previous day
next day
all days

View: session overviewtalk overview


This session features the most recent methodological advances that are pushing forward venom research: from gene editing to biophysics, from high throughput experimentation and bioinformatics to cell biology.

14:05-14:35 Session 2: POSTER 1
Evroula Hapeshi (University of Nicosia, Cyprus)
Batuhan Orman (Ege University, Turkey)
Didem Şen Karaman (Izmir Katip Celebi University, Turkey)
Ayşe Nalbantsoy (Ege University, Turkey)
Cytotoxic Activity Investigation of Anatolian Bee (Apis mellifera anatoliaca) Venom Loaded Mesoporous Silica Nanoparticles
PRESENTER: Batuhan Orman

ABSTRACT. Aim Since cancer is one of the major problems of our time, the search for new treatments has increased the studies of bee venom on cancer in recent years and it has been shown that it can be effective. Mesoporous silica nanoparticles (MSNs) arose as promising drug carrier platforms because of their outstanding biocompatibility, loading capacity provided by pores, great chemical and biological robustness, and ability to reach tumors. This study is aiming to increase the potential cytotoxic effect of bee venom with the aid of mesoporous silica nanoparticles drug delivery system. Methods In the present study, fluorescently labeled mesoporous silica nanoparticles were synthesized and SEM, zeta potential, and DLS characterization studies were carried out. The cytotoxicity of venom and venom-loaded MSNs were determined on MDA-MB 231, PC3, and HeLa cancer cells, and their biocompatibility was investigated on HEK-293 and CCD34-Lu normal cell lines. It was investigated by fluorescence microscopy and flow cytometry whether the venom peptides were transported into PC3 and MDA-MB 231 cells. The generally desired apoptotic effect on tumor cells was examined by flow cytometry. After that, the effects of bee venom and venom MSN combinations on mitochondrial membrane potential were examined. Results Positively charged MSNs were obtained at pH:7.2 by zeta potential analysis. The cytotoxic effect of the venom was generally enhanced or maintained by MSNs. High cellular uptake MSNs to PC3 and MDA-MB 231 cells. 1.5 fold increament in apoptotic effect of venom was observed on PC3 cells nice combined with MSN compared to alone introducing cells. Main Conclusions In conclusion, MSNs and the venom of Apis mellifera anatoliaca grown in our country have the potential to offer cost-effective, easily accessible, and innovative treatment options for treatments of cancers by eliminating potential, side effects, and cancer resistance

Yanning Yong (Monash University Malaysia, Malaysia)
Michelle Khai Khun Yap (Monash University Malaysia, Malaysia)
Gene Cloning and Expression of Cobra Venom Cytotoxin using Bacterial Expression System
PRESENTER: Yanning Yong

ABSTRACT. Aim To overcome the geographical and species variations of cobra venom cytotoxin (CTX), a conserved sequence of cobra cytotoxin was synthesized using the E. coli expression system for downstream characterization and functionality studies. Methods Cloning: The plasmid, pET-22b(+), and the CTX gene were digested with restrictive enzyme pairs, MscI and XhoI; NdeI and XhoI. The digested plasmid and insert were subjected to gel purification before ligation with T4 DNA ligase. The ligated recombinant plasmid was then transformed into Dh10b via the heat shock method. The transformed colonies were screened with antibiotic selection with ampicillin and streptomycin as well as colony PCR. The plasmids from the transformed colonies were extracted and sent for sequencing to verify the orientation of the insert. Expression: The recombinant plasmid was extracted and transformed into BL21 competent cells. The expression of the CTX gene was induced with a range of IPTG concentrations (0.1, 0.5, 1.0 mM), with varying temperatures (16, 24, 30 ℃) and time-points (8, 16, 24 hr). Four different fractions (total, periplasmic, soluble, insoluble) were extracted from the expressed culture for SDS-PAGE and Western blot analysis to examine the expression levels of CTX. Results The sequencing results indicated the start codon, promoter, 6X His-tag, and the CTX gene were in the correct orientation for both MscI and NdeI clones. Preliminary optimization found that the MscI/XhoI clone exhibited the optimum expression of a soluble CTX at 24 ℃ for 16 hours with 0.5 mM IPTG, whereas no soluble CTX was expressed in the NdeI/XhoI clones. The periplasmic fraction of the MscI/XhoI clone was thus chosen for downstream purification in terms of abundance and solubility of the protein. Main Conclusions The bacteria culture can be upscaled at the optimum conditions for downstream purification using immobilized metal affinity chromatography (IMAC) resin or column.

Konrad Hus (Rzeszow University of Technology, Poland)
Łukasz Marczak (Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland)
Vladimír Petrilla (Department of Physiology, University of Veterinary Medicine and Pharmacy, Slovakia)
Monika Petrillová (Department of General Education Subjects, University of Veterinary Medicine and Pharmacy, Slovakia)
Jaroslav Legáth (Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy, Slovakia)
Aleksandra Bocian (Rzeszow University of Technology, Poland)
How your research strategy can influence your data

ABSTRACT. Aim The dynamic development of venomics in recent years has resulted in a significant increase in publicly available proteomic data. These results are frequently used for comparisons between different datasets and to draw biological conclusions therefrom. However, the differences in the research strategies among various research groups can greatly influence the final results, making any data comparisons very difficult. Therefore, we wanted to show how the final results may vary when different research strategies are used. Methods We applied two software solutions (PeptideShaker and MaxQuant) to process data from shotgun LC-MS/MS analysis of Naja ashei venom and collate it with our previous 2DE-MS report concerning this species. Results We were able to provide new information regarding the protein composition of Naja ashei venom but also point out the qualitative and quantitative limitations of currently used proteomic methods. We have shown how the applied research strategies influence the final data but also how the results of the same LC-MS analysis may differ after using different methods for data processing. Main Conclusions Current high-throughput analytical methods produce enormous amounts of experimental data, which then have to be analyzed with the use of sophisticated bioinformatics tools. Therefore, it is important to plan the experiment with full knowledge concerning the pros and cons of the applied analytical techniques as well as tools used for the data processing. Our results draw attention to the necessary caution in the interpretation of data from current proteomic experiments, with particular emphasis on the comparisons of data derived from studies that greatly differ in the applied research strategy.

Naira Ayvazyan (Orbeli Institute of Physiology NAS RA, Armenia)
Gevorg Ghukasyan (Orbeli Institute of Physiology NAS RA, Armenia)
Lusine Ghulikyan (Orbeli Institute of Physiology NAS RA, Armenia)
Gayane Kirakosyan (Orbeli Institute of Physiology NAS RA, Armenia)
Gohar Sevoyan (Orbeli Institute of Physiology NAS RA, Armenia)
Armen Voskanyan (Orbeli Institute of Physiology NAS RA, Armenia)
Zaruhi Karabekian (Orbeli Institute of Physiology NAS RA, Armenia)
PRESENTER: Naira Ayvazyan

ABSTRACT. The epithelium is a primary target for injected viper venom toxic substances, and therefore a focus in modern toxinology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for the analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during the intoxication is a reason for concern and requires a critical review. Here we use the Vero cell as a model to assess the action of complete Macrovipera lebetina obtusa venom and the action of the same venom with inhibited two key enzymatic components (phospholipase A2, PLA2; and metalloproteinases) on the bioenergetic cellular response. In addition to the rate of free radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data witnesses that viper venom driving an increase in both glycolysis and respiration in Vero cells, while blocking the PLA2 or/and metalloproteinases exclusively pushed only oxphos. PLA2 blocking in venom also increases the cytotoxic activity and overproduction of the reactive oxygen species.

