previous day
all days

View: session overviewtalk overview


In this session major applications of venom research are presented, including developments in the biomedical, agrochemical, cosmeceutical, and antimicrobial fields. Clinical aspects of envenomation and antivenom interventions are featured as well.

14:05-14:45 Session Keynote: Glenn KING - Institute for Molecular Bioscience - University of Queensland
Gregor Anderluh (National Institute of Chemistry, Slovenia)
Glenn King (Institute for Molecular Bioscience - University of Queensland, Australia)
Deadly cures: a spider-venom peptide for treating ischemic injuries of the heart and brain

ABSTRACT. Cardiovascular disease is the leading cause of death worldwide. Within this disease category, myocardial infarction (MI) and stroke account for most deaths, making these two diseases alone responsible for 27% of all deaths worldwide. In spite of this massive disease burden, there are no drugs available to protect the heart and brain from the tissue injury caused by MI and stroke, respectively. The reduced supply of oxygen to affected regions of the heart and brain during MI and stroke, respectively, induces a switch to fuel production via anaerobic glycolysis, which leads to lactic acidosis. The resultant drop in pH activates acid-sensing ion channel 1a (ASIC1a), a proton-gated sodium channel. Activation of ASIC1a appears to promote death of neurons and cardiomyocytes (heart muscle cells) by exacerbating intracellular calcium overload and directly activating programmed cell death pathways. We recently isolated a disulfide-rich peptide (Hi1a) from venom of the K’gari funnel-web spider that inhibits ASIC1a with picomolar potency and exceptional selectivity. Hi1a dramatically reduces infarct size and improves behavioural outcomes even when administered up to 8 hours after ischemic stroke in rats1. More recently, we demonstrated that genetic ablation of ASIC1a leads to improved functional recovery in an in vivo mouse model of MI, and that this effect can be recapitulated by therapeutic blockade of ASIC1a using Hi1a2. Collectively, our data provide compelling evidence that ASIC1a is a novel target for neuroprotective and cardioprotective drugs to reduce the burden of MI and stroke, and that venom-peptide Hi1a is an exciting lead compound for these indications.

1. Chassagnon et al. (2017) Potent neuroprotection after stroke afforded by a double-knot spider toxin that inhibits acid-sensing ion channel 1a. Proc. Natl. Acad. Sci. USA 114, 3750–3755 2. Redd et al. (2021) Therapeutic inhibition of acid sensing ion channel 1a recovers heart function after ischemia-reperfusion injury.

14:45-15:45 Session 3: ORAL 1
María Ikonomopoulou (Imdea-food institute, Spain)
Goran Gajski (Institute for Medical Research and Occupational Health, Croatia)
Tamara Čimbora-Zovko (Ruđer Bošković Institute, Croatia)
Sanjica Rak (Ruđer Bošković Institute, Croatia)
Maja Osmak (Ruđer Bošković Institute, Croatia)
Vera Garaj-Vrhovac (Institute for Medical Research and Occupational Health, Croatia)
Mutual anticancer effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines
PRESENTER: Goran Gajski

ABSTRACT. Aim Regardless of major scientific and technological advancements in combinatorial chemistry, drugs derived from natural products still make a huge contribution to drug discovery today. Therefore, we investigated the possible mutual anticancer ability of bee venom and cisplatin towards two cancer cell lines: parental cervical carcinoma HeLa cells and their cisplatin-resistant HeLa CK subline, as well as laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CK2 subline. Methods Cytotoxicity of bee venom alone and in combination with cisplatin was investigated using MTT test. Additionally, we examined morphological changes and the type of cell death using vital staining as well as by the induction of PARP cleavage using Western blot analysis. Results Bee venom applied alone in concentrations of 30-60 μg/mL displayed dose-dependent cytotoxicity against all cell lines tested. Cisplatin-resistant cervical carcinoma cells were more sensitive to bee venom than their parental cell lines (IC50 values were 52.50 μg/mL for HeLa vs 47.64 μg/mL for HeLa CK cells), whereas opposite results were obtained for cisplatin-resistant laryngeal carcinoma cells (IC50 values were 51.98 μg/mL for HEp-2 vs > 60.00 μg/mL for CK2 cells). Treatment with bee venom alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium-bromide and a lack of cleavage of apoptotic marker PARP. Combined treatment of bee venom and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, indicating that bee venom could enhance the killing effect of selected cells when combined with cisplatin. Main Conclusions The obtained results indicate that mutual treatment with bee venom could be useful from the point of minimizing cisplatin concentration during chemotherapy, thus reducing and/or postponing the development of drug resistance. Our data suggest that bee venom could be used in the development of a new strategy for cancer treatment.

Javier Moral-Sanz (Imdea-food institute, Spain)
Manuel Alejandro Fernandez-Rojo (IMDEA-Food Institute, Spain)
Gonzalo Colmenarejo (Imdea-food institute, Spain)
Sergey Kurdyukov (University of Sydney, Australia)
Andreas Brust (The University of Queensland, Australia)
Lotten Ragnarsson (The University of Queensland, Australia)
Asa Andersson (The University of Queensland, Australia)
Sabela Vila (University of Santiago Compostela, Spain)
Pablo Cabezas (University of Santiago Compostela, Spain)
Patrick Wilhelm (The University of Queensland, Switzerland)
Yanni Chin (The University of Queensland, Australia)
Yaiza Lopez (IMDEA-Food Institute, Spain)
Taylor Smallwood (The University of Queensland, Australia)
Richard Clark (The University of Queensland, Australia)
Bryan Fry (The University of Queensland, Australia)
Glenn King (The University of Queensland, Australia)
Grant Ramm (QIMR Berghofer, Australia)
Paul Alewood (The University of Queensland, Australia)
Richard Lewis (The University of Queensland, Australia)
John Miles (James Cook University, Australia)
Maria Ikonomopoulou (Imdea-food institute, Spain)
The anti-tumoral profile of an octopus peptide in BRAF-melanoma cells is mediated by the conformation of the neurokinin 1 receptor-binding domain

ABSTRACT. Aim Targeted therapies & immunotherapy have vastly enhanced survival in patients with metastatic BRAF-mutated melanoma. However, drug resistance hinders the overall treatment success. Therefore, new and safe regimes are still needed for melanoma patients. In this study, we aimed to characterise the antiproliferative profile of Octpep-1, a tachykinin peptide derived from Octopus kaurna and to dissect the role of neurokinin 1 receptor-binding domain in reducing the proliferation of human BRAF melanoma. Methods We conducted viability, apoptotic assays and examination of cell cycle phases to determine whether Octpep-1 targets melanoma cells. To dissect the significance of mitochondria in this process, we assessed mitochondrial membrane potential, generation of reactive oxygen species and inhibitors of mitochondrial complexes coupled with seahorse metabolic flux analyser. To showcase Octpep-1’s mechanism of action we carried out structure activity relationship, including molecular modelling and CRISPR/Cas9 editing of the targeted gene. Lastly, we used mouse and zebrafish melanoma xenograft models to validate its therapeutic potential. Results We observed that Octpep-1 diminishes the proliferation of melanoma cells with negligible effects on healthy neonatal foreskin fibroblasts and without leading to cell death. Octpep-1 increased the reactive oxygen species with unaltered mitochondrial membrane potential or changes in cell cycle phases. Moreover, Octpep-1 modified the rate of oxygen consumption by enhancing the non-mitochondrial and mitochondrial respiration with an inefficient ATP coupling in melanoma-treated cells. Neurokinin receptor knock-out melanoma cells suggested that Octpep-1 acts via a neurokinin-independent mechanism. Molecular modelling and in vitro experiments supported that the antiproliferative profile of Octpep-1 depends upon a mixture of α-helix and polyproline conformation in the C-terminal region. Finally, as a proof-of-concept we confirmed that Octpep-1 diminishes the tumor progression in two independent xenograft animal models. Main Conclusions We characterised the antiproliferative activities and showed the therapeutic potential of a tachykinin octopus peptide in melanoma of BRAF mutation.

Mathilde Wells (University of Mons, Belgium)
Stéphanie Hambye (University of Mons, Belgium)
Bertrand Blankert (University of Mons, Belgium)
Identifying new potential antimalarial compound in toad venoms through a bio-guided fractionation process
PRESENTER: Mathilde Wells

ABSTRACT. Aim This work aims to develop a fractionation process that allows to isolate and identify compounds displaying a potential antiplasmodial activity from toad venom extracts. Methods The strategy combines analytical and separative techniques with biological evaluations of the obtained samples. Three species were considered: Rhinella marina (RM), Bufo bufo (BB), Incillius alvarius (IA). Crude extracts were obtained through sonication-assisted solvent extractions of dried venoms. The different crude extracts were characterized by TLC and LC-MS and fractionated using flash chromatography and preparative TLC. Each crude extract and the subsequently obtained fractions were tested for their antiplasmodial activity on two Plasmodium falciparum strains (3D7 and W2) using the SYBR-Green I assay. Their cytotoxicities were also assessed on FHs74int and HUVEC cell lines using the MTT and Crystal Violet assays. The hemolytic activity was evaluated. Results Among the tested samples, several displayed an interesting antiplasmodial activity, mainly those originating from the venoms of RM and IA. One of the fractions obtained from the dichloromethane extract of RM displayed an activity against in vitro cultures of Plasmodium falciparum (IC50(3D7) = 49 µg/mL; IC50(W2) = 40 µg/mL). The main component of this sample was identified as being resibufogenin. The antiplasmodial activity of this isolated compound seems promising (IC50(3D7) = 37 µg/mL; IC50(W2) = 30 µg/mL). No hemolytic activity was highlighted. Cytotoxicity assays on human cells are still ongoing but have already pointed out the toxicity of several samples including resibufogenin. Main Conclusions This work has demonstrated that toad venoms could constitute an interesting source for new antimalarial drug candidates. Resibufogenin, a cardiotonic steroid, was identified as being the main protagonist. This compound being exempt of hemolytic activity still, however, displays a certain level of toxicity against human cells. However, many perspectives are possible to reduce toxicity while maintaining antiplasmodial activity (chemical modifications through hemi-synthesis, nanoencapsulation).

Ehsan Alirahimi (University Hospital of Cologne, University of Cologne, Germany)
Marianne van Cann (University Hospital of Cologne, University of Cologne, Germany)
Jörg Isensee (University Hospital of Cologne, University of Cologne, Germany)
Alexey Kuzmenkov (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia, Russia)
Andreev-Andrievskiy Alexander (Biological Faculty, Lomonosov Moscow State University, Russia, Russia)
Steve Peigneur (Toxicology and Pharmacology, KU Leuven, Belgium, Belgium)
Georgii Khusainov (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia, Russia)
Antonina Berkut (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia, Russia)
Jan Tytgat (Toxicology and Pharmacology, KU Leuven, Belgium, Belgium)
Vanessa O. Zambelli (Laboratório de Dor e Sinalização, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil, Brazil)
Gisele Picolo (Laboratório de Dor e Sinalização, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil, Brazil)
Queren Apuque Alcantara (Laboratório de Dor e Sinalização, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil, Brazil)
André C. Morandini (Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, 05508-220, Brazil., Brazil)
Antonio Carlos Marques (Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, 05508-220, Brazil, Brazil)
Juliana Mozer Sciani (Multidisciplinary Research Laboratory, São Francisco University (USF), Avenida São Francisco de Assis,, Brazil)
Alexander Vassilevski (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia, Russia)
Tim Hucho (Translational Pain Research, University Hospital of Cologne, University of Cologne, Germany, Germany)
High Content Microscopy Screening for Potentially Pain Modulating Toxins
PRESENTER: Ehsan Alirahimi

ABSTRACT. Aim Pain-initiating nociceptive neurons are highly heterogenous and respond to a vast variety of stimuli such as ionotropic depolarization, prostaglandins, growth factors, cytokines, hormones, pH, ROS, and metabolites. Thereby, they are able to detect a huge variety of potentially pain modulating toxins. A challenge is how to monitor such heterogeneous sensory neurons to allow stepwise fractionation-based purification of potentially subgroup-specific ionotropic as well as metabotropic acting toxins. Methods We established a High Content Microscopy approach for the analysis of primary sensory neuron cultures on a single cell basis for higher throughput screening. We established phenotypic assays to monitor activation as well as inhibition of pain-initiating PKA-II and/or Erk signaling in 50-100 µl volumes in 96-well plates. Venoms, can then be tested for potentially pain-initiating or analgesic activity, sequentially fractionated and retested until purity, sequenced and recombinantly produced. Furthermore their cellular mechanism can be e.g. pharmacologically characterized and verified in behavioral animal pain tests. Results We tested 123 crude venoms from different species. Thirty four percent (42 out of 123) of the tested venoms showed pain-initiating signaling whereas 13.8% (17 out of 123) showed signaling-inhibiting, i.e. potentially analgesic activity. Interestingly, among all the species tested, only the venom of spiders showed de-sensitizing activity. First attempts identified MeuNaTxa1 as a pain initiating toxin acting via Nav1.2. Identification of further initiating and/or potentially analgesic toxins is ongoing. Main Conclusions We show a first High Content microscopy based screening approach suitable for identification not only of potentially pain initiating but also of inhibiting toxins on highly heterogeneous primary sensory neurons in a higher throughput manner.

James Boncan (Patrick G Johnston Centre for Cancer Research, UK)
Samantha Bann (School of Chemistry and Chemical Engineering, Queen's University Belfast, UK)
Stephen Cochrane (School of Chemistry and Chemical Engineering, Queen's University Belfast, UK)
Diego Cobice (Mass Spectrometry Centre, Ulster University, UK)
Stephen McClean (School of Biomedical Sciences, Ulster University, UK)
Anne Jordan (Patrick G Johnston Centre for Cancer Research, UK)
Karen McCloskey (Patrick G Johnston Centre for Cancer Research, UK)
Ken Mills (Patrick G Johnston Centre for Cancer Research, UK)
Purification of novel phospholipase A2 enzymes with anticancer activity from Pseudechis australis snake venom
PRESENTER: James Boncan

ABSTRACT. Aim Precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) is a haematological malignancy characterised by immature proliferation of B-cell lineage blasts. Current treatment for pre-B ALL involves regimens of chemotherapy which despite high curative rates are often not tolerated well by patients. Previously our group has identified phospholipase A2 (PLA2) as a novel, anticancer component in Pseudechis australis (PA) snake venom. Here we purify this component and elucidate its mechanism of action in a panel of pre-B ALL cell lines. Methods Crude PA snake venom was purified using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC), with the identity of our compound of interest (CoI) confirmed using MALDI-ToF mass spectrometry, and a shotgun protein ID approach. For functional studies, normal peripheral stem cells (PSC) along with a panel of pre-B ALL cell lines were used with; viability and cytotoxicity assays, Caspase 3/7 and 8-Glo activity assays, Annexin V 7-AAD and propidium iodide cell staining undertaken. Results The CoI from crude PA snake venom was purified by RP-HPLC, eluting as three overlapping peaks. MALDI-ToF and sequencing analysis revealed these to be three PLA2 homologues. All three homologues were pooled together and denominated, PA-PLA2. A panel of pre-B ALL cell lines treated with PA-PLA2 generated IC50 values in two cell lines (Reh; 0.74µg/mL and SD-1; 13.58µg/mL) after 24 hours. No decrease in PSC viability was observed following 24, 48 and 72 hours treatment with PA-PLA2. In functional assays, after 24 hours PA-PLA2 treatment (Reh; 0-5µg/mL and SD-1; 0-50µg/mL), caspase-independent cell death along with significant increases in necrotic cells as well as cells in sub-G1 were observed (p<0.05). Main Conclusions This study has identified and purified a cluster of PLA2 enzymes from crude PA venom with anticancer activity. The purified PA-PLA2 was shown to selectively target pre-B ALL cancer cells inducing a caspase-independent mode of cell death.

15:45-16:15 Session 3: POSTER 1
Beata Halassy (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Carole Yaacoub (Aix marseille université, France)
Ziad Fajloun (Université Libanaise , EDST, Centre AZM, rue Miaten, 1300 Tripoli, Liban, Lebanon)
Coutard Bruno (UVE-UMR 190 Unité des Virus Emergents , IHU, 19-21 Boulevard Jean Moulin 13005, Marseille, French Guiana)
The Cytotoxic Effect of Apis mellifera Venom with a Synergistic Potential of Its Two Main Components-Melittin and PLA2-On Colon Cancer HCT116 Cell Lines
PRESENTER: Carole Yaacoub

ABSTRACT. Bee venom (BV) is one of the most natural compounds studied in recent years due to its richness in bioactive molecules that possess a wide range of biological activities. Recently, it has been demonstrated to have an anti-cancer effect against several types of cancer cells mainly due to its two main compounds Melittin (MEL) and phospholipase A2 (PLA2). The present work shows the strong cytotoxic effect of Apis mellifera venom on human colon carcinoma cells (HCT116) and demonstrates the synergistic effect of MEL and PLA2 on these cells. The proportions of Mel and PLA2 on BV, were analyzed through high-pressure liquid chromatography, then a cell viability assay was done to study the effect of BV, MEL, PLA2 and a mixture of MEL and PLA2 on the HCT116 cells. The A. mellifera venom showed a strong cytotoxicity against HCT116 cells, and to a lower extent MEL or PLA2 alone. In addition, the obtained results showed clearly the improvement of the cytotoxic effect of MEL and PLA2 when they were added together. These results confirm the cytotoxic effect of the A. mellifera venom and demonstrate the synergistic potential activities between MEL and PLA2 on probably disturbing the membrane of HCT116 cancer cells. Altogether, these findings could serve for the development of new anti-cancer drugs.

Alexey Averin (Institute of Cell Biophysics, Russian Academy of Sciences, Russia)
Miroslav Nenov (Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Russia)
Vladislav Starkov (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia)
Tatyana Andreeva (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia)
Victor Tsetlin (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia)
Yuri Utkin (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia)
Cardiovascular effects of snake toxins: new data for known toxins

ABSTRACT. Aim The cardiovascular system is one of the main targets of snake toxins. Cobra cardiotoxins exert a direct effect on the heart and blood vessels. Our aim is to compare effects of two cardiotoxins, representing S- (CT1) and P-types (CT2), from cobra Naja oxiana. Methods The effects of CT1 and CT2 on the rat papillary muscle and aorta were studied. As mammalian heart and aorta contain potassium channels, we also investigated the effect of β-bungarotoxin, a potassium channel blocker, on the contraction of papillary muscle and aorta. Results Both cardiotoxins at low concentration changed the force of contraction and the character of force–frequency relationship in the rat myocardium. In the presence of cardiotoxins, the positive relationship in the region of 1–3 Hz, characteristic of healthy mammalian myocardium disappears and the relationship becomes completely negative. Both cardiotoxins induced slowly developing aortic ring contraction that reached a maximum in 40-50 minutes. Cardiotoxins showed a similar profile of action on the contractile properties of the myocardium and aorta, as well as on the development of effects under conditions of blockade of calcium current. In phenylephrine-precontracted rat aorta, acetylcholine produced relaxation, which was partly inhibited by 4-aminopyridine, an inhibitor of voltage-dependent potassium channels. At the concentration of 10 μg/ml, β-bungarotoxin induced a slight decrease in the vasorelaxant acetylcholine effect. On the rat papillary muscle, β-bungarotoxin at concentrations up to 10 μg/ml did not affect the force of contraction and the level of diastolic tension, the kinetic parameters of contraction also being unchanged. Main Conclusions A comparison of the activity of cardiotoxins showed that the P-type CT2 has a higher activity than S-type CT1. At rat aorta, β-bungarotoxin partially blocked vasorelaxant effect of acetylcholine, that depended on potassium channel.

Adrián Marcelo Franco Vásquez (Instituto de Química, Mexico)
Fernando Lazcano Pérez (Unidad Académica de Sistemas Arrecifales Puerto Morelos, Mexico)
Giovanny Blandón Marín (Universidad Católica de Manizales, Colombia)
Roberto Arreguín Espinosa (Instituto de Química, Mexico)
Isolation, identification and characterization of phospholipases A2 from Lachesis acrochorda snake venom with activity on central nervous system glioma cancer cells

ABSTRACT. Aim This work aims to isolate and characterize phospholipases A2 from Lachesis acrochorda venom. Methods In order to isolate the phospholipases A2 (PLA2), an ultrafiltration was carried out as first step with an Amicon8050® equipment using membranes of 30 and 10 kDa cut off. The sample with the activity of interest was subjected to two consecutive steps of reverse phase high performance liquid chromatography (RP-HPLC) using Zorbax RX and Zorbax Eclipse XDB C18 columns. The purified fraction was analyzed through a 2D electrophoresis gel and the spots were identified by mass spectrometry (MS/MS). SDS-PAGE electrophoresis gels and enzymatic activity tests were performed using the EnzChek® Phospholipase A2 Assay. Finally, to characterize the fraction, tests for calcium dependence were performed with chelating agents EDTA and EGTA, as well as a SRB cytotoxicity assay on six cancer lines. Results A fraction with PLA2 activity of 583 (Ua/µg) was identified. The 2D electrophoresis gel showed three components with molecular masses close to 14 kDa and isoelectric points of 4.7, 5.0 and 5.3. Mass spectrometry analysis showed molecular masses of 13,969 and 14,300 Da, which correspond to this family of enzymes. Tryptic digestion and mass spectrometry (MS/MS) confirmed the presence of three Asp49-type proteins with coverage percentages ≥ 70% which correspond with PLA2s reported for organisms of this genus. The fraction inhibited the growth of U251 cells by 19.98%, whereas it was not cytotoxic to other cells. Finally, both EDTA and EGTA inhibited the activity of the fraction by 99%. Main Conclusion L. acrochorda venom contains calcium-dependent Asp49-type PLA2 isoforms with high levels of enzymatic activity capable of partially inhibiting the growth of glioma cancer cells that can be used for biotechnological developments.

Sanja Mateljak Lukacevic (University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Croatia)
Tihana Kurtovic (University of Zagreb, centre for Research and Knowledge Transfer in Biotechnology, Croatia)
Marija Brgles (University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Croatia)
Stephanie Steinberger (Institute of Chemical Technologies and Analytics, Austria)
Martina Marchetti-Deschmann (Institute of Chemical Technologies and Analytics, Austria)
Beata Halassy (University of Zagreb, centre for Research and Knowledge Transfer in Biotechnology, Croatia)
Quality-related properties of the whole IgG snake antivenoms obtained by different refinement protocols

ABSTRACT. Aim Whole IgG antivenoms represent a complex mixtures of both venom-specific and therapeutically irrelevant antibodies of various IgG subclasses. They are extracted from hyperimmune animal plasma by diverse refinement principles that most probably have different impact on their functional and/or structural properties. The aim of this work was to compare the influence of five purification protocols on purity, aggregate content, IgG subclass distribution, venom-specific protective efficacy and thermal stability of the final IgG preparations. Methods IgG fraction was extracted from unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma by each of the following purification approaches: ammonium sulphate precipitation (ASP), caprylic acid precipitation (CAP), anion- (AEX) and cation-exchange chromatography (CEX) and affinity chromatography (AC). Purity and aggregate content were monitored by size-exclusion-HPLC, impurities were identified by mass spectrometry, venom neutralization potency by ED50 assay in mice, thermal stability by thermal shift assay and IgG subclass distribution by in-house ELISA. Results The highest purity was achieved by CAP and AC, while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated sample from AEX-based sample. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparations. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. AC-derived sample was the only one whose thermal stability was reduced. Main conclusion Our results showed that employed refinement protocols have noticeable impact on IgG features we have studied which might affect both safety and effectiveness of the final product.

Tihana Kurtović (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Maja Lang Balija (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Miran Brvar (Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana, Slovenia)
Mojca Dobaja Borak (Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana, Slovenia)
Sanja Mateljak Lukačević (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Beata Halassy (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Comparison of preclinical properties of several available antivenoms in the search for effective treatment of Vipera ammodytes and Vipera berus envenoming
PRESENTER: Tihana Kurtović

ABSTRACT. Aim Snakebites in Europe are mostly caused by Vipera ammodytes, Vipera berus and Vipera aspis. Among eight available antivenoms, only Zagreb antivenom, Viperfav and ViperaTAb have been used almost exclusively for decades. Zagreb antivenom and Viperfav are considered clinically efficient against envenoming caused by all three medically relevant species, while ViperaTAb is indicated for the treatment of V. berus bites solely. When the production of Zagreb antivenom was discontinued and a shortage of Viperfav occurred, other potentially suitable antivenoms were implemented into clinical practice, but without comparative assessment of their eligibility. Due to a lack of the product indicated for the treatment of V. ammodytes bites, other antivenoms were implemented into clinical practice without comparative assessment of their eligibility. The aim of our work was to identify at preclinical level the most promising candidate that might ensure the successful treatment of V. ammodytes and V. berus bites. Methods A thorough preclinical analysis of the safety-related properties and efficacy of a panel of anti-Vipera spp. antivenoms that are currently available, or in development for the European market, was performed in a comparative manner. Purity and aggregate content were evaluated by size-exclusion chromatography. Neutralisation potencies were determined by lethal toxicity neutralisation assay in mice. Results Since Zagreb antivenom is no longer available on the European market, Viperfav emerged as the highest-quality product and the only one whose neutralisation potency against V. ammodytes and V. berus venoms was above regulatory requirements. Main conclusion Although monitoring of effectiveness is of outmost importance in the decision-making process, the presented findings may serve as a starting point for guidance to clinicians when choosing the most appropriate antivenom for the treatment of envenoming in Southeastern Europe.

Iuliana Codreanu (University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania)
Ștefania Raita (University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania)
Mario Darius Codreanu (The General Association of Romanian Veterinarians, Romania)
Simona Nicolae (University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania)
Ion Rădoi (University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Romania)
Ciprian Ilie Ivas (University of Life Sciences Iași, Faculty of Veterinary Medicine, Romania)
Implications of bee venom administration on some biochemical and haematological parameters in Wistar rats

ABSTRACT. Aim The aim of this study is to establish the bee venom inoculation’s implications on some haematological and blood biochemical parameters dynamics in Wistar rats, when a non-lethal dose of bee venom is administered. The bee venom’s controversial properties assessment represent the base of this study. Methods The experiment was performed on 3 groups of Wistar rats injected with 0.1 ml bee venom. The blood tests’ results obtained for each group were compared to a control group. Blood samples were harvested at 3, 6 and 9 hours after inoculation. Results Total proteins value increases to 6.76 g/dL at 3 hours after inoculation, decreasing to 6.43 g/dL after 9 hours. ALP increased at 9 hours after inoculation (504.6 IU/L). Serum amylase significantly increased (1355.2 IU/L) 9 hours after inoculation. Creatinine values increased at 3 and 9 hours (25.9 mg/dL, respectively, 61 mg/dL). Hemoglobin values increased from 10.26 g/dL to 13.75 g/dL. The hematocrit increased at 3 hours after inoculation to 49.1% and after 9 hours decreases to 45%. RBC show an increase (8.60x103/μL) at 6 hours and a decrease (7.12x103/μL) at 9 hours after inoculation. Main Conclusions Total proteins values increase due to hemoconcentration, three hours after inoculation and decrease after 9 hours due to defense-compensation reactions, respectively tissue repair processes. ALT increases due to the damage of the liver, kidneys and pancreas. ALP activity increase due to venom’s toxic effect on the liver. The amylase activity increase shows a potential pancreatic damage, due to bee venom’s melittin cytotoxicity. The urea and creatinine increase indicate cytotoxic effects, leading to kidney function alteration. Although hemoconcentration occurs, hemolysis leads to decreased hematocrit values. The progressive hemoglobin increase and RBC decrease correlate with bee venom’s hemolytic effect - due to the presence of hemolysin, containing alpha-toxins that cause erythrolysis.

Nika Žibrat (National Institute of Chemistry, Slovenia)
Marjetka Podobnik (National Institute of Chemistry, Slovenia)
Gregor Anderluh (National Institute of Chemistry, Slovenia)
Is mechanistic action of NLP-induced plant membrane damage universal?
PRESENTER: Nika Žibrat

ABSTRACT. Aim Necrosis- and ethylene-inducing 1-like proteins (NLPs) form a superfamily of proteins found in various phyla of plant-associated microorganisms, such as bacteria, oomycetes, and fungi. To date, more than 1,700 homologs of these apoplastic effectors have been identified. Numerous NLPs are cytotoxic and contribute to the facilitation of infections against a variety of crops. The target for NLP interaction with the plant plasma membrane is glycosyl inositol phosphoramide (GIPC), a major sphingolipid found almost exclusively in the plant plasma membrane. Numerous genera of bacteria (e.g., Pectobacterium), oomycetes (e.g., Phytophthora), and fungi (e.g., Moniliphtora) with diverse lifestyles secrete NLPs, and their precise roles in the infection cycle remain enigmatic. Structural and functional characterization of NLPs from evolutionarily distant organisms is essential to understand the evolution and mechanistic action of these proteins. Our goal is to investigate the GIPC binding of additional NLPs.

Methods After recombinant expression of NLP proteins, we will use structural approaches, such as X-ray crystallography. Also, infiltration into the tobacco apoplast, conductivity measurements, surface plasmon resonance, liposome sedimentation assay to assess binding to the eudicot sphingolipid GIPC and membrane damage of NLPs.

Results We expressed and examined two NLPs, NPPPp from Phytophthora parasitica and MpNEP2 from Moniliphtora perniciosa. Both proteins showed a necrotic effect on tobacco plants after infiltration into the apoplast, but at different concentration ranges. Moreover, sedimentation assay showed that the binding of MpNEP2 to GIPC-containing liposomes was strong, but this does not seem to be the case for NPPPp. We obtained crystals of NPPPp and the diffraction was measured, however, the data showed twinning which prevented us from solving the crystal structure.

Main Conclusions Preliminary results show that both NLPs cause necrosis on tobacco plants, but the mechanism might be different. This needs further investigation.

16:15-16:30Coffee Break
16:30-17:30 Session 3: ORAL 2
María Ikonomopoulou (Imdea-food institute, Spain)
Kity Požek (Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia, Slovenia)
Adrijana Leonardi (Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia, Slovenia)
Igor Križaj (Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia, Slovenia)
Milan Kojić (University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Laboratory for Molecular Microbiology, Serbia)
VaaMPIII-3, a disintegrin-like/cysteine-rich protein from the Vipera a. ammodytes venom, and its toxic effects on blood

ABSTRACT. Aims VaaMPIII-3, a glycoprotein from the venom of the nose-horned viper (Vipera a. ammodytes), is a representative of the novel P-IIIe subclass of snake venom metalloproteases. Two principal aims of this study are (1) further structural and biochemical characterization of VaaMPIII-3 and (2) analysis of its effects on blood. Methods To purify VaaMPIII-3 from crude venom, we employed size-exclusion, cation-exchange, and covalent chromatography. We determined its biochemical properties using isoelectric focusing, differential scanning fluorimetry, CD spectrometry, mass spectrometry, and Edman sequencing. To predict its key structural properties, we constructed a 3D model of its structure using Modeller 9v14 homology modeling software. The effects of VaaMPIII-3 on human blood were studied using turbidimetry and flow cytometry. MBP-tagged VaaMPIII-3 was expressed in E. coli and purified by amylose-affinity chromatography. Recombinant VaaMPIII-3 was purified by RP-HPLC after proteolytic removal of the MBP-tag. Results VaaMPIII-3 is an acidic and highly N-glycosylated protein. It is stable in Ca2+-containing buffers with pH above 6. Although it contains a free Cys at position 6, VaaMPIII-3 appears in the venom as a 21 kDa monomer. Structural modeling revealed that VaaMPIII-3 possesses an SECD motif on its surface, common to disintegrin-like/cysteine-rich proteins that inhibit platelet aggregation. Indeed, it hindered platelet aggregation stimulated by ADP, collagen or arachidonic acid. VaaMPIII-3 did not interfere with platelet agglutination. Accordingly, it did not bind the platelet receptors CD61, CD41, CD42a, or CD42b involved in this process. VaaMPIII-3 did not affect blood coagulation as well. Main Conclusions We optimized the protocol for purification of the natural VaaMPIII-3 and devised the protocol for production of its recombinant form. We thoroughly characterized the protein biochemically and structurally. We demonstrated that VaaMPIII-3 inhibits platelet aggregation aiding at the overall anticoagulant effect of the venom. It is therefore a suitable substance to develop original antithrombotics.

John Dunbar (National University of Ireland Galway, Ireland)
Damien Redureau (University of Liège, Belgium)
Neyaz Kahn (National University of Ireland Galway, Ireland)
Antoine Fort (National University of Ireland Galway, Ireland)
Ronan Sulpice (National University of Ireland Galway, Ireland)
Aoife Boyd (National University of Ireland Galway, Ireland)
Loic Quinton (University of Liège, Belgium)
Michel Dugon (NUI Galway, Ireland)
New kids on the block: Envenomation occurrence, medical significance and venom profile of the Noble false widow spider Steatoda nobilis
PRESENTER: Michel Dugon

ABSTRACT. Aim The Noble false widow Steatoda nobilis is a medium size spider belonging to the family Theridiidae and is closely related to the “true” widows of the genus Latrodectus. Steatoda nobilis is thought to originate from the Atlantic Macaronesian archipelago, but the species is now present in most of Western Europe and in localised areas of North and South America. In recent years, envenomations have been reported from the United Kingdom, Ireland and the Pacific Coast of South America. The symptomatic resulting from envenomation by spiders from the genus Steatoda is referred to as Steatodism.

Methods We investigated 16 confirmed cases of envenomations by Steatoda nobilis that occurred in the United Kingdom (N=1) and Ireland (N=15) between 2018 and 2021. In addition, we investigated the venom composition of female Steatoda nobilis using a combination of transcriptomic and proteomic cutting-edge approaches and explored the potential for Steatoda nobilis to vector pathogenic bacteria during bite events.

Results Results show that victims experienced a range of symptoms reminiscent of latrodectism, including prolonged intense debilitating pain, sweating, tremors, cramping and skin lesions. In five cases, victims sought medical attention and hospitalisation was required for two cases. We also confirm that Steatoda nobilis carries and can vector a range of potentially pathogenic bacteria, including Staphylococcus epidermidis, Kluyvera intermedia, Rothia mucilaginosa and Pseudomonas putida, and antibiotic resistant strains of Pseudomonas putida, Staphylococcus capitis and Staphylococcus edaphicus.

Main Conclusions The venom of Steatoda nobilis includes the main toxins present in Latrodectus, including alpha-latrotoxins, delta-latroinsectotoxins and latrodectins, all of which were first characterised from black widow venoms and enzymes including metalloproteinases, serine proteases, and chitinases are present in large quantities. Our data indicates that Steatoda nobilis is an emergent species of medical importance, and its disruptive potential is enhanced by its preference for synanthropic habitats, adaptability, and competitiveness.

Adrijan Ivanušec (Jožef Stefan Institute, Slovenia)
Jernej Šribar (Jožef Stefan Institute, Slovenia)
Peter Veranič (Faculty of Medicine, University of Ljubljana, Institute of Cell Biology, Slovenia)
Maja Zorović (Faculty of Medicine, University of Ljubljana, Institute of Pathophysiology, Slovenia)
Marko Živin (Faculty of Medicine, University of Ljubljana, Institute of Pathophysiology, Slovenia)
Igor Križaj (Jožef Stefan Institute, Slovenia)
A presynaptically neurotoxic secreted phospholipase A2 group IIA from snake venom and its mammalian orthologue bind to the same mitochondrial receptor

ABSTRACT. Aim Ammodytoxin (Atx), a presynaptically neurotoxic secreted phospholipase A2 (sPLA2) from the venom of the nose-horned viper (Vipera a. ammodytes), was previously shown to target cytochrome c oxidase subunit II (CCOX-II) in neuronal mitochondria. The aim of this study was to determine whether the rat group IIA (GIIA) sPLA2, the mammalian orthologue of Atx, also interacts with the same mitochondrial receptor as Atx.

Methods The binding affinity of the rat GIIA sPLA2 to CCOX-II was tested with competition binding assay using radioiodinated Atx and mitochondrial membranes extract from porcine cerebral cortex. The GIIA sPLA2 effect on CCOX enzymatic activity was measured spectrophotometrically on mitochondria, isolated from PC12 cells, and histochemically on rat brain tissue sections, while its effect on mitochondrial membrane potential in PC12 cells was investigated using flow cytometry. For localization studies, GIIA sPLA2 was labelled with a fluorescent dye and tracked in cells with confocal microscopy.

Results Rat GIIA sPLA2 inhibited 50% of binding of radioiodinated Atx to CCOX-II at about 1 µM concentration, roughly 100-fold higher concentration than Atx itself. Nevertheless, GIIA sPLA2 substantially inhibited the oxidase activity of CCOX on isolated mitochondria and the inhibition was independent of its phospholipase activity. On the other hand, GIIA sPLA2 had only subtle effects on mitochondrial membrane potential in PC12 cells and CCOX activity on rat brain tissue sections. Exogenously added fluorescently labelled GIIA sPLA2 internalized into PC12 cells and colocalized with mitochondria.

Main Conclusions Rat GIIA sPLA2 binds to CCOX-II and inhibits CCOX oxidase activity, indicating a possible regulatory role of mammalian GIIA sPLA2 in mitochondria and opening an important direction of study to advance the understanding of the involvement of the mammalian GIIA sPLA2 in mitochondrial function and dysfunction.

Alexander Vassilevski (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Russia)
Design and production of subtype-selective ligands of voltage-gated potassium channels

ABSTRACT. Potassium channels are the most diverse members of the voltage-gated ion channel superfamily. They play major physiological roles such as controling the resting potential and shaping the action potential. In addition, several diseases are caused by the disfunction of potassium channels. Subtype-selective ligands of these channels are therefore of high interest and can be used in both fundamental and applied research. Animal venoms are particularly important sources of potassium channel ligands, which are mostly disulfide-rich peptides and can bind to their targets with nanomolar or even subnanomolar affinity. Previously, we have created Kalium, a database that contains comprehensive data on all known peptide ligands of potassium channels, and found that more than half of those originated from scorpion venom. Next, we undertook a systematic analysis of the selectivity of scorpion toxins blocking potassium channels. This was done by building models of channel–toxin complexes and computing the interaction parameters. Using the models, we were able to fine-tune the selectivity of several peptides. The respective derivatives of natural toxins were produced recombinantly, and their activity was checked on a panel of channels. I will discuss examples of selective blockers of Kv1.1, 1.2, 1.3, and 1.6 channel isoforms. We believe that our systematic approach will eventually provide us with a toolkit of selective ligands to all major isoforms of voltage-gated potassium channels. This work was supported by the Russian Science Foundation and Research Foundation — Flanders, grant nos. 20-44-01015 and G0E7120N.

Yasmine Boughanmi (UMR7273 CNRS-LATOXAN SAS, France)
Harold de Pomyers (LATOXAN, France)
Kamel Mabrouk (UMR7273 CNRS, France)
From venom to bioactive peptide: Identification, Synthesis and Structure-Activity Relationship Study.


The use of peptide drugs offer advantages over conventional synthetic or semi-synthetic organic chemicals, in particular their low-dose efficacy, low toxicity, harmless metabolites, high capacity for structure modification and high target specificity. Animal venoms contain numerous bioactive peptides, sometimes rich in disulfide bridges, of different lengths, potent, stable and exerting various multipharmacological actions. Our projects have aimed at isolating, identifying and characterizing bioactive molecules from animal venoms in several fields of health and cosmetics. The main research and therapeutic investigations concern the fields of anti-tumor, antibiotic resistance, neurodegenerative diseases, pain and depigmentation.

Methods Several dozen animal venoms have been screened for pharmacological activity. Venoms with biological activity are fractionated by HPLC. The active fractions are sub-fractionated. After several sub-fractionations, the active molecules are isolated and their purity confirmed by HPLC. Once their mass and sequences have been determined, structure-activity relationship studies are performed using synthetic peptides.

Results Thus, we have identified: i) for the first time a polyamine, isolated from spider venom, which inhibits both enzymes involved in white and black skin depigmentation; ii) a peptide from wasp with antibacterial (gram-) anti-inflammatory activity. The activity and stability of this peptide was improved by a structure-activity relationship study using more than 70 synthetic analogues; iii) several potentially anti-tumor molecules from snake venoms.

Main Conclusions

This communication describes the identification and purification of several bioactive molecules isolated from animal venoms. After isolating several bioactive venoms, the fractionation strategy of these latters allowed the identification of several molecules that possess biological activities, such as antiproliferative, depigmentation inhibitory or antibacterial activity. Many new molecules have yet to be identified from active fractions and other venoms have not yet been tested. Several patents have been filed and recently published.

17:30-18:00 Session 3: POSTER 2
Beata Halassy (Centre for research and knowledge transfer in biotechnology, University of Zagreb, Croatia)
Javier Moral-Sanz (Imdea-food institute, Spain)
Ana Vela-Sebastian (Imdea-food institute, Spain)
Manuel Fernandez-Rojo (Imdea-food institute, Spain)
Jeremy Potriquet (QIMR Berghofer Medical Research Institute, Australia)
Andreas Brust (The University of Queensland,, Australia)
Patrick Wilhelm (The University of Queensland, Australia)
Taylor B Smallwood (The University of Queensland, Australia)
Richard J Clark (The University of Queensland, Australia)
Bryan G. Fry (The University of Queensland, Australia)
Paul F. Alewood (The University of Queensland, Australia)
John J. Miles (QIMR Berghofer Medical Research Institute, Australia)
Jason P Mulvenna (QIMR Berghofer Medical Research Institute, Australia)
Maria Ikonomopoulou (Imdea-food institute, Spain)
Unraveling the cytotoxic mechanism of the Octpep-1 venom-derived peptide in BRAF(V600E) mutated melanoma and its synergy with mTORC1 and ERK inhibitors

ABSTRACT. Aim 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 constitutive activation of the mitogen-activated protein kinases (MAPK) signaling, 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 mTOR signaling are key elements linked to drug resistance, cell survival and tumorigenesis.

This study aims to investigate the metabolic and molecular targets of the Octopus kaurna-derived peptide (Octpep-1) and its synergic interactions with targeted therapies in human BRAF(V600E) -mutated melanoma cells.

Methods We performed Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra method (SWATH-MS) analysis to identify the affected cell signaling pathways after Octpep-1 treatment in MM96L melanoma cells. Synergies between Octpep-1 and targeted therapies were identified using cell viability assays and characterized by seahorse metabolic flux analyser and western blot.

Results Proteomics revealed that Octpep-1 targets the PI3K/AKT/mTOR signaling in BRAF(V600E) melanoma cells. Interestingly, Octpep-1 potentiated the cytotoxic mechanism of rapamycin and ERK1/2 inhibitors. The synergy between Ocpep-1 and ERK1/2 inhibitors is partly explained by the bioenergetics, in which the treated melanoma cells show compromised mitochondrial respiration combined with reductions in glycolysis. On the other hand, Octpep-1 potentiates rapamycin by downregulating the mTORC1 regulatory subunit raptor and its downstream effector S6K.

Main Conclusions Octpep-1 through the regulation of the PI3K/AKT/mTOR signaling pathway represents an optimal candidate for combined therapies against melanoma of BRAF(V600E) mutations

Magdalena Kulma (National Institute of Chemistry, Slovenia)
Aleksandra Šakanović (National Institute of Chemistry, Slovenia)
Simon Caserman (National Institute of Chemistry, Slovenia)
Marjetka Podobnik (National Institute of Chemistry, Slovenia)
Gregor Anderluh (National Institute of Chemistry, Slovenia)
Equinatoxin II-D4 fusion protein as a promising tool to study organization of plasma membrane lipids in mammalian cells during viral infection
PRESENTER: Magdalena Kulma

ABSTRACT. Aim Many pore-forming toxins produced by various organisms specifically recognize a particular lipid of the target cell, i.e. sphingomyelin and cholesterol. Blockage of these lipids could prevent cellular processes involved in the viral entry. The aim of our study was to produce and characterize biophysical properties of a fusion protein that binds sphingomyelin and cholesterol simultaneously and could protect mammalian cells from viral infection by interfering with the lipid composition of cellular membranes. Methods A recombinant fusion protein was generated through the joining of genes encoded non-lytic mutant (V8C/K69C) of equinatoxin II (Eqt8-69C) and the D4 domain of clostridial toxin- perfringolysin O. After expression in the bacterial expression system, toxic activity of purified Eqt8-69C-D4 was verified by hemolysis assay. Then lipid-binding properties were characterized by lipid/protein overlay assay and the vesicle co-sedimentation method. To determine the plasma membrane pool of sphingomyelin and cholesterol accessible for binding of Eqt8-69C-D4, detergent extraction of membranes from mammalian epithelial (VeroE6) and macrophage-like (Raw 264.7) cell lines was employed. Results Our results showed that the fusion Eqt8-69C-D4 was efficiently expressed as a soluble disulfide bonded protein with impaired hemolytic activity that was restored to near the wild type EqtII level under reducing conditions. The lipid–protein binding analysis revealed that Eqt8-69C-D4 specifically recognizes both sphingomyelin and cholesterol. Liposome cosedimentation assay confirmed stronger binding of Eqt8-69C-D4 to sphingomyelin/cholesterol-containing liposomes than liposomes consisting of sphingomyelin and cholesterol separately. Furthermore, analysis of binding to mammalian cells showed that Eqt8-69C-D4 selectively bound to the sphingomyelin/cholesterol mixture in the lipid raft fraction of the plasma membrane. This interaction was more sensitive sphingomyelin than cholesterol level. Main Conclusions We have demonstrated that the nontoxic Eqt8-69C-D4 selectively binds sphingomyelin/cholesterol complexes in lipid rafts of mammalian cells. This suggests that Eqt8-69C-D4 may concern an alternative approach to prevent cell infection with viruses.

Ana Vela-Sebastian (IMDEA FOOD INSTITUTE, Spain)
Javier Moral-Sanz (IMDEA FOOD INSTITUTE, Spain)
Sergey Kurdyukov (University of Sydney, Australia)
Andreas Brust (The University of Queensland, Austria)
Patrick Wilhelm (The University of Queensland, Australia)
Paul F. Alewood (The University of Queensland, Australia)
G. Gregory Neely (University of Sydney, Australia)
Evelyne Deplazes (The University of Queensland, Australia)
María Ikonomopoulou (IMDEA Food Institute, Spain)
Cholesterol modulates the cytotoxicity of Gomesin in melanoma of BRAF mutation.

ABSTRACT. Aim Gomesin is an antimicrobial peptide isolated from the South American spider Acanthoscurria gomesiana that shows potent and selective antitumoral effect in BRAF-mutated melanoma. Gomesin has a CRAC domain, known to favor cholesterol interactions with cell membranes. The aim of this study is to elucidate upon the occurring interactions between gomesin and lipids present in cellular membranes, focusing on the mediation of cholesterol in the cytotoxic profile of gomesin. Methods Cell viability was analyzed by MTT assays comparing BRAF-mutated melanoma cells and healthy fibroblasts, treated with gomesin in the presence of increasing concentrations of cholesterol or modifiers of the cellular content of cholesterol. Electrochemical impedance spectroscopy (EIS) techniques and tethered bilayer lipid membranes (tBLM)/EIS assays were used to analyze protein binding to the membrane or membrane disruption induced by gomesin, respectively. Results Gomesin variants show a poor binding to POPC membranes. The presence of POPS and cholesterol significantly improved peptide binding and reduced membrane disruption. Moreover, the cytotoxicity of gomesin was blunted by increasing concentrations of cholesterol in both melanoma cells and fibroblast. Surprisingly, cholesterol depletion potentiates the cytotoxicity in fibroblast but not in melanoma cells, suggesting a specific role of cholesterol in the selective cytotoxicity of gomesin. Main Conclusions Our results support the selective antitumoral effect of gomesin in melanoma of BRAF mutation. We observed that the mechanism of action of gomesin differs between cancerous and non-cancerous cells and confirmed that cholesterol plays a key role in the binding and biological membrane disruption.

Hakan Caliskan (Eskisehir Osmangazi University, Turkey)
Figen Caliskan (Eskisehir Osmangazi University, Turkey)
Adolfo Rafael de Roodt (Primera Cátedra de Toxicología, Facultad de Medicina, Universidad de Buenos Aires, Argentina)
Horse selection and immunization for effective production of polyclonal antibodies against venom from venomous animals
PRESENTER: Hakan Caliskan

ABSTRACT. Aim Equine immunoglobulin and their fragments are used as the only specific medical treatments against envenomation. The aim of the study is to describe important aspects of preparation and selection that should be considered for obtaining horses that produce high levels of polyclonal antibodies to venom from venomous animals.

Methods The available data and experience in systems for the production of antivenoms for therapeutic use, in use in the different health systems, were analyzed, from the experience of authors and available information.

Results Effective production of equine hyperimmune sera is depending on several methods and factors among which the selection and breeding criteria of the horses and the immunization schedule, usage of antigen and adjuvant and monitoring of horses, are fundamental. The most important “factory of antibodies” or “bio-reactor” for venom neutralization used by the industry in the world is predominantly the horse. With the correct selection and management, they may be maintained in production with good health status for about 20 years. This depends not only on selection and management but also on the system of production. The mixed management or the extensive management seems to be better regarding the length of the life of the horse as a producer.

Main Conclusion In this study, important points from our experience in our institutions and other institutions around the world that can help those who have to deal with the selection, maintaining and management of horses to be used for antivenom production are evaluated.

Adam Jacobsen (National University of Ireland, Galway, Ireland)
Alannah Forde (National University of Ireland, Galway, Ireland)
Kevin Healy (National University of Ireland, Galway, Ireland)
Michel Dugon (National University of Ireland, Galway, Ireland)
Exploring global trends in the clinical manifestations of scorpion envenomations
PRESENTER: Adam Jacobsen

ABSTRACT. Aim Despite the development of area-specific venom antisera over the past six decades, scorpion envenomations still claim 3000+ lives globally every year, mainly among children living in rural parts of developing nations in Asia, Africa, and South America. In addition, many survivors suffer long-lasting, debilitating sequelae. In the literature, the clinical manifestations of scorpion envenomation (scorpionism) are often treated as a homogeneous, mostly neurological syndrome, irrespective of where it occurs. However, the diverse ecology, behaviors, and venom composition found across scorpions suggests that the symptoms and effective treatments, associated with scorpionism are unlikely to be homogeneous. Here we investigate if scorpionism may be a polymorphic disease that depends on species, geographical location, and the age of clinical patients. Methods Clinical symptoms associated with scorpionism worldwide were collected from the literature (40 papers published between 1985 and 2019) along with the number and age group of patients experiencing each symptom or combination of symptoms. Symptoms were graded on a severity scale from 1 to 4 and a binomial regression was performed to infer how symptom type and severity differed depending on locality and the age profile of patients. Results Results suggest that the severity and the overall outcome (i.e. patient survival rate) following medically significant scorpion envenomations differ significantly depending on where in the world the envenomation occurred. While mortality rates were significantly higher in African regions, cardiological symptoms were highest in India. Other symptoms, such as neurological and respiratory were also more common in other regions. Main Conclusions The results of our study indicate that scorpionism is a polymorphic syndrome, with species-specific, geographically distinct manifestations, that vary significantly depending on the age of patients.

Blerina Vrenozi (Research Center of Flora and Fauna, Faculty of Natural Sciences, University of Tirana, Albania)
Venomous spiders of Albania – Does the increase of the air temperature influence the toxicity of the spider venom?

ABSTRACT. Aim Venomous spiders encountered in Albania belong to the families of Sicariidae: the Mediterranean brown recluse Loxosceles rufescens (Dufour, 1820), and Theridiidae: the black widow Latrodectus tredecimguttatus Rossi, 1790. Although their venom is rarely fatal, their bite is medically significant, raising questions about the influence of the higher air temperatures on their toxicity. Therefore, this retrospective study will highlight the importance of the spiders’ venom severity and raise awareness to prevent their morbidity.

Methods The description of the 125 case studies during a decade, from the villages of Fier County, hospitalized in the regional hospital, is presented. Detailed data on the localities, age, gender, bitten location, symptoms, supportive treatment, bite date, and hospitalized time, are analyzed. The spider bites were during the seasonal fieldwork, from April till November. Spiders were identified as black widows or simply as a spider bite.

Results The hospitalized patients from the spider bites were mainly farmers from the agricultural areas in the Western Lowland. Based on the fieldwork time, the venom severity is higher in August (29.6%). The spider bite localities were particularly the lower and upper limbs of the age groups of 15-59 years old, although there were also kids helping their parents (5.6%). The main symptoms were limb pain followed by abdominal pain, swelling, itching, redness, vomiting, tiredness, and difficulties in breathing. Patients were discharged mainly 72h after their hospitalization, although 8% of the case studies had more than three days of hospitalization.

Main Conclusions The morbidity of the spider venom in Albania raises awareness to consider the distribution map of the venomous spiders and detailed data on their venom severity based on the air temperature, including black widows and brown recluses. Nevertheless, other medically significant spiders are identified in Albania, although there is no clear evidence from the patients.

Gülay Sezer (Erciyes University, School of Medicine, Department of Pharmacology, Turkey)
Figen Çalışkan (Eskişehir Osmangazi University, Science and Art Faculty, Biology Department,, Turkey)
Tuncay Türkeş (Niğde Ömer Halisdemir University, Science and Art Faculty, Biology Department,, Turkey)
Ertuğrul Şahmetlioğlu (Kayseri University, Safiye Çıkrıkçıoğlu Vocational School, Turkey)
Anticancer activity of Argiope bruennichi spider venom
PRESENTER: Gülay Sezer

ABSTRACT. Aim We aimed to investigate the anticancer activity of Argiope bruennichi spider venom on the triple negative breast cancer cell line.

Methods For this purpose, venoms obtained from Argiope bruennichi spiders collected from the Black Sea Region in August 2020 were applied to reverse phase High Pressure Liquid Chromatography (RP-HPLC) and a total of 20 fractions were collected every 3 minutes for 60 minutes. Each lyophilized fraction was applied to human breast cancer cell line, MDA.MB.231 cells, and viability was analyzed by MTT assay. The effects of the fractions, which caused a significant decrease in cell proliferation compared to the control group, on the production of intracellular ROS and the expression of genes related to apoptosis were investigated.

Results Fractions collected at 12, 33, 42 ve 45. minutes caused a statistically significant decrease in cell viability compared to the control group at both 24 and 48 h. Of these, the 45. minute fraction significantly increased intracellular ROS production. The fractions caused an increase in mRNA expressions of some apoptosis-related genes.

Main Conclusions We think that our findings will enable the development of in vitro and in vivo studies that aim to purify the active fractions of the venom with advanced methods, to identify the peptide or proteins responsible for the activity, and to further investigate their effects and mechanisms.

Quirino Attilio Vassalli (Scripps Research institute La Jolla, CA, United States)
Zaverio Marcello Ruggeri (Scripps Research institute La Jolla, CA, United States)
Maria Vittoria Modica (Stazione Zoologica Anton Dohrn, Naples, Italy, Italy)
Marco Oliverio (Sapienza University Roma, Italy)
Manuela Cervelli (Roma Tre University, Italy)
Jonathan Reinoso Sanchez (Roma Tre University, Italy)
Yunfeng Chen (Scripps Research institute La Jolla, CA, United States)
The anti-platelet activity of VWA1-like proteins from the vampire snail Cumia reticulata

ABSTRACT. Aim Platelets play a central role in haemostasis and contribute to pathological thrombosis. Endogenous subendothelial von Willebrand factor (VWF) as well as plasma VWF initiate platelet adhesion at sites of vascular damage. This process depends on VWF A1 domain (VWFA1) binding to the platelet membrane glycoprotein (GP) Ibα. Subsequently, soluble plasma VWF recruits circulating platelets creating larger aggregates on the vessel wall. These interactions are affected by shear stress in the circulating blood. Given its relevance in haemostasis, hematophagous animals evolved a variety of inhibitors of platelet aggregation, generally produced in salivary glands and injected into the host. Here we investigate the potential platelet aggregation inhibitory activity of salivary VWFA1-like proteins recently identified in the vampire snail Cumia reticulata.

Methods We produced recombinant forms of two polypeptide sequences deduced from transcripts identified in Cumia, Seq5 and WD6. We used Surface Plasmon Resonance (SPR) to analyze the binding of these proteins to purified GPIbα, and aggregometry to test their ability to inhibit dimeric VWFA1 (dA1)-mediated platelet agglutination.

Results In SPR, Seq5 and WD6 bound to human GPIb with 100-times lower affinity than human VWFA1. In the aggregometry assay, regardless of the dA1 concentration used, both Seq5 and WD6 caused dose-dependent inhibition of platelet agglutination, indicative of ability to interfere with the VWFA1-GPIb interaction.

Main Conclusions Our results support the hypothesis that salivary VWFA1-like proteins from the vampire snail bind to human platelet GPIb and inhibit human GPIb-VWFA1 interaction. Their lower affinity compared to human VWFA1 could be explained by structural differences between their native target (GPIb on fish thrombocytes) and the human GPIb used in the test. A down-tuned binding may also ensure the balance between impairing haemostasis to facilitate blood suction and survival of the host. To differentiate between these hypotheses, further investigation will be carried out.


Posters will be further discussed in 3 parallel live Q&A sessions

Evroula Hapeshi (University of Nicosia, Cyprus)
19:25-19:45 Session CLOSING: Congress closing
Figen Caliskan (Eskisehir Osmangazi University, Turkey)
Maria Vittoria Modica (Stazione Zoologica Anton Dohrn, Italy)
Manuel J. Tenorio (University of Cadiz, Spain)