linked chitosan nanoparticles significantly decreased the biofilm formation of clinical yeast sepsis of C. tropicalis and C. krusei, as well as clinical yeasts of vulvovaginal candidiasis of C. albicans. In this study, biodegradable nanocarriers prepared using simple and reproducible methods demonstrated the ability to deliver the TistH peptide from T. stigmurus and improve its antifungal activity. Pros and cons of paraspecificity and cross-reactivity of different monospecific anti-Vipera antivenom products towards the Middle East vipers’ venoms

Naira M Ayvazyan, Arsen V Kishmiryan, Armen V. Voskanyan

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 23 Orbeli Institute of Physiology of the National Academy of Sciences of RA, Orbeli 22, Yerevan, Armenia

In spite of some new research trends for snakebites envenoming neutralization, antibody technologies still remain the best treatment applied in the field of toxinology. Here we try to discuss the benefits and drawbacks of ability to neutralize toxins of some species and subspecies of snakes of the genus Vipera, Montivipera and Macrovipera, by few available monovalent antisera, which has been raised against the venoms of Vipera berus berus , Vipera ammodytes ammodytes , Macrovipera lebetina turanica and Macrovipera lebetina obtusa . Taking into consideration different molecular formats each of these products has their own therapeutic characteristics as toxin- targeting antibodies, pharmacokinetic features, a propensity to cause adverse reactions, etc. We found an important degree of paraspecific protection within each genera, which let us conclude that in many cases there are more preferable to have the monovalent antivenom which has cross- reactivity with the venom of other vipers of the region rather than polyvalent, for which the adverse reactions are more expected. With the current situation in the Middle East, where polyvalent antivenoms are usually not always available in all countries, this information may be of use to clinicians charged with the treatment of Vipera or Monti/Macrovipera envenomations with non- specific antivenoms. NUCC-39 activates the CXCR4 receptor and accelerates the recovery from the peripheral neuroparalysis induced by Taipan snake envenomation

Marco Stazi 1§ , Giorgia D’Este 1, Andrea Mattarei 2, Samuele Negro 1, Florigio Lista 3, Michela Rigoni 1, Aram Megighian 1 and Cesare Montecucco 1,§

1Department of Biomedical Sciences and 2Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova 35131, Italy 3Center of Medical and Veterinary Research of the Ministry of Defense, Rome

Envenomation by snakes is a major neglected human disease that takes many lives and leaves permanent deficits in a large number of people each year. Animal antisera are available as primary therapeutic support, but there is a consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We found that a small molecule of rather simple synthesis, named NUCC-390, accelerates the recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. This beneficial effect follows the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12 α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12 α. In this presentation we will describe the results obtained in a murine model of neuroparalytic envenoming by a Papuan Taipan ( Oxyuranus scutellatus ) which causes a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, which is a major toxic component of this venom. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. NUCC-390 is currently under test in the envenomation by other snake venoms that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings,

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 24 that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery. Progress towards next-generation antivenoms

Andreas Hougaard Laustsen

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

For more than 125 years, plasma-derived antivenoms have been the mainstay of treatment against animal envenomings. Since then, these medicines have been instrumental in saving lives and limbs of an innumerable number of victims. Nevertheless, antivenoms are based on heterologous antibodies or antibody fragments and therefore possess a number of drawbacks that may be addressed by the application of biotechnology and medicinal chemistry. In this talk, an overview of the newest developments within next-generation antivenoms will be presented. The therapeutic utility, manufacturability, and feasibility of development will be discussed for a range of different antitoxin scaffolds, including monoclonal antibodies and nanobodies, alternative binding proteins, peptides, aptamers, small molecule inhibitors and chelators, as well as antivenoms produced via innovative immunization techniques. A special focus will be given to the newest developments within engineering of broadly-neutralizing antibodies, culminating in a discussion on the pros and cons of the “top down” and “bottom up” design approaches for recombinant antivenoms. Why is skeletal muscle regeneration impaired in viperid snakebite envenomings?

José María Gutiérrez

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

The regeneration of skeletal muscle is largely impaired after myonecrosis induced by viperid snake venoms, resulting in the development of permanent sequelae. Skeletal muscle regeneration after myonecrosis involves the activation, proliferation and fusion of myogenic cells, and a coordinated inflammatory response encompassing phagocytosis of necrotic cell debris, and the concerted synthesis of cytokines and growth factors. When myonecrosis is induced by purified myotoxins, or by venoms that do not inflict damage to the microvasculature, the regenerative process develops successfully. Thus, it has been proposed that viperid snake venom metalloproteinases (SVMPs), which cause hemorrhage and general degradation of the extracellular matrix, is likely to be the main factor affecting muscle regeneration in viperid envenomings. The microvasculature is disrupted as a consequence of the hydrolysis of key components of the microvascular basement membrane, particularly type IV collagen, affecting blood perfusion to muscle cells and generating ischemia. This impacts on the proliferation and fusion of myoblasts since an adequate blood supply is a key requirement of muscle regeneration. The disruption of the microvascular network also precludes the timely recruitment of inflammatory cells, with consequences in the removal of necrotic debris and in the synthesis of growth factors. Moreover, SVMPs directly affect the viability of the myogenic satellite cells and hydrolyze the basement membrane of muscle cells, which provides a structural scaffold for regenerating cells. The effects of local ischemia on intramuscular nerves also contributes to deficient regeneration. Furthermore, venom components remaining in the affected tissue directly affect myoblast proliferation and fusion. Understanding the basis of Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 25 these phenomena will pave the way for the development of novel therapeutic interventions aimed at improving muscle regeneration after viperid snakebite envenomings. A spider-venom peptide for treatment of chronic visceral pain

Yan Jiang 1, Joel Castro 2, Akello Agwa 3, Linda Blomster 1, Volker Herzig 4, Jessica Maddern 2, Gudrun Schober 2, Christina I Schroeder 1,5 , Stuart Brierley 2 & Glenn F King 1

1Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia 2Flinders University & South Australian Health and Medical Research Institute, Adelaide SA 5000, Australia 3Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK 4School of Science and Engineering, University of the Sunshine Coast, Sippy Downs QLD 4556, Australia 5National Cancer Institute, National Institutes of Health, Frederick MD 21702, USA

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterised by abdominal pain, discomfort, bloating, and altered bowel habits that affects ~11% of the global population. 30– 40% of IBS patients exhibit enhanced sensitivity to colonic distension, and the chronic visceral hypersensitivity of colonic afferents is implicated in development and maintenance of chronic visceral pain in these patients. A lack of suitable treatments for this disorder is a major contributing factor to its debilitating nature. Loss-of-function mutations in the human voltage-gated sodium channel Na V1.7 lead to a condition known as congenital indifference to pain, in which individuals cannot sense noxious stimuli. It has proved difficult to recapitulate this condition pharmacologically, with most Na V1.7 inhibitors providing little or no analgesia in rodent models of pain. However, the role of Na V1.7 in chronic visceral pain has not been explored. From a screen of arachnid venoms we isolated a novel Na V1.7 inhibitor (Tsp1a) from the venom of a Peruvian tarantula. Tsp1a is a gating modifier that potently inhibits Na V1.7 (IC 50 10 nM) by inducing a hyperpolarising shift in the voltage dependence of inactivation and slowing recovery from fast inactivation. It has >100-fold selectivity over Na V1.3–Na V1.6, 45-fold selectivity over Na V1.2, and 25- fold selectivity over Na V1.1. Determination of its structure using NMR revealed a classical inhibitor cystine knot (knottin) fold, and like many other spider knottins it proved to be highly stable in human serum (t 1/2 >> 24 h). Remarkably, intra-colonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of IBS without affecting colonic compliance. Our data suggest that Na V1.7 is intimately involved in the development of chronic visceral hypersensitivity in IBS, and that Tsp1a is an exciting lead compound for development of an orally active peptide analgesic for treatment of chronic visceral pain. SNAKEBITE CONTROL PROJECT(Myanmar)

Yi Yi Khine

Department of Nephrology, Naypyitaw General Hospital, Myanmar

Snakebite control project (Myanmar) was first established in 1994 to combat snakebites which is one of the important occupational hazards in Myanmar. In 1996 this project was collaborated with WHO. First project manager was Professor May Mya Win ;1996 to 2002 .From 2002 to 2017 Professor Khin Thida Thwin had performed the job and handed over to Professor Yi Yi Khine in 2017 uptil now. This project is under the guidance of Prevention of Noncommunicable disease Unit, Ministry of Health and Sports(MOHS). According to the national health plan( five yearly plan )

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 26 the ultimate goal is to provide universal health coverage to the community so all the projects has been looking forward to cooperate into UHC in the coming years.The objectives of the SBCP are to provide capacity building to the basic health staffs to provide proper management and timely referral at the primary healthcare level and to provide proper knowledge, attitude and practice at the community level and to collaborate with other ministries to improve public awareness and ASV productions for the whole country.With these objectives the project has been performed training of trainer workshops in high incident areas of the snakebite in major township and divisional levels.Hospital in patients data are collected by health management information unit in MOHS and distributed to the respective project annually. The high incident areas of snakebites are Bago, Ayeyarwaddy and Yangon regions in Lower Myanmar and Magwe,Mandalay and Sagaing regions in upper Myanmar. In the past 12 years average number of 10000 cases has been suffered from snakebite annually mainly by Russell’s viper comprising about 80% and the remaining by Cobra, green snakes and non-poisonous snakes. In 2018 ,10068 cases of snakebite with case fatality of 483(4.7%). Regarding the clinical research, SBCP has been carried out researches in collaboration of Department of Medical Research and Medical Universities. In 2002 seminars on papers done in snakebites was held in Yangon. Ongoing researches have been done and papers are presented in annual DMR conference. Regarding the ASV production there were shortage ASV productions due to technical and financial issues in the previous days. In 2014 Australian Government has established Myanmar Snakebite project which is done by Adelaide University and MOHS Myanmar 2014-2018. That project has contributed the technical support in ASV productions in MOI. Now ASV productions is adequate for the demand and lyophilized forms are produced so it is good for storage in tropical weather. However many areas needs to be improved especially in data registry and ongoing research programs both in clinical and public health areas as well as collaborations with other ministries to improve mortality, morbidity and public awareness. A retrospective descriptive study of snakebite patients admitted to Yangon General Hospital in Myanmar, in 2018

Moe Moe San 1, May Zabe 1, Ei Thwe Phyo 1, Saw Eh Pho Htoo 1, Cho Cho Nwe 1, Bethany S Moos 2

1Tropical and Infectious Diseases Department, University of Medicine (1) Yangon, Myanmar 2Hedena Health, Headington, Oxford, OX3 9JA, UK

Predominantly a disease of the rural poor and an occupational hazard of farming, snakebite is common in the tropics. Snakebite remains a major health concern in Myanmar, which is primarily an agricultural country and Yangon is one of its six high incidence regions. There remain many barriers to timely and effective treatment and many people seek help from traditional healers. Inappropriate first aid methods to treat snakebite are commonplace and associated with detrimental outcomes. Data was recently published by the Myanmar Snakebite Project about events surrounding snakebite in Mandalay, in the north of Myanmar. This new study provides additional data for Yangon further south, to contribute to the essential development of a strategy to reduce morbidity and mortality from snakebite in Myanmar. All snakebite patient charts in Yangon General Hospital, from 1 st January to 31 st December 2018, were reviewed. Data was collected about the circumstances of the bite and initial management. The study included 499 patients (73.1% male). Yangon General Hospital received the most cases of snakebite in May and June, with 13.2% and 12.8% respectively. The majority of patients were admitted from referring Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 27 hospitals within the Yangon region (53.5%). Snakes were identified from a combination of either a dead specimen, photograph, description or clinical syndrome. Russell’s viper was responsible for 55.3% of bites, Cobra for 11.2%, Green pit viper 5.8%, Krait 0.4% and Sea snake 0.2%. Most bites occurred whilst doing farm duties (29.3%) and the majority were on the foot or ankle (69.9%). Bites were noted to be more common between 1600 and 2200 (41.5%). Appropriate first aid was rarely used; tourniquet application was the most commonly used traditional treatment (31.7%). This study describes the snakebite events and highlights the need for urgent community education on prevention of snakebite and the appropriate first-aid management. Updates in Venomous Snakes of Thailand : An Issue of Protobothrops kelomohy venom and its envenomation

Lawan Chanhome , Taksa Vasaruchapong, Panithi Laoungbua, Orawan Khow, Narongsak Chaiyabutr

Queen Saovabha Memorial Institute, The Thai Red Cross Society, Bangkok 10330, Thailand

Thailand is in the tropical climate suitable for the living of diverse snakes. Within a decade the number of snakes are increased from 208 to 248 species and subspecies. 72 of 248 species and subspecies are qualified as venomous snakes in Families Elapidae and Viperidae, of which 37 species and subspecies are terrestrial and arboreal. Three new Viperid species, one new Elapid and one new Viperid reports have been described since 2011. Protobothrops kelomohy is amongst the best known by hill tribe people in northern Thailand and probably a medically important venomous snake. It is, therefore, interesting to investigate the venom biological characteristics including the symptoms of envenomation. The lethal toxicity (LD 50 ) was 0.67 (0.58 – 0.78) g/g mouse body weight by intravenous route. The enzymatic activities exhibited markedly higher in protease and arginine ester hydrolase than that of Daboia siamensis venom. The phospholipase A 2 and phosphodiesterase activities were significantly higher when compared with that of Calloselasma rhodostoma venom. The clinical observation of a victim bitten by Protobothrops kelomy on the right leg showed signs of progressive swelling and developed coagulopathy, acute kidney injury, rhabdomyolysis within a day. A total of eight doses of 30-ml Hemato Polyvalent antivenom were given. The coagulation test returned to normal on Day 5 and the clinical symptoms and laboratory findings progressively improved within one week of hospitalization. The myth of venom cytotoxin – more than just cytolytic actions

Teoh Shun Qi 1, Hiu Jia Jin 1, Yap Michelle Khai Khun* 1,2

1School of Science, Monash University Malaysia, Bandar Sunway, Malaysia 2Tropical Medicine and Biology Platform, Monash University Malaysia, Bandar Sunway, Malaysia

Cytotoxin (CTX) is a highly basic three-finger toxin found predominantly in cobra venom. It has 60- 63 amino acid residues in a single polypeptide chain comprises anti-parallel β-pleated sheets. Its three-finger functional loops interact with cell membrane and induce cell death by cell lysis. However, the actual molecular mechanisms of cytotoxicity are not conclusive due to a few contradicting hypotheses besides direct lytic action. The present work reports the cytotoxicity of CTX from the venom of Equatorial spitting cobra ( Naja sumatrana ). Its cytotoxic effects in MCF-7 cells were examined by cell-based apoptotic assays, membrane permeabilisation assay, HMGB1 Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 28 and label-free quantitative (LFQ) proteomics. It was found that CTX induced cytotoxicity before 24 h due to activation of caspase and mitochondrial membrane depolarisation. Furthermore, the absence of necrosis marker HMGB1 indicated apoptosis was the profound cell death independent of its direct cytolytic actions. The LFQ proteomics revealed the overexpression of proteins involved in glucose metabolism, cell-cell adhesion, inflammatory responses and necroptosis at higher toxin concentration. The necroptosis was presumably the initial stress response in MCF-7 cells when exposed to the high concentration of CTX. Altogether, these findings concluded CTX induced caspase-dependent mitochondrial-mediated apoptosis which was independent of its cytolytic effects. The cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis when concentration of CTX increased. The cytotoxicity of CTX was thus a concentration-dependent but not a time-dependent process. Horizontal gene transfer is a key mechanism of centipede venom evolution

Ronald A Jenner 1, Eivind A B Undheim 2

1Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK 2Centre for Biodiversity Dynamics, Department of Biology, NTNU, N-7491 Trondheim, Norway

Horizontal gene transfer (HGT) is currently not recognized as a major mechanism of venom evolution. HGT is a well-supported hypothesis for only three protein families found in the venoms of arachnids and hymenopterans, while other possible instances currently lack robust phylogenetic and genomic support. We show that HGT has contributed to the expansion of venom arsenals in all five centipede orders. We used phylogenetic analyses of venom proteome-annotated venom gland transcriptome data, assisted by genomic analyses, to show that centipede venoms have recruited at least five gene families from bacterial and fungal donors. This involved at least nine HGT events, including three bacterial and one fungal gene family that were each transferred twice into centipede venoms. Three of the bacterial gene families encode an enzyme and two pore-forming toxins that are known virulence factors. This suggests that HGT can provide a fast track channel for the evolution of novelty by the exaptation of bacterial weapons for new functions in animal venoms. Discovery strategies for novel antiplasmodial compounds from the venom of Bufo toads

Mathilde Wells , Stéphanie Hambÿe, Bertrand Blankert

Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons, Place du Parc 20, 7000 Mons, Belgium

Malaria remains a major concern for health organizations around the world. In 2018, the World Health Organization reported more than 228 million cases and 405.000 deaths. With more than 80 countries affected, approximately 3 billion people are at risk of infection. The emergence and transmission of resistances to most antimalarial drugs are a real worry. The need for new therapeutic candidates represents an absolute necessity. In more recent years, animal venoms and secretions have sparked a growing interest for scientists. In fact, toad venoms constitute a rich source of molecules with many potential therapeutic activities. The goal of this work is to develop a Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 29 bio-guided fractionation process and the subsequent discovery of new drug candidates against malaria from toad venom. Several Bufo species are considered during this work. Three species are currently being studied: Rhinella marina , Bufo bufo and Incillius alvarius . The extraction process from the air-dried venom is based on a four-solvent system assisted by sonication. After each extraction, the crude extracts are analyzed by TLC and LC-MS to obtain a chemical profile. For the fractionation step, flash chromatography is considered as a first approach to obtain rough fractions that will also be characterized and then biologically studied. In the first fractionation round, 3 to 4 fractions are obtained. The following step will consist in producing subfractions of the fractions displaying interesting therapeutic properties. For this purpose, further preparative techniques will be considered such as flash chromatography and semi-preparative LC. Each crude extract and the subsequently obtained fractions are tested for their antiplasmodial activity (3D7 and W2 strains) using the SYBR-Green I assay and microscopy. Their cytotoxicities are also assessed on a panel of human cell lines. The samples that display antiplasmodial activities will be further analyzed and structurally characterized by MS and NMR. Toxin expression in snake venom evolves rapidly with constant shifts in evolutionary rates

Agneesh Barua 1, Alexander S Mikheyev 2

1Ecology and Evolution Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa-ken, Japan 904-0495 2Evolutionary genomics group, Australian National University, Canberra ACT 0200, Australia

Key innovations provide ecological opportunity by enabling access to new resources, colonization of new environments, and are associated with adaptive radiation. The most well-known pattern associated with adaptive radiation is an early burst of phenotypic diversification. Venoms evolve rapidly, facilitate prey capture, and are widely believed to be key innovations leading to adaptive radiation. However, few studies have estimated their evolutionary rate dynamics. Here, we test for patterns of adaptive evolution in venom gene expression data from 52 venomous snake species. By identifying shifts in tempo and mode of evolution along with models of phenotypic evolution, we show that snake venom exhibits the macroevolutionary dynamics expected of key innovations. Namely, all toxin families undergo shifts in their rates of evolution, likely in response to changes in adaptive optima. Furthermore, we show that rapid-pulsed evolution modelled as a Lévy process better fits snake venom evolution than conventional early burst or Ornstein–Uhlenbeck models. While our results support the idea of snake venom being a key innovation, the innovation of venom chemistry lacks clear mechanisms that would lead to reproductive isolation and thus adaptive radiation. Therefore, the extent to which venom directly influences the diversification process is still a matter of contention. What's in a mass?

Juan J. Calvete 1, Libia Sanz 1, Diana Mora-Obando 1, Bruno Lomonte 2, Anita Tanaka-Azevedo 3, Karen de Morais-Zani 3, Sávio Sant'Anna 3 & Cleópatra A.S. Caldeira 4

1Evolutionary and Translational Venomics Laboratory, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain 2Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 30 3Laboratório de Herpetologia, Instituto Butantan, São Paulo, Brasil 4Centro de Estudos de Biomoléculas Aplicadas a Saúde (CEBio), Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brasil

Arguably, the amino acid sequence is the most distinctive molecular feature of a protein. The advent at the turn of the XXI century of biological mass spectrometry, and its constantly increasing sensitivity, mass-to-charge range, efficiency and speed, has open the door to the accurate and unambiguous identification of all toxin components through database-assisted top-down mass profiling. The key concept that allows using the intact protein mass for locus-resolved protein species identification is the theoretical demonstration that the binomial monoisotopic mass and isotopic distribution is a uniquely distinctive feature of any polyatomic molecule composed of polyisotopic elements. These parameters differentiate a given analyte from any other differing in their molecular formula, represented by the kind (C, H, N, O, S...) and number (n) of atoms of each element present in that compound, e.g., C nHnNnOnSn. In the case of proteins, this means that molecules departing in their polypeptide length and/or in their amino acid composition have molecular formulas that differ by at least one neutron. Conversely, two polypeptide with the same number and type of residues, even if they are arranged in different order, have identical molecular formulae and isotopic distribution. However, molecular evolution is intrinsically a process that creates molecular diversity, and while structural divergence represents the substrate for molecular evolution, the convergent evolution of isogenes to produce identical copies has never been documented. In this presentation, we will reflect on the applied importance of the isotopic distribution of molecular ions and will discuss how accurate mass profiling, particularly of toxins lacking posttranslational modifications other than disulfide bonds, may aid in evolutionary ecological venomics, as well as for reconstructing phylogeographic dispersal patterns of medically relevant snake species in the context of translational venomics investigations. Complement inhibition: a possible therapeutic strategy for controlling cobra envenomation

Felipe Silva de França 1, Isadora Maria Villas-Boas 1, Bruno Cogliati 2, Trent Woodruff 3, Edimara da Silva Reis 4, John D. Lambris 4, Denise V. Tambourgi 1

1Immunochemistry Laboratory, Butantan Institute, Brazil 2School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil 3School of Biomedical Sciences, University of Queensland, Australia 4Perelman School of Medicine, University of Pennsylvania, USA

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. In this study, we explored complement’s contribution to the physiopathogenesis of Naja annulifera envenomation. Here, we have shown that Naja venom triggers complement activation in vitro and in vivo , followed by the release of inflammatory mediators. By performing pharmacological interventions in a human whole-blood model of inflammation, we have also demonstrated that blockade of C3 or C5 cleavage and C5aR1 signaling inhibition reduce several inflammatory parameters associated with envenomation immunopathology. Furthermore, in various mouse models of envenomation, we have shown that C5a-C5aR1 axis activation is crucial for local and systemic inflammation and that changes induced by the venom, including pulmonary injury (ALI), can be abrogated by the use of PMX205, a C5aR1 Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 31 cyclic peptide antagonist. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms. Financial support: CETICS/FAPESP # 2013/07467-1. Paraspecific neutralization of venoms

Elda E. Sánchez 1, 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

A most recent snakebite occurring at the National Natural Toxins Research Center (NNTRC) at Texas A&M University-Kingsville by a Bothrops moojeni resulted in the need to test the efficacy of antivenoms on venoms not used in their manufacture. Not knowing if specific antivenoms for which venoms from species not used in their production would neutralize effectively is a concern not only to zoos and other venomous snake facilities but also to the treating physicians. This work aims to determine the paraspecific neutralization of venom from snakes kept by zoos and other facilities. In vivo assays were used to determine the effective dose 50 (ED50) of several antivenoms on various snake venoms. A total of five antivenoms (Antivipmyn, South African Institute of Medical Research (SAIMR) Polyvalent, SAIMR Echis carinatus , CroFab®, Suero Antiofidico Polivalente (SAP)) where used in this study. Antivipmyn and CroFab® were tested on venoms of Crotalus vegrandis , C. durissus terrificus , Bothrops colombiensis , and B. moojeni . SAIMR Polyvalent was tested on venoms of Atheris squamigera , Naja naja karachiensis , N. philippineansis , and N. samarensis . SAIMR Echis carinatus was tested on A. squamigera venom, and SAP was tested against B. colombiensis venom. The preliminary data suggest paraspecific neutralization of snake venoms, which may prove useful and allow peace of mind to those facilities carrying snakes for which no specific antivenom is allotted. This study also aims to create an extensive database of paraspecific neutralization of snake venoms by various antivenoms that will afford snake facilities the information they need when acquiring new snakes or when attaining an unfortunate snakebite. Revisiting the big antivenom crisis in Brazil

Hui Wen Fan 1, João Ricardo N Vissoci 2, Jacqueline Sachett 3, Wuelton Monteiro 3

1Instituto Butantan, São Paulo, Brazil 2Duke University School of Medicine, Duke University, USA 3Fundação de Medicina Tropical, Manaus, Amazonas, Brazil

A serious antivenom shortage occurred in Brazil in mid 1980s when three public antivenom- manufacturing laboratories were not able to provide the necessary amount to meet the national demand while a private manufacturer, responsible for 70% of the market, decided to quit vaccines and antivenoms production due to quality rejection of their products. A rapid reversal of the shortage was possible due to huge investments of the Ministry of Health in the modernization of the industrial plants. A national program for snakebite control also included the standardization of the production and quality control processes, and a model epidemiological surveillance system has been implemented since then. However, 35 years after, there are still gaps in the availability and

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 32 accessibility to treatment, as antivenom is available in less than 50% of the municipalities. The epidemiological scenario of snakebite envenoming has been changing, revealing regional and specific aspects, mainly in the Amazon region. Deaths and lethality remain unchanged, and there is no reliable data of sequels or disabilities. In parallel, a significant increase in scorpion sting envenomings have been reported. Climate changes and poor sanitary infrastructure of the cities seem to be associated, and mitigating measures have been failed in curbing the spread of the scorpion population in urban areas. Strategies to reduce deaths and disabilities due to envenomings caused by venomous animals may be at risk if there is no more investment and commitment with this goal. To achieve this goal, initiatives to train health professionals in the management of patients and to improve access to antivenom treatment are a priority. Bothrops lanceolatus update

Dabor Resiere

Critical Care Unit, University Hospital of Martinique, Fort-de-France, Martinique

Abstract unavailable

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 33 Poster abstracts

(Unedited) Discovery of broadly-neutralizing recombinant human monoclonal antibodies against cobra cytotoxins using cross-panning phage display

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)

Frederico Almada 1, José Neto 1, Américo G. Duarte 2, Francisca Rodrigues 1, Luisa Maia 3, José J.G. Moura 3, Stephane Besson 3, Juan Calvete 4

1MARE—Marine and Environmental Sciences Centre, ISPA Instituto Universitário, Rua Jardim do Tabaco 34, 1149-041 Lisboa, Portugal 2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal 3LAQV-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 4Evolutionary and Translational Venomics Laboratory, CSIC, Jaume Roig 11, 46010 Valencia, Spain Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 34 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

Edgar Barajas-Ledesma 1, Ignazio Avella 2, Nicholas R. Casewell 3, Robert A. Harrison 3, Paul Rowley 3, Edouard Crittenden 3, Wolfgang Wüster 4, Riccardo Castiglia 5, Chris Holland 1 and Arie van der Meijden 2

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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 35 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)

Verônica Lacerda de Barros 1, Anita de Moura Pessoa 2, Raimundo Nonato Leite Pinto 3, Raphael Ladislau de Alcântara 4, Rafael Stuani Floriano 5, and Nelson Jorge da Silva Jr. 2,3,6

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,

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 36 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)

Verônica Lacerda de Barros 1, Anita de Moura Pessoa 2, Raimundo Nonato Leite Pinto 3, Raphael Ladislau de Alcântara 4, Rafael Stuani Floriano 5, and Nelson Jorge da Silva Jr. 2,3,6

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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 37 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

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.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 38 Taxonomic identity analysis of Bothrops atrox snake from Peru and Brazil using enzymatic markers

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 species. 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

John P. Dunbar 1, Antoine Fort 2, Damien Redureau 3, Aiste Vitkauskaite 1, Ronan Sulpice 2, Michel M. Dugon 1 & Loïc Quinton 3

1Venom Systems & Proteomics Lab, School of Natural Sciences, Ryan Institute, National University of Ireland Galway, Galway, Ireland 2Plant Systems Biology Lab, Plant and AgriBiosciences Research Centre, School of Natural Sciences, Ryan Institute, National University of Ireland Galway, Galway, Ireland 3Mass 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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 39 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), - 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%), -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?

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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 40 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

Maike L Fischer 1, Natalie Wielsch 2, Sol A Yepes Vivas 1, David G Heckel 1, Andreas Vilcinskas 3, Heiko Vogel 1

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

Parviz Ghezellou, 1 Wendell Albuquerque, 2 Vannuruswamy Garikapati, 1 Nicholas R. Casewell, 3 Seyed M. Kazemi, 4 Alireza Ghassempour, 4 Bernhard Spengler 1

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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 41 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

Manuel A. Fernandez-Rojo 1,2 , Javier Moral-Sanz 1, Jeremy Potriquet 2, Joshua Daley 2, Yaiza A. López 1, Pamela Mukhopadhyay 2, Andreas Brust 3, Patrick Wilhelm 3, Taylor Smallwood 3, Richard Clark 3, Nic Waddell 2, Bryan Fry 3, Paul Alewood 3, Jason Mulvenna 2, John Miles 2,4 & Maria P. Ikonomopoulou 1,2

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 & 6h, 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 Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 42 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

Daniel H Jestrzemski 1,2 , Parviz Ghezellou 3, Ulrich Kuch 1

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 3Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Giessen, 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)

Daniel H Jestrzemski 1,2 , Parviz Ghezellou 3, Maria Athanasiadou 4, Frank Gessler 5,6 , Bernhard Spengler 3, Ulrich Kuch 1

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 3Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Giessen, Germany 4Health Monitoring Unit, Ministry of Health, Nicosia, Republic of Cyprus

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 43 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

Adrijana Leonardi 1, Kity Požek 1,2 , Igor Križaj 1

1Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia 2University 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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 44 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

Gisela L Lopez 1, Andrea Van de Velde 1, David Hernandez 2, Laura C Leiva 1, Luciano S Fusco 1

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 (15-30-45 μg) and BaV/Na2EDTA (45-90-135 μg). Blood samples were collected on days 14-29-41 for antibody 4 analysis. Sera from BaV/Na2EDTA protocol have a titer (5.1x10 ) higher than those treated with BaV (1.3x10 4). The neutralizing ability of both antivenoms was tested against proteolytic, coagulant and

PLA 2 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

Henri Mathé , Raul Soria

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 45 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 TM dedicated for sub-Saharan Africa (Inoserp PAN-AFRICA ) 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 LD 50 . 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-AFRICA TM 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 LD 50 of venom of Echis ocellatus whereas the neutralization potency has been evaluated to 279.9 LD 50 and

109.8 LD 50 for Antivenom B and Antivenom C respectively. When reported to the quantity of proteins per vial, the neutralization potency for Inoserp PAN-AFRICA TM 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

David Meléndez-Martínez 1, José Manuel Aguilar-Yáñez 1,2 and Cuauhtémoc Licona-Cassani 1

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., México Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 46 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

Javier Moral-Sanz 1, Yaiza A. López 1, Andreas Brust 2, Patrick Wilhelm 2, Glen Boyle 3, Bryan Fry 2, Paul Alewood 2, John Miles 3,4 , Manuel A. Fernandez-Rojo 1,3 & Maria P. Ikonomopoulou 1,3

1Madrid Institute for Advanced Studies in Food, Madrid, E28049, Spain 2Institute for Molecular Bioscience, The University of Queensland, Queensland, 4072, Australia 3 QIMR Berghofer MRI, Queensland, 4006, Australia 4James 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 BRAF V600E 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 BRAF V600E - mutated melanoma cells. Octpep-I was tested in human non-transformed Neonatal Foreskin Fibroblast (NFF) and in several lines of melanoma cells carrying the BRAF V600E mutation (i.e., MM96L, A2058, HTT144, JA, SKMEL28, A02). The MTT colorimetric assay revealed a dose-dependent Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 47 cytotoxicity for Octpep-I. Most importantly, 48h treatment with 200 µg/mL Octpep-I selectively reduced the cellular viability in BRAF V600E 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 BRAF V600E melanoma, showcasing its potential as a melanoma candidate to prevent drug resistance and relapse in combinatorial treatments with MAPK inhibitors. Preliminary Evidence for Mimicry of the Spectacled Cobra ( Naja naja ) in the Javan Slow Loris ( Nycticebus javanicus ) including spaces

Anna R Watkins 1, Thais Q Morcatty 1, K Anne-Isola Nekaris 1

1Nocturnal 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. Death from naja nigricollis poisoning and use of traditional therapy in Benguela – Angola

Paula Oliveira 1, Guilherme Kilembeketa 2, Mercedes Bardaji 2, Audreys Rosário 2, Signey Coimbra 3, Eduardo Kedisobua 2

1Faculty of Medicine, Katyavala Bwila University, Angola 2Benguela General Hospital, Angola 3National Center for Scientific Research, Angola

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 48 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

Yadanar Oo 1, Katrin Hampe-Krone 2, Sibylle Pagel-Wieder 2, Ulrich Kuch 3, Frank Gessler 2,4

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 Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 49 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

Adriana M S Parente 1, Bruno A Carmo 1, Jarbas M Resende 2, Renata M Araujo 1, Matheus F F Pedrosa 1

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. 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

Júlia G R Passos 1, Jacinthia B X Santos 1, Jacyra A S Gomes 1, Fabiana O Yamashita 1, Juliana F Silva 1,2 , Arnóbio A S Júnior 1, Matheus F F Pedrosa 1

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 50 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

Carina V Pires 1, Aline G Leão-Torres 1, Amanda C Ribelato 1, Maria C Zerbinatti 1, Rogério Giuffrida 2, Rosa MB Nogueira 2, Inês C Giometti 2, Nelson J Silva Jr 3, Rafael S Floriano 1

1Graduate Program in Health Sciences, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2- 205, 19.067-175, Presidente Prudente, SP, Brazil 2Graduate Program in Animal Science, University of Western São Paulo, Rodovia Raposo Tavares km 572, B2- 205, 19.067-175, Presidente Prudente, SP, Brazil 3Graduate 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)

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 51 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. Inhibitory potential of rosmarinic and chlorogenic acids against the effects of envenomation with the Bothrops leucurus snake venom

Diana Pontes da Silva 1, Sarah de Sousa Ferreira 1, Manoela Torres do Rêgo 1, Allanny Alves Furtado 1, Fabiana de Oliveira Yamashita 1, Juliana Felix da Silva 2, Karla Patricia de Oliveira Luna 3, Matheus de Freitas Fernandes Pedrosa 1

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. Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 52 Functional characterization of a phospholipase A2 from Bothrops atrox snake venom and its neutralization by three types of polyclonal antibodies

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 A 2 (PLA 2s), 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 PLA 2s. 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-PLA 2 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 PLA 2. The commercial antibothropic serum produced by INS (Peru) was also used for neutralization tests. The isolated PLA 2 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. Potential application from Harpalyce brasiliana roots extract for auxiliary treatment of snakebites

Enos Emanuel Azevedo Rocha 1, Manoela Torres-Rêgo 1,2 , Felipe França Cavalcanti 1, Fabiana de Oliveira Yamashita 2, Diana Pontes da Silva 2, Sarah de Sousa Ferreira 2, Ana Karoline Silva de Aquino 1, Renata Mendonça Araújo 1, Matheus de Freitas Fernandes-Pedrosa 2

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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 53 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

Jasmin Weisemann 1, Nadja Krez 1, Uwe Fiebig 2, Sylvia Worbs 2, Martin Skiba 2, Tanja Endermann 2, Martin B. Dorner 2, Tomas Bergström 3, Amalia Muñoz 4, Ingrid Zegers 4, Youssef Fikri 5, Christian Müller 6, Stephen P. Jenkinson 6, Marc-Andre Avondet 6, Laurence Delbrassinne 5, Sarah Denayer 5, Reinhard Zeleny 4, Heinz Schimmel 4, Crister Åstot 3, Brigitte G. Dorner 2, Andreas Rummel 1

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/A 1, B 1 and E 1, 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/A 1, B 1, E 1 and F 1 were successfully

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 54 detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD 50 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

1E L Saaiman , 2 P J C Buys

1Anaesthetist, Private practice, Windhoek, Namibia; Member of the Namibian Snakebite Interest Group 2Ear, Nose and Throat Surgeon, Private practice, Windhoek, Namibia; Namibian snakebite management expert; Founding member and head of the Namibian Snakebite Interest Group

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, 1 rst and 2 nd 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 A 2 from Bothrops diporus venom inhibits cancer cell adhesion and migration

Daniela J. Sasovsky 1, Laura D. Galarza 1, Laura C. Leiva 1, Elisa D. Bal de Kier Joffé 2, Soledad Bustillo 1

1Grupo de Investigaciones Biológicas y Moleculares (GIByM) IQUIBA-NEA, CONICET. Northeast National University, Corrientes, Argentina

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 55 2Universidad 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 PLA 2 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 (3×10 4/well) were preincubated for 30 min at 37°C with

PLA 2 (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

PLA 2 (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 PLA 2 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 PLA 2 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 PLA 2 isolated from B. diporus venom. Evaluation of the multifunctional therapeutic potential of chitosan nanoparticles containing Crotalus durissus cascavella snake venom

Fiamma G Silva 1, Karla S R Soares 2, Arnóbio A S Júnior 1,2 , Matheus F F Pedrosa 1, 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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 56 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

Ibrahim Khalil Al Haidar 1, Mohammed Noman 1, Sohanur Rahman Sohan 2, Mahedi Hasan 2, M. Monirul Islam 3, Mohammad Abdul Wahed Chowdhury 3, Aniruddha Ghose 1, Abdullah Abu Sayeed, 1 Md. Abu Reza 2*

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 LD 50 of T. albolabris venom which was found to be 1.2 mg/kg in mice by i.p. injection, significantly lower than the reported LD 50 from other countries which implies that the venom composition is different, measured the PLA 2 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

Md. Jahangir Alam 1, Sohanur Rahman Sohan 1, Md. Mahmudul Hasan Maruf 1, Khaled Mahmud Sujon 1, Masum Miah 2, Archana Deka 2, Robin Doley 2 and Md Abu Reza 1*

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 Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 57 action of its venom components. LD 50 value was found to be 0.12mg/kg which is significantly lower than the reported LD 50 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

Montamas Suntravat 1, 2 , Oscar Sanchez 1, Armando Reyes 1, Abcde Cirilo 1, Emelyn Salazar 1, Peter Davies 3, Elda E Sanchez 1, 2

1National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA 2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA 3Institute 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.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 58 Identification and molecular comparative study of basic phospholipase A2 of three snake venoms of the genus Bothrops from Perú

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). The effect of disulfide bond mimetics on the structure-activity relationship for conotoxins, specifically αGI and µKIIIA

Charlotte, Whitmore 1, Astrid, Knuhtsen 2, Rachel Whiting 1, Thomas Piggot 1, Brian O. Smith 3, Christopher M. Timperley 1 A. Christopher Green 1, & Andrew G. Jamieson 2

1Chemical, Biological and Radiological Division, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ 2School of Chemistry, University of Glasgow, Joseph Black Building, Glasgow, G12 8QQ UK 3Institute 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

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual 59 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

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 are 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.

Venoms and Toxins 2020 (Oxford) - Virtual | 16-17 September 2020 | http://lpmhealthcare.com/venoms-and-toxins-2020-virtual