Gašper Šolinc (Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia)
Nada Žnidaršič (Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia)
Gregor Anderluh (Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia)
Marjetka Podobnik (Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia)
Bryoporin from moss Physcomitrium patens is structurally related to pore-forming actinoporins
PRESENTER: Gašper Šolinc

ABSTRACT. Aim: Actinoporins are one of the most studied families of toxins that form pores in target lipid membranes, first discovered in sea anemones but can be found in other cnidarians as well. Actinoporins are involved in defence against predators and aid in prey capture as they are highly lethal to small crustaceans, molluscs and fishes. Bryoporin is a protein from a moss Physcomitrium patens and shares 34 % sequence identity with actinoporins. It displays haemolytic activity and was shown to play a role in water stress [1], but its exact biological function remains unknown. The aim of our study was to further characterize bryoporin and its structure/function relation to actinoporins. Methods: For more detailed functional analysis of the bryoporin pore-forming activity, we performed the calcein release experiments on membranes of various lipid compositions, planar lipid membrane experiments and haemolytic assays, and compared it to actinoporins. We determined the X-ray structure of the water-soluble form of monomeric bryoporin and visualized and reconstructed the model of the bryoporin pore using electron microscopy. Results: Bryoporin displayed haemolytic activity comparable to typical actinoporins: In calcein release experiments it specifically bound and perforated membranes with sphingomyelin. Moreover, the presence of cholesterol significantly increased the pore-forming activity. Planar lipid membrane experiments confirmed sphingomyelin specificity and showed that bryoporin forms homogeneous pores. The structure of monomeric bryoporin highly resembles typical actinoporins and imaging of the bryoporin oligomers on lipid vesicles with both, negative-stain TEM and cryo-EM showed, that bryoporin forms transmembrane pores similar to those found in actinoporins. Main Conclusions: Despite the phylogenetic distance between land plants and cnidarians, bryoporin exhibits highly conserved actinoporin fold and shares many functional characteristics with actinoporins, including formation of predominantly octameric pores in sphingomyelin containing membranes. While bryoporin functionally behaves like a toxin, its biological function in the moss remains unknown.

Zafer Sancak (Kastamonu University, Turkey)
Idris Yazgan (Kastamonu University, Turkey)
Mehmet Ali Demir (Kastamonu University, Turkey)
Antibacterial properties of Androctonus crassicauda (Oliver, 1807) venom synthesized gold nanosturctures
PRESENTER: Idris Yazgan

ABSTRACT. Aim Scorpion venom contains enzymes, peptides, free amino acids, inorganic salts, amines, lipids and nucleotides. Despite of their detrimental effects, they have been used in treatment of a variety of diseases and symptoms including infections, metabolic disease, and pain. The aim of this study, we used venom from Androctonus crassicauda is synthesis of gold nanoparticles in the presence of a modified sugar ligand (cellobiose p-aminobenzoic acid, CpAB) as the reducing agent. Methods In the electrical stimulation method, the scorpions are fixed with the help of a mechanism. 12 volts of electricity is given to the body and wire of the scorpion by touching it at intervals of 5-10 seconds. The venom from the scorpion was dissolved in 5 mL of 1 mM NaCl aqueous solution and then added to the medium containing the sugar ligand. The 5 mg sugar ligand used is Cellobiose p-aminobenzoic acid (CB-pAB). By adding HAuCl4 salt solution to the medium, vortexing is performed and AuNP synthesis is performed by incubation at room temperature. UV-Vis, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), infrared (IR) spectroscopies were used to characterize the gold nanostructures (Au NSs). Results At high venom to Au3+ ratio nanosized as 5 ±2.5 nm Au NSs while at lower venom to Au3+ ratio micro- to nano-sized plates and aspherical Au NSs were obtained. Larger Au NSs gave single crystalline structure while nanosized Au NSs gave polycrystalline nature. Main Conclusions Bench top centrifugation system was used to isolate larger and smaller Au NSs for IR analyzes. For both type of Au NSs proteins played the key roles to stabilize the nanostructures. Smaller Au NSs were tested against Pseudomonas aeruginosa, which showed the Au NSs can synergistically advance erythromycin’s antibacterial and anti-film potential against the tested bacteria.

Florence Jungo (SIB | Swiss Institute of Bioinformatics, Switzerland)
Edouard de Castro (SIB | Swiss Institute of Bioinformatics, Switzerland)
Elisabeth Coudert (SIB | Swiss Institute of Bioinformatics, Switzerland)
Alan James Bridge (SIB | Swiss Institute of Bioinformatics, Switzerland)
The Uniprot Consortium (UniProt Consortium, Switzerland)
Two web resources on venoms and their proteins: the UniProt ToxProt program and VenomZone
PRESENTER: Florence Jungo

ABSTRACT. Venoms are complex mixtures composed of numerous proteins and are mostly studied for their potential as drug leads or to develop new treatments against envenomation. Making it possible to quickly find a structured summary of these studies, or simply to access protein sequences, is one role of web resources. Here, we present both the UniProt animal toxin annotation program, also known as ToxProt (, and VenomZone (, a website dedicated to venoms. The UniProtKB ToxProt program provides continuously updated information extracted from the literature on about 7,000 protein entries (release 2021_03) from all venomous taxa. Information on venom proteins mostly consists of sequences, function/activity post-translational modifications, and links to relevant databases such as the 3D structure database PDB, the DDBJ/EMBL/GenBank nucleotide sequence database, or venom organism-specific databases. At a broader level, VenomZone aims to provide a venom overview from the six major venomous taxa (snakes, scorpions, spiders, cone snails, insects and sea anemones). In addition, it permits a quick access to UniProtKB/Swiss-Prot venom protein entries by using the UniProtKB search tool with appropriate terms. Key features of VenomZone include information such as venom composition, protein family descriptions, as well as the classification of toxins by pharmacological targets. Contributions from the community, by giving comments/suggestions via the contact forms at or, or by submitting protein sequences (obtained by MS/MS de novo sequencing and Edman degradation) at are very welcome.

Grigoris Panayi (University of Nicosia, Cyprus)
Christos Petrou (University of Nicosia, Cyprus)
Yiannis Sarigiannis (University of Nicosia, Cyprus)
Linear Scorpion Peptides: An unexplored pool for peptide hydrogels
PRESENTER: Grigoris Panayi

ABSTRACT. Blood Brain Barrier (BBB) crossing is among the major challenges in drug development because BBB controls the transfer of all substances from blood to brain tissues. We have recently shown that linear scorpion peptides are an unexplored pool for forming hydrogels under physiological pH conditions, a fast - growing field in drug delivery. Here, we present in silico studies of short linear antimicrobial scorpion peptides (13-19 amino acids) for their ability to cross the blood-brain barrier and fight infections of the central nervous system and for their hemolytic activity, one of the main drawbacks of these peptides. Results Two online applications were used to calculate the ability of the linear scorpion peptides to cross the BBB. Peptides that appeared to cross the BBB were collected and tested for their hemolytic activity. Moreover, an in-silico Ala – scan test was performed to find the responsible amino acids for their hemolytic activity. New peptides that did not exhibit hemolytic activity were re-tested for their ability to cross the BBB. Our results reveal that the peptides found to cross the BBB were the BmKn2 (NDBP-4.4) and the UyCT2 (NDBP-4.17). The LR platform of the B3Pred application showed that only Pantinin-1 (NDMP-4.20) was able to cross the BBB. On the contrary XGB platform showed that no peptide was able to cross the BBB. However, the RF platform showed that the only peptide that was able to cross the BBB was the Pantinin-2 (NDMP-4.21). Of the peptides found to cross the BBB, Pantinin-1 and UyCT2 peptides did not exhibit any hemolytic activity in their native structure. In conclusion the results extracted from these experiments varied between the two online applications. Further wet chemistry studies are needed to test the ability of the linear scorpion peptides to cross the blood-brain barrier exhibiting low hemolytic activity.

14:35-15:35 Session 2: ORAL 1
Agostinho Antunes (University of Porto, Portugal)
José Ramón Pardos-Blas (Museo Nacional de Ciencias Naturales (CSIC), Spain)
Iker Irisarri (Institute for Microbiology and Genetics, University of Goettingen, Germany)
Samuel Abalde (Swedish Museum of Natural History, Sweden)
Carlos.M L Afonso (Centre of Marine Sciences (CCMAR), Universidade do Algarve, Portugal)
Manuel.J Tenorio (Departamento CMIM y Química Inorgánica-INBIO, Facultad de Ciencias, Universidad de Cádiz, Spain)
Rafael Zardoya (Museo Nacional de Ciencias Naturales (CSIC), Spain)
Genome organization and structure of venom genes of the Mediterranean cone snail Lautoconus ventricosus (Gmelin, 1791)

ABSTRACT. Aim In recent years, transcriptomics has revolutionized the detection and classification of conotoxins from the venom gland of cone snail species. However, the processes underpinning the generation of venom complexity at the gene level remains barely unexplored. The main goal of this work was to shed light on the genome organization and structure of venom genes. Methods By generating PacBio long reads as well as Chicago and HiC libraries, we were able to assemble a highly contiguous genome for the cone snail Lautoconus ventricosus. In parallel, we also assembled the transcriptomes of the venom gland and foot of this species. All newly generated data were used to locate and annotate the venom genes in the genome. Results The assembled genome was 3.59 Gb size with 86.6% of the genome assembled into 35 pseudochromosomes. Venom gland reported 289 transcripts of conotoxin precursors, hormones, and other venom proteins. The venom genes were scattered in the different pseudochromosomes. The majority (62.6%) of conotoxin precursor genes had three exons and two introns. The ratio of conotoxin precursor genes and transcripts was close to 1, suggesting that toxin diversity is produced through gene duplication and later through post-translational modification of peptides but not during transcription. Surprisingly, we found transcripts of conotoxins precursors in the foot tissue, being their expression about one order of magnitude lower than in the venom gland. Main Conclusions The new genome will be a reference for future comparative genomics studies on venom evolution within cone snails. The venom diversity in L. ventricosus is owing to an expansion of the conotoxin gene family and genes are distributed widespread across the chromosomes. The low expression of conotoxin precursor transcripts outside the venom gland points out to a neofunctionalization from housekeeping genes at the origin of venom genes.

Mirijam Kozorog (National Institute of Chemistry, Slovenia)
Marc-Antoine Sani (The University of Melbourne, Australia)
Martina Lenarčič Živković (National Institute of Chemistry, Slovenia)
Janez Plavec (National Institute of Chemistry, Slovenia)
Frances Separovic (The University of Melbourne, Australia)
Gregor Anderluh (National Institute of Chemistry, Slovenia)
The use of one-dimensional fluorine (19F) NMR in membrane-protein studies of pore-forming toxin listeriolysin O
PRESENTER: Mirijam Kozorog

ABSTRACT. Aim Listeriolysin O (LLO) is 57 kDa protein and a main virulence factor of bacterium Listeria monocytogenes. It binds to cholesterol-rich membranes, where it oligomerizes and forms large biological pores. The process itself is still not explained in the detail at the molecular. Our aim was to investigate the role of highly conserved LLO tryptophan residues in the above mentioned process. Methods We expressed recombinant LLO that was uniformly labeled with 5-fluorotryptophan (5F-Trp) using E. coli expression system. Produced LLO with all seven tryptophan residues labelled with 19F (19F-LLO) was incubated with cholesterol preparations in solution or with cholesterol-rich multilamellar vesicles. Solution and solid state 19F NMR spectra of bound and unbound 19F-LLO were recorded and fluorine signals were unambiguously assigned by using single point Trp → Phe LLO mutants. Results Circular dichroism spectra showed no structural changes in 19F-LLO or Trp mutants compared to the unlabeled wild-type protein. 5F labelled tryptophan residues also had no measurable effect on the protein’s pore-forming activity. Solution 19F NMR experiments indicated that none of the tryptophan residues were directly involved in cholesterol binding in solution. On the other hand, significant chemical shift changes were observed upon 19F-LLO binding to cholesterol rich vesicles. Along with the results of measured activity of individual Trp → Phe LLO mutants we concluded that several tryptophan residues are involved in membrane interactions (W512) and oligomerization (W189 and W489). Main Conclusions Our results show the attractiveness of 19F NMR spectroscopy use in membrane-protein interaction studies also for higher molecular weight toxins. Although fluorine is not present in naturally occurring biological molecules, it can be selectively incorporated into proteins by using simple bacterial expression system and without causing major structural perturbations in labelled molecule.

Maik Damm (Technische Universität Berlin, Germany)
Benjamin-Florian Hempel (Charité Berlin, Germany)
Bayram Göcmen (Ege University, Turkey)
Ayse Nalbantsoy (Ege University, Turkey)
Roderich D. Süssmuth (Technische Universität Berlin, Germany)
Unveiling the Old World Mysteries - High Scale Proteomics of Turkish Viperine Venoms

ABSTRACT. Aim Snake venoms of the Old World vipers, Viperinae subfamily, are composed of >16 different toxin families, which can be grouped from major abundant to more rare ones. Their proteomic compositions vary strongly between genera and show clear trends regarding different distribution areas. With more than ten species and numerous subspecies, particularly the territory of Turkey is a hotspot for a multitude of vipers. Our ongoing project about Turkish snakes sheds light on the complex venom compositions in this taxonomic highly discussed clade of Eurasian vipers. In this study, we investigated six Turkish viper venoms within the three genera Daboia, Vipera, and Montivipera. Methods Therefore, the venoms were analysed by high-resolution mass spectrometry (MS), combining bottom-up and top-down proteomics approaches. This includes a detailed intact mass profiling, especially for the diverse peptide parts. Finally, the venoms were compared by their relative quantification based on the approved three-level snake venomics protocol regarding the reversed-phase HPLC peak area, 1D SDS-PAGE band intensity and if necessary, TOP3 relative MS ion intensity. Results All six venoms consist of toxin families, which are representative for the Old World vipers, but are, like expected, divers in their individual compositions. While the Montivipera venoms are similar among each other, the Vipera berus barani and V. darevskii venoms clearly show the high variability within the Vipera genus. Furthermore, the Turkish Daboia palaestinae underlines differences among the western Mediterranean and an eastern tropical Asian group of Daboia. Main Conclusions Over the past decade, we characterized several snake venoms through different state-of-the-art mass spectrometry methods and with this study expand the range of investigated Turkish viper venoms. We highlight the compositional trends amid Old World viper venoms, especially in comparison to the newly described Daboia, Vipera, and Montivipera venom proteomes.

Benjamin-Florian Hempel (Charité – Universitätsmedizin Berlin, Germany)
Aida Verdes (Museo Nacional de Ciencias Naturales, Spain)
Eivind Andreas Baste Undheim (Norwegian University of Science and Technology, Norway)
Ronald Jenner (Natural History Museum, UK)
Spatial venomics: Toxin production and functional biology of glycerid toxins by mass spectrometry imaging

ABSTRACT. Aim Throughout evolution, toxic secretions have independently evolved as biochemical weapons in association with highly specialized venom delivery systems across a broad phylogenetic range of venomous animals. Despite initial research, we have a poor understanding of the venom production and storage systems for most venomous animals. Methods Spatial venomics uses techniques such as non-targeted mass spectrometry imaging (MSI) and spatial transcriptomics, and can help to fill the gap of venom storage/production and functional morphology constraints in venom systems. In this study, we apply a spatial venomics approach to study the venom system of glycerids, a group of marine polychaetes, also known as bloodworms. Results Glycerids have a tapering, ringed proboscis and greatly elongated eversible pharynx tipped with four jaws directly connected to putative venom glands that produce complex venoms comprised of large neurotoxic proteins as well as numerous other proteinaceous and peptidic toxins. A newly recognized part of the venom system is a number of pharyngeal lobes, which are poorly understood structures that have recently been shown to be the main site for neurotoxin production in Glycera tridactyla. To shed further light on the functional organization of the venom system of glycerids, we applied non-targeted mass spectrometry imaging (MSI) in combination with standard omics approaches, and were able to spatially identify a number of putative toxins that were restricted to the pharyngeal lobes or venom glands in these poorly known venomous organisms. Main Conclusion Here, we performed a spatial venomics analysis of the venom system of G. dibranchiata by combining transcriptomics, proteomics, and MSI approaches. The application of integrative omics tools for the spatial identification of various toxins in the venom system allowed us to shed new light on the functional biology of glycerid venoms, and put them in a morphological context.

Aida Verdes (Museo Nacional de Ciencias Naturales MNCN-CSIC, Spain)
Sami Saarenpää (KTH Royal Institute of Technology, Sweden)
Eivind Undheim (Norwegian University of Science and Technology, Norway)
Juan Junoy (Universidad de Alcalá, Spain)
Ana Riesgo (Museo Nacional de Ciencias Naturales MNCN-CSIC, Spain)
Stefania Giacomello (Norwegian University of Science and Technology, Spain)
Combining Spatial Transcriptomics and Mass-Spectrometry Imaging to identify and visualize novel nemertean toxins within histological sections
PRESENTER: Aida Verdes

ABSTRACT. Aim Ribbon worms (Nemertea) are active predators that use a proboscis to inject toxins into prey and defend themselves with a toxic epidermal mucous secretion. Nemerteans do not have distinct multicellular glands, instead toxins are secreted by cells lining the body wall and proboscis epithelia. The lack of a distinct gland has prevented the use of traditional venomics approaches to investigate their venom. Methods Spatial Transcriptomics (ST) is a recently developed technology that allows to visualize and quantify transcriptomes within histological sections at 100 m resolution. It facilitates the identification of toxins and their distribution within the tissue, revealing venom composition and producing tissue simultaneously. We adapted the method to analyze cryosections of 3 biological replicates from the species Cerebratulus marginatus and Lineus longissimus, identifying a number of gene clusters linked to specific histological features. Proboscis and epidermis were the most distinct, suggesting their profiles might be driven by toxin genes not expressed elsewhere. We mapped the expression of previously identified mucus toxins such as nemertide alpha-1 and cytotoxin A-III on the tissue, revealing their distribution is not restricted to the epidermis. We also identified genes differentially expressed in the glandular epithelium of the proboscis, which may represent novel predatory toxins. Additionally, we used mass-spectrometry imaging (MSI) to identify and visualize peptides distributed in the proboscis and glandular epithelia of Lineus longissimus. Main Conclusion Our findings suggest there is a hidden diversity of nemertean toxins and illustrate the great potential of spatial methods such as ST and MSI to investigate challenging venomous organisms, such as those without distinct venom glands or where venom cannot be milked.

15:35-15:45Coffee Break
15:45-16:15 Session 2: POSTER 2
Evroula Hapeshi (University of Nicosia, Cyprus)
Sophie Laugesen (University of Copenhagen, Denmark)
Helena Safavi-Hemami (University of Copenhagen, Denmark)
Lars Ellgaard (University of Copenhagen, Denmark)
Lea Cecilie Christensen (University of Copenhagen, Denmark)
Recombinant expression of cone snail venom insulins
PRESENTER: Sophie Laugesen

ABSTRACT. Some species of fish-hunting cone snails produce specialized insulins to induce dangerously low blood sugar in their prey. These insulins activate the human insulin receptor, although at lower potency than human insulin, and exhibit several properties that make them ideal compounds for the development of drug leads for diabetes. Since the discovery of the first venom insulins in Conus geographus, we have identified approximately 120 additional venom insulin sequences for biomedical exploration. In this project, we will set up an expression system that allows for production of cone snail insulins and hybrid analogs of venom and human insulins for functional evaluation and biochemical characterization.

To efficiently produce the insulins as soluble proteins, we utilize a modified version of the CyDisCo expression system. This system involves co-expression of enzymatic catalysts of disulfide bond formation and isomerization. Thereby, it allows for correct folding of the protein, without the comprehensive isolation and refolding steps, typically applied for recombinant production of insulin.

Preliminary results using insulin from Conus geographus show that the modified CyDisCo system can provide fully folded insulin. To establish the robustness of the system, we expressed several additional insulins derived from diverse cone snail species. Functional testing of these insulins is underway.

The modified CyDisCo expression system has potential for expression of cone snail insulins as well as its hybrid analogs.

Fiorella Tonello (CNR of Italy, Istituto di Neuroscienze, Padova, Italy, Italy)
Maria Lina Massimino (CNR of Italy, Istituto di Neuroscienze, Padova, Italy, Italy)
Julian Fernandez (Instituto Clodomiro Picado, Universidad de Costa Rica, San José, 11501, Costa Rica., Costa Rica)
Barbara Spolaore (Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy, Italy)
Enzymatic conjugation with transglutaminase to label venom toxins: method and applications
PRESENTER: Fiorella Tonello

ABSTRACT. Aim To study the cellular localization and molecular interactions of animal venom toxins, it is necessary to attach them coloured labels to observe them under the microscope, and molecular hooks to use them as bait to isolate their interactors. For this purpose, we used the method of enzymatic conjugation by transglutaminase (TGase), which allows attaching a ligand to side chains of Lys or Gln residues of proteins, under native conditions. This type of conjugation is one of the most reproducible and delicate methods to modify proteins and it allows a site-specific derivatization leading to the production of homogenous derivatives1. We applied TGase to perform labelling of poisonous toxins and then we characterized the conjugated proteins and evaluated their functionality. Methods Toxins extracted from snake venom or produced by expression in E. coli and folded in vitro, were conjugated by reaction with TGase to glutamine-containing peptides carrying fluorescent dyes or biotin as label. The modified proteins were isolated by RP-HPLC and analysed by ESI-Q-TOF mass spectrometry. The labelled proteins were then tested for their enzymatic activity, if any, on synthetic substrates, and for their toxicity on cellular models. We then used fluorophore-conjugated proteins to observe their intracellular localization by fluorescence microscopy and biotin-conjugated proteins to perform pull-down experiments from extracts of their target cells. Results Labelled proteins maintained intact their enzymatic and/or toxic activity on cellular models. Proteins modified with a peptide conjugated to a fluorophore allowed us to investigate their intracellular localization by fluorescence microscopy, that has been confirmed also in vivo by immunofluorescence. Biotin-conjugated proteins were useful as bait to isolate their molecular interactors. Main Conclusions Enzymatic conjugation by TGase is a technique suitable for the modification of venom toxins because it does not alter their activity and, with the same method, allows attacking different types of molecular tags.

Rafael Amigot-Sánchez (Complutense University of Madrid, Spain)
Diego Heras-Márquez (Complutense University of Madrid, Spain)
Ana Robles (Barcelona Supercomputing Center, Spain)
Sergi Roda (Barcelona Supercomputing Center, Spain)
Laura Fernandez-Lopez (Institute of Catalysis, CSIC, Spain)
Cristina Coscolín (Institute of Catalysis, CSIC, Spain)
Álvaro Martínez-del-Pozo (Complutense University of Madrid, Spain)
Manuel Ferrer (Institute of Catalysis, CSIC, Spain)
Víctor Guallar (Barcelona Supercomputing Center and Institució Catalana de Recerca i Estudis Avançats, Spain)
Sara García Linares (Complutense University of Madrid, Spain)
Engineering of the actinoporin FraC confers hydrolytic activity to the pore channel: building the foundations towards pore-based nanoreactors

ABSTRACT. Aim Actinoporins are pore-forming proteins from sea anemones. Fragaceatoxin C (FraC), from Actinia fragacea, is one of the most studied ones and its three-dimensional structure, both the monomer and the transmembrane pore, are known at the atomic level. The possibility of extending the properties of these biological nanopores from mere analyte capture to a versatile biocatalytic system that integrates the advantages of the nanopores and the recognition and transformation of analytes, has not been evaluated to date. Here, the knowledge accumulated while designing PluriZymes, enzymes with multiple reactive groups introduced by modern protein engineering techniques, is applied to evaluate the possibility of designing actinoporins with biocatalytic properties. Methods Using PELE software and molecular dynamics, which allows mapping ligand diffusion and binding, we performed an exploration of the structure of FraC to find potential sites to accommodate a catalytic triad for ester-hydrolysis, by introducing the appropriate amino acid mutations. We have produced and purified both wild-type and engineered FraC, in solution and reconstituted in nanodiscs, and further tested against a large battery of esters. Results PELE software successfully identified a potential binding site in FraC for accommodating an ester substrate commonly transformed by most ester-hydrolases. This site involved three amino acids of two different monomers. When a mutant nanopore was reconstructed on nanodiscs, FraC turned catalytic, being able to hydrolyze a number of ester substrates at diverse pH and temperatures. Main Conclusions The preliminary results herein presented suggest that it is feasible to introduce catalytic activity into FraC. This result opens the door to the use of actinoporins as versatile biocatalyst agents because of the possibility to combine and modulate their physical, chemical, and biological properties.

Ayse Nalbantsoy (Ege University, Turkey)
İsmail Hakkı Akgun (Ege University, Turkey)
Ayse Bercin Barlas (Ege University, Turkey)
Gülşah Sunal (İzmir Katip Çelebi University, Turkey)
Gunnur Onak Pulat (İzmir Katip Çelebi University, Turkey)
Ozan Karaman (İzmir Katip Çelebi University, Turkey)
Fulya Unalp (Ege University, Turkey)
Batuhan Orman (Ege University, Turkey)
Cytotoxic Effects of Selected Cyclic Peptides Based on Venom Mimetics on Glioblastoma (U87MG) Cells
PRESENTER: Ayse Nalbantsoy

ABSTRACT. Aim Snake venoms are a natural biological source that has potential therapeutic value with various active protein compounds that are traditionally used in the treatment of many diseases among the population. Venom ingredients contain neurotoxins, cardiotoxins, nerve growth factor, lectins, disintegrins and various different enzymes. Improvements in bioengineering and modelling studies have led snake venoms are important natural sources for the development of peptide-based, molecules with high bioactivity. In this study, it was achieved through the identification of the strongest cytotoxic effect crude venom and its fractions in small amounts in glioblastoma (U87MG) cells from endemic Vipera anatolica snake species. Methods The amino acid sequences of bioactive disintegrin structures isolated from venom belonging to V. anatolica and some other snake species were studied. Based on the information obtained, interactions of designed cyclic peptides with integrins were investigated by molecular modeling studies and two cyclic peptides, cyc-GRGDD, cyc-RGDNN were selected as promising candidates. As a result of molecular modeling studies two cyclic peptides were synthesized by solid phase peptide synthesis method. The structures of purified disintegrin molecules determined by LC - MS and the bioactivity screening test (cell proliferation-cytotoxicity test) carried out by MTT assay. Results According to the results obtained from cell proliferation-cytotoxicity tests IC50 values of cyc-GRGDD determined as 6.27 ± 1.10 µg/ml and for cyc-RGDNN higher than 31.23 ± 3.49 µg/ml. Main Conclusions As a result of the bioactivity studies guided using a molecule isolated from snake species in Turkey with a biomimetic activity-modeling approach, cyclic peptides with bioactivities were achieved and the studies on this subject continuous.

Purification and evaluation of a novel analgesic peptide isolated from the venom of Centruroides exilicauda

ABSTRACT. Aim The aim of this work is the isolation of an analgesic peptide found in the venom of Centruroides exilicauda, an endemic scorpion of Baja California, Mexico.

Methods The purification of the analgesic peptides found in the venom of Centruoides exilicauda consisted of two parts. Starting with exclusion chromatography (Sephacryl® S-100) in which we isolated the active fraction. After this, we used RP-HPLC (Zorbax C18) to further purify the components into isolated peptides which we tested to find the one responsible for the analgesic effect. To test the analgesic effect of the venom, the Hargraves model was performed in mice (female; 20-25g). To induce thermal hyperalgesia the mice received CFA (Complete Freund's Adjuvant) injection into the right paw while the left paw was used as a control. We measured the paw withdraw latency in 30 min intervals over a 5-hour time period. The effects of the peptides found in the venom were compared to other analgesics (Dexamethasone, 2mg/kg) to determine their potential as an analgesic for medical use.

Results The purification process of the venom produced various fractions which where all tested for analgesic effects, at different doses (0.03mg/kg, 0.3 mg/kg, 3 mg/kg iv) to see if there was a dose-dependent nature to its activity. From these fractions only one produced analgesic effects and was further purified into its individual peptide responsible for the analgesic effect. Having the best analgesic effect of 65.32% at the highest dose.

Main Conclusions The intravenous injection of the peptide found in the venom of C. exilicauda produced analgesic effects in the Hargraves mice model without noticeable side effects.

Evelyne Benoit (Service d’Ingénierie Moléculaire pour la Santé, ERL CNRS/CEA 9004, Gif-sur-Yvette, France, France)
Michel De Waard (Institut du Thorax, Inserm UMR 1087 / CNRS UMR 6291, Nantes, France, France)
Rémy Béroud (Smartox Biotechnology, Saint-Egrève, France, France)
Michel Partiseti (Sanofi R & D, Integrated Drug Discovery – High Content Biology, Vitry-sur-Seine, France, France)
Denis Servent (Service d’Ingénierie Moléculaire pour la Santé, ERL CNRS/CEA 9004, Gif sur Yvette, France, France)
Multi-scale evaluation of spider toxins as potential anti-nociceptive agents: Example of Cyriotoxin-1a
PRESENTER: Evelyne Benoit

ABSTRACT. Aim Our expertise is the identification and structural and functional characterization of original natural toxins targeting receptors and ion channels, and which may have applications in human health. In this context, and in collaboration with the Institut du Thorax, Smartox Biotechnology and Sanofi R & D, we are interested in the multi-scale evaluation (from the cell in vitro to the organism in vivo) of spider toxins as potential anti-nociceptive agents, as illustrated by the example of Cyriotoxin-1a (CyrTx-1a). NaV1.7 channel subtype is highly expressed in dorsal root ganglia (DRG) of the sensory nervous system and plays a central role in the pain signaling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. Methods We used high-throughput screening of the venom library, using automated patch-clamp experiments on human voltage-gated sodium channel subtypes, and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced and chemically synthesized. Analgesic effects were evaluated in mice in vivo. Results We identified and further characterized CyrTx-1a, a novel peptide isolated from Cyriopagopus schioedtei spider venom. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV1.1-1.3 and 1.6-1.7 in the low nanomolar range, compared to the micromolar range for hNaV1.4-1.5 and 1.8. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse DRG neurons in vitro, and approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability in vivo. CyrTx-1a exhibited an analgesic effect in mice by significantly increasing reaction time in the hot-plate assay. Main Conclusions The pharmacological profile of CyrTx-1a paves the way for further engineering studies aimed to optimize the potential anti-nociceptive properties of this peptide.

Figen Caliskan (Eskisehir Osmangazi University, Turkey)
A new approach to antivenom preparation: Using Androctonus crassicauda venom loaded biodegradable nanoparticles as an immunoadjuvant.

ABSTRACT. Aim Androctonus crassicauda is a widely distributed species of scorpion in the Middle East, including Turkey, Iran, Iraq, Syria, Jordan, Saudi Arabia and Yemen. The common treatment approach used in envenomation caused by these scorpions is antivenom therapy. The aim of the study is the development of a new approach to the production of antivenom with the use of biodegradable chitosan nanoparticles (CNPs) containing A.crassicauda venom (ACV) as an immunoadjuvant.

Methods A.crassicauda venom extracted by electrical stimulation method. CNPs were obtained by ionic gelation method and A.crassicuda venom-loaded CNPs prepared under optimum conditions. Intraperitoneally and subcutaneously immunization studies were conducted in the CD1 strain mice. Venom-loaded CNPs and venom containing traditional adjuvants were used during the immunisation procedure, ELISA assays were used to monitoring the evolution of the immunoreactivity.

Results The results of the study show that CNPs loaded with A.crassicauda venom can be used in immunization processes. Study results show that venom-loaded CNPs administration produces a strong immune response in mice. The use of biodegradable nanoparticles instead of more aggressive and injurious traditional adjuvants has been demonstrated with this study and a protocol has been created that can be evaluated in antivenom production processes.

Main Conclusion These findings suggested that A.crassicauda venom-loaded CNPs as immunoadjuvants could be an alternative to traditional adjuvants for generating polyclonal antivenoms.

Jia Jin Hiu (Monash University Malaysia, Malaysia)
Shun Qi Teoh (Monash University Malaysia, Malaysia)
Michelle Khai Khun Yap (Monash University Malaysia, Malaysia)
The transition of apoptosis to necroptosis by cobra venom cytotoxin is associated with concentration-dependent membrane permeabilization.

ABSTRACT. Aim Cytotoxin (CTX) is a highly basic three-finger toxin found predominantly in cobra venom. Its three-functional loops interact with the cell membrane and induce cell death. However, its molecular mechanism in cytotoxicity is poorly understood, especially in the transition of cell death mode. This work aimed to investigate the transition mechanism of cell death caused by a CTX-I isoform from the venom of Equatorial spitting cobra (Naja sumatrana). Methods CTX-I was purified from N. sumatrana venom using cation-exchange chromatography, its purity and identity were confirmed by LC-MS. The cytotoxic effects of CTX-I were then investigated by cell apoptotic assays. The degree of membrane permeability was measured by enzyme lactate dehydrogenase activity and calcein-AM fluorescence intensity. Then, label-free quantitative (LFQ) secretome analyses were conducted to determine the alteration of the intracellular proteome by CTX-I. Results CTX-I was found to induce cell death before 24 h through activation of apoptotic caspase, deprivation of mitochondrial membrane potential, and remarkable percentage of AV+ apoptotic cells. The absence of necrotic markers, HMGB1 suggested apoptosis was the profound cell death independent of membrane permeabilization. Nevertheless, the degree of membrane permeabilization increased following a concentration-dependent manner. The LFQ secretomes demonstrated the presence of 105 significantly distinguished proteins involved in carbon metabolism, structural supports, inflammation, and necroptosis. The exposure of high toxin concentrations triggered stress-induced necroptosis, followed by membrane permeabilization preceding the release of intracellular proteins, which ultimately caused metabolites deprivation. Main Conclusions Our findings concluded that, at low concentrations, CTX-I induced caspase-dependent mitochondrial-mediated apoptosis. As the toxin concentrations increased, a transition of apoptosis to necroptosis was associated with increasing membrane permeabilization.

16:15-17:15 Session 2: ORAL 2
Agostinho Antunes (University of Porto, Portugal)
Richard Stenner (University of Bristol, UK)
Huan Sun (University of Bristol, UK)
Imre Berger (University of Bristol, UK)
Christiane Berger-Schaffitzel (University of Bristol, UK)
Using Ribosome Display to Generate a Safe, Efficient and Affordable Antivenom to treat Snakebite Envenoming
PRESENTER: Richard Stenner

ABSTRACT. Aim Snakebite envenoming is a Neglected Tropical Disease that annually causes up to 138,000 deaths and 400,000 disabilities in surviving victims. Current anti-venoms, manufactured from hyperimmunised animals, are weakly effective as only 10-15% of the pooled anti-venom neutralizes toxins. The ADDovenom project aims to use state-of-the-art protein engineering and display technology to generate novel, high-affinity, and safe anti-venoms from naïve libraries.

Methods Ribosome Display is an in vitro selection and evolution method for generating high-affinity antibodies from naïve libraries. Using our nanobody and ADDobody libraries, ribosome display will be used to generate high-affinity binders against a diverse range of snake venom antigens. The high-affinity binders will be isolated, biophysically characterized, and their efficacy evaluated in cell-based assays at the Liverpool School of Tropical Medicine (in collaboration with Prof. Nicholas Casewell). The snake venom antigens used in the selections include recombinantly expressed venom proteins (in collaboration with Dr. Renaud Vincentelli) and the EpiString constructs developed by Prof. Robert Harrison.

Results To-date, phospholipase A2s (PLA2s), metalloprotease EpiStrings, and disintegrins have been recombinantly produced from bacteria and their activities have been confirmed (a novel assay for measuring disintegrin activity was also developed). All the antigens have been biotinylated prior to ribosome display, with this modification having no deleterious effect on activity. A nanobody library has been generated in a format suitable for ribosome display and is actively being employed in selections to generate high-affinity binders against purified PLA2s and metalloprotease EpiStrings. In addition, a naïve ADDobody library has also been generated.

Main Conclusions The primary goal of the ADDovenom project is to combat snakebite envenomation by producing safe, effective, and affordable antivenoms. The preliminary data indicate that ribosome display is an effective methodology for accomplishing this objective by generating high-affinity binders against an assortment of pertinent snake venom targets.

Joshua Torres (Department of Biomedical Sciences University of Copenhagen, Denmark)
The King’s Gambit: Small Molecule Arsenal of Conus imperialis

ABSTRACT. Cone snails rely on venoms for predation and protection Their ability to generate conopeptides and use them in sophisticated strategies allowed them to survive for millions of years. However, peptides are not the only active components of cone snail venoms. In this work, we demonstrate that Conus imperialis contain small molecules that are biologically active. We further propose how two of the small molecules, genuanine and conazolium A mimic the mating pheromones of their prey and use them as part of a worm hunting strategy. This work reveals that small molecules are important components of venom and that their chemical diversity can provide new aspects in the areas of chemical ecology and drug discovery.

An initial metabolomic survey was done on several species of C. imperialis venom to assess the diversity of its small molecule venom components. We purified target masses to elucidate the structures of these new compounds, including conazolium A. We mined venom duct transcriptomes and heterologously expressed the enzyme, ConA and show how cone snail biochemically synthesize conazolium A. Conazolium A and genuanine were tested for their ability to induce mating behaviors in marine worms using the model organism Platynereis dumerilii.

In this work, we sought out the non-peptidic small molecule components in Conus imperialis venom. We show that conazolium A is biochemically synthesized by the snail and provide evidence on how this compound along with genuanine A can both act to mimic the mating pheromones of the marine annelid, Platynereis dumerilii.

The ability of cone snails to diversify their venom components have tremendously helped these ancient animals create effective hunting strategies for predation. Our work demonstrates, for the first time, how small molecules act as important constituents of venoms in Conus imperialis.

Fernanda Gobbi Amorim (University of Liège, Belgium)
Damien Redureau (University of Liège, Belgium)
Nicholas Casewell (Liverpool School of Tropical Medicine, UK)
Loïc Quinton (University of Liège, Belgium)
Application of Multi-Enzymatic Limited Digestion for inventorying the snake venom arsenal

ABSTRACT. Aim Multi-Enzymatic Limited Digestion (MELD) is a new methodology that applies synergic and time-limited digestion of multiple enzymes, representing a versatile yet straightforward approach for a new generation of proteomics methodology for defining venom composition. The generation of a higher number of peptides per protein during the MELD digestion increases the quality of protein sequencing and identification. We applied the MELD strategy for the venomics of two snake species: Echis ocellatus (EoV) and Dendroaspis polylepis (DpV). Methods 10 µg of each venom were reduced/alkylated followed by two different digestion protocols: (1) trypsin and (2) MELD (Trypsin/GluC/Chymotrypsin). The digested materials were analysed in a Q-Exactive™Plus Mass Spectrometer with protein identification performed by PeaksStudio X+. Results MELD showed more peptides/proteins identified for both venoms compared to the trypsin protocol. In EoV, 82.3% were identified as toxins and 17.2% as non-toxins, compared to the trypsin protocol which resulted in 69.2% toxins, 24% non-toxins and 6.9% for cellular components (CC). In DpV, MELD showed a coverage of 26.2% for toxins, 39.9% for non-toxins and 33.9% for CC, while for trypsin we obtained 23.3% toxins, 37.4% non-toxins and 39.3% CC. MELD was able to identify new components in both venoms. 51% of the EoV were metalloproteinases while DpV showed a high content of nerve growth factor (22%). The highest number of mass spectras (MS) were obtained for metalloproteinases (tr|Q2UXQ4) for EoV, in which MELD obtained 4-time more MS. For DpV, dendrotoxin I (P00979) showed the greatest number of MS, and trypsin yield 2-times more MS. Main Conclusions MELD presented a different coverage according to the presence of high molecular mass content in the venom arsenal. This strategy can be applied to identify new groups of venom components. It represents an innovative strategy for venomics, opening new perspectives for sequencing and inventorying the venom arsenal.

Steven Trim (Venomtech Limited, UK)
Improving the utility of venoms in high throughput screening for drug discovery.

ABSTRACT. The birth of High Throughput Screening (aka HTS) in the early 1990’s came from a need to screen more compounds in a shorter time to speed up discovery of drug like molecules. This need was driven, and supported by, the enormous growth in capacity of organic synthesis of small molecules. In order to perform experiments faster, screening had to be simplified, miniaturised and carried out by robotic systems. The first 96-well plate was developed in 1952 but it wasn’t until the early 90’s that robotic liquid handling could cope with these and then in 1992 came the 384 well plate, followed by the Society of Biomolecular Sciences (SBS) standard in 1995. This meant that any SBS standard plate could be used in any SBS standard instrument and lead to the rapid growth of HTS platforms in the pharmaceutical industry. This capacity is perfect to find the useful, drug like venom compounds within the complex mixtures of hundreds of thousands of venomous species. However, venom screening did not keep up with the needs of the pharmaceutical HTS platforms and thus appeared to fall from favour. In the early 2000’s there were only a few pioneers using venoms in 96-well formats and it took until 2017 to see the 384 well plates, the mainstay of HTS screening, published with venom fractions in the wells. There is still a total lack of publications with venoms being used in mainstay HTS assay systems such as ALPHAscreen and HTRF (Homogeneous Time Resolved Fluorescence). This talk will deliver the evidence and data to bridge the gap between the amazing potential of venoms and the needs of modern drug discovery.

Alexis Panagiotopoulos (Foundation for Research & Technology Hellas, Patras 26504, GREECE, Greece)
George Mitsainas (University of Patras, Patras 26504, GREECE, Greece)
Maria Klapa (Foundation for Research & Technology Hellas, Patras 26504, GREECE, Greece)
Snake venom metabolomics: Comparative analysis between Vipera ammodytes meridionalis and Malpolon insignitus fuscus in the Peloponnese

ABSTRACT. Aim The variety of small molecules in the snake venom has not been vastly explored. Further studies are needed as small molecules may play a substantial role in the venom activity. Profiling the snake venom over a geographic region is also important from an evolutionary perspective too. In this context, major objective of this study was to develop the protocol for the venom metabolic profiling of snakes captured in the field and apply it for the comparative analysis of the solenoglyphous Vipera ammodytes meridionalis and opisthoglyphous species Malpolon insignitus fuscus collected in the Peloponnese. Methods Twelve Vipera and 3 Malpolon snakes of both sexes were captured from two locations in the Peloponnese, Strofylia Forest (sea-level) and Mount Chelmos (mountain level). Venom was collected in the laboratory, after drug administration for increased venom release in the case of Malpolon. The venom metabolic profiles were quantified by Gas Chromatograpy-Mass Spectrometry, normalized and filtered using our in-house M-IOLITE ( software and analyzed using the omic data analysis software TM4/MeV. Results The venom metabolic profile of both species was rich, with 170 and 150 metabolite peaks detected in the Vipera and Malpolon samples, respectively. While Vipera venom profile was richer than that of Malpolon, a large fraction of the detected metabolites were in small concentration. Citrate is the most abundant metabolite in the venom of both species, in larger concentration in the Vipera species. Further qualitative and quantitative differences between the two species were observed, indicating a variety in the snake venom composition that needs to be further explored. Main Conclusions This study further supports the richness of the snake venom metabolic profile. Exploring the variety and richness of the snake venom metabolic profile in relation to other omics is important for evolutionary biology, but also for the identification of substances of pharmacological interest.

17:15-17:30Coffee Break
17:30-18:05 Session Keynote: Mandë Holford - City University of New York / American Museum of Natural History
Manuel J. Tenorio (University of Cadiz, Spain)
Mande Holford (City University of New York - American Museum of Natural History, United States)
Killer Snails: Agents of change and innovation

ABSTRACT. Venomous animals are found in the seas, on land, and in the skies. Their ubiquitous presence demonstrates the evolutionary success of venom in shaping fundamental biological processes related to defense, predation, and competition. My lab uses venoms and venomous animals as agents of change and innovation. We are interested in how venoms direct the evolution of organisms and how they can improve human lives. Considerably, venoms are the prototype of precision medicine: inducing a highly specific and immediate response. Venom peptides, and the genes from which they are derived, are a resource for investigating biological processes pertaining to organismal evolution (adaptive radiation, diversification), gene development (duplication, neofunctionalization), and cellular physiology involving ion channels (activating/inhibitory ligands). This talk will demonstrate the scientific path from mollusks to medicine examining how venom evolved over time in the terebrid snails (Terebridae), and how we can use this evolutionary knowledge as a roadmap for discovering and characterizing new compounds with therapeutic potential for treating pain and cancer. A venomics strategy, combining genomics, transcriptomics, and proteomics, has been applied to the discovery, characterization and optimization of terebrid venom peptides (teretoxins). However, identifying bioactive venom peptides remains a significant challenge. The Holford lab uses inventive tools from chemistry and biology to: (1) investigate the evolution of venom in the 600 known species of predatory marine snails, (2) discover disulfide-rich peptides from a venom source, (3) develop high-throughput methods for characterizing structure-function peptide interactions, and (4) deliver novel peptides to their site of action for therapeutic application.

18:15-18:45 Session 2: POSTER 3
Evroula Hapeshi (University of Nicosia, Cyprus)
Joana S Rodrigues (University of Lisbon, Portugal)
Andreia Faria-Pereira (University of Lisbon, Portugal)
Sérgio P Camões (University of Lisbon, Portugal)
Vanessa Morais (University of Lisbon, Portugal)
Joana P Miranda (University of Lisbon, Portugal)
Towards a physiologically relevant human stem cell-derived hepatic in vitro model for evaluating venom pharmacology, toxicology and pathology

ABSTRACT. Aim In vitro models able to recapitulate the hepatic physiology are fundamental for studying venom pharmacology and toxicology. However, most hepatic in vitro models maintain cells in non-physiological conditions, i.e., high concentrations of glucose, insulin and dexamethasone, hampering result translation to the clinical setting. This work focused on the development of a functional hepatocyte-like cell (HLC)-based in vitro model under more physiological conditions. Methods HLCs were derived from human stem cells (hSCs) through a 3-step protocol lasting 21 days. From day 21 (D21) to D34, 4 media compositions were tested: i) medium with 25 mM of glucose, 1.72 µM of insulin and 1 µM of dexamethasone (Diff); ii) Diff with 5 mM of glucose (Diff -glu); iii) medium with 5 mM of glucose, 1 nM of insulin and 100 nM of dexamethasone (Physiol); and iv) Physiol with 25 mM of glucose (Physiol +glu). HLCs were characterized regarding the expression of hepatic-specific genes (qPCR); urea, albumin and ATP production; phase I/II activities; glycogen storage ability (PAS staining); and mitochondrial activity (Seahorse XF Cell Mito Stress Test). Results HLCs maintained in all conditions were evaluated regarding the presence/activity of hepatic-specific markers to evaluate cells functionality. All media induced albumin production, glycogen storage and overexpression of Alb, Cyp3a4 and Hnf4a relative to hSCs. Importantly, Physiol increased urea production (p < 0.001) and phase I (p < 0.01) and phase II (p < 0.001) activities when compared to Diff. The energy metabolism status of cells via Seahorse Test was also assessed. Results show that there is a higher mitochondrial activity in HLCs maintained in Physiol, contrasting with higher glycolytic activity in HLCs in Diff, associated to a diabetic phenotype and therefore less physiological. Main Conclusions This work suggests the potential use of HLCs for the development of human relevant hepatic in vitro models.

Ewa Ciszkowicz (Rzeszow University of Technology, Poland)
Aleksandra Bocian (Rzeszow University of Technology, Poland)
Anna Miłoś (Rzeszow University of Technology, Poland)
Konrad Hus (Rzeszow University of Technology, Poland)
Justyna Buczkowicz (Rzeszow University of Technology, Poland)
Vladimir Petrilla (University of Veterinary Medicine and Pharmacy, Slovakia)
Monika Petrillova (University of Veterinary Medicine and Pharmacy, Poland)
Jaroslav Legath (Rzeszow University of Technology, Poland)
Cytotoxicity of Phospholipases A2 and 3FTxs from Naja ashei venom

ABSTRACT. Aim Snake venoms are mixtures of a large group of biologically active proteins, including highly catalytically active phospholipases A2 (PLA2s) and non-enzymatic proteins named three-finger toxins (3FTxs). These two groups of compounds are most responsible for toxicity observed in envenomation by snakes. The aim of this study was to evaluate the cytotoxicity of Naja ashei venom fractions against breast and prostate cancer cell lines. Methods Naja ashei venom fractions obtained in earlier studies were tested against breast (MCF-7, ATCC HTB-22™) and prostate (DU145, HTB-81™) cancer cell lines for their cytotoxicity. In order to evaluate the effectiveness of each venom fraction in inhibiting a specific biological or biochemical function in proliferating cells, the half-maximal inhibitory concentration (IC50) was calculated (0.5 mg/mL MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; dissolving solution 100% DMSO; absorption measurement λ=570 nm). Results The results of cytotoxicity experiments on two cancer cell lines have shown that F3 and F9 fractions have the highest statistically significant activity, being the most effective against the DU145 and MCF-7 cell lines. The fraction containing 79.1% of PLA2 (F3) revealed IC50=148 μg/mL (R2=0.98) and IC50=167 μg/mL (R2=0.82) against, respectively, DU145 and MCF-7 cancer cell line. Higher cytotoxic properties were obtained by fraction F9 (98% of 3FTx) against both cell lines, DU145 (IC50=43 μg/mL; R2=0.93) and MCF-7 (IC50=67 μg/mL; R2=0.91). Main Conclusions The results clearly show that PLA2s and 3FTxs play a crucial role in dose-dependent cytotoxicity against two tested cancer cell lines. In order to evaluate the pharmacological and industrial potential of those two groups of proteins, additional experiments on normal cell lines are needed, as well as identification of signaling pathways modulated by PLA2s and 3FTxs.

Nadia Caram Salas (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Joaquín López Carrillo (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Natalia Morales Cardona (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Alexei F. Licea Navarro (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Erick Huerta Romero (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Samanta Jiménez Flores (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Johanna Bernáldez Sarabia (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
Miryam Ochoa Agredano (Centro de Investigación Científica y de Educación Superior de Ensenada, Mexico)
A new w-conotoxin from Californiconus californicus with analgesic effect after systemic administration in chronic inflammatory pain.

ABSTRACT. Aim The main objective of this work is to evaluate the systemic analgesic effect of a new w-conotoxin (O1_cal6.4b) obtained from the venom of the cone snail Californiconus californicus. Methods Wistar rats (120-140 g) were kept in controlled conditions of light (12/12 h), food and water (ad libitum), relatively humidity (50 5°C) and temperature (22 ± 2°C). Chronic inflammatory pain was induced by injecting 100 µl of complete Freund’s adjuvant (CFA) into a right paw. Two days after CFA injection and before the administration of the drugs, O1_cal6.4b (0.001-1 mg/kg), saline (1 ml/kg, vehicle), dexamethasone (2 mg/kg), morphine (1 mg/kg) and w-MVIIA (0.5 mg/kg) were administered intraperitoneally (i.p). The paw withdrawal latency was measured in both paws at 30 min intervals over 8 h for the analgesic evaluation. Results The s.c. administration of O1_cal6.4b (0.001–1 mg/kg) reduced in a dose-dependent manner the inflammatory pain (p< 0.05). At 1mg/kg, O1_cal6.4b reversed the thermal hyperalgesia at the peak response (1 h) by 80%, this effect was maintained during for more than 3 h, after which the effect returned to control values until 8 h of evaluation. No differences in % of analgesia between the doses of 0.1 mg/kg (∼47%) or 1 mg/kg (∼48%) after systemic administration were observed. Main conclusions To our knowledge, this is one of the first w-conotoxin with analgesic activity by systemic administration and could be one of the first conotoxin that can be administered without catheter implantation, improving the quality of life of patient suffering chronic inflammatory pain.

Ana Crnkovic (National Institute of Chemistry, Slovenia)
Gašper Šolinc (National Institute of Chemistry, Slovenia)
Marija Srnko (National Institute of Chemistry, Slovenia)
Marjetka Podobnik (National Institute of Chemistry, Slovenia)
Gregor Anderluh (National Institute of Chemistry, Slovenia)
Upgrading biological nanopores with unnatural amino acids
PRESENTER: Ana Crnkovic

ABSTRACT. Aim Biological nanopores are ideally suited for the detection of low-abundance molecules, such as various biomarkers or toxic compounds. Unnatural amino acids can be introduced by genetic code expansion (GCE) to enrich the arsenal of available side chain chemistries in protein targets. The aim of this study is to examine whether nanopores can be synthesized with unnatural amino acids to modulate the sensitivity of nanopore biosensing experiments. Methods 4-propargyloxy-L-phenylalanine (pPpa), a tyrosine mimic with an additional alkyne group, was introduced into an actinoporin via GCE. Synthesis of the pPpa-containing actinoporin relies on the pPpa-specific aminoacyl-tRNA synthetase and its cognate suppressor tRNA to facilitate insertion of pPpa at the position of an in-frame stop codon. We targeted five amino acid residues in the stalk region of the actinoporin for UAG-mediated pPpa insertion. Results All five residues facing the lumen could be reassigned to pPpa; three of them also retained a high level of hemolytic activity. In single-channel measurements, the active pPpa-containing actinoporins retained stable open pore currents and low noise comparable to that of the unmodified nanopore. Importantly, competitive protein yields were achieved when using GCE for the production of pPpa-containing actinoporins. Main Conclusions Unnatural amino acids containing alkyne groups can help detect common pollutants such as inorganic azides via click chemistry. The facile introduction of unnatural amino acids into nanopores via GCE technology could prove very useful in tailoring protein nanopores specifically for certain types of analytes.

Simona Saponara (University of Siena, Italy)
Amer Ahmed (University of Siena, Italy)
Maria Frosini (University of Siena, Italy)
Beatrice Gorelli (University of Siena, Italy)
Fabio Fusi (University of Siena, Italy)
In vitro assessment of venom cardiovascular pharmacology and safety: a trip from single cells to isolated organs
PRESENTER: Simona Saponara

ABSTRACT. Aim Venoms have always represented an inestimable and almost inexhaustible source of bioactive molecules, some being currently used as pharmacological tools or therapeutic agents in the area of cardiovascular diseases. A systematic approach based on in vitro functional assays was developed to identify the cardiovascular activity and safety of both well-known and novel toxins and characterize their molecular mechanism. Methods and Results Freshly isolated or cultured vascular and cardiac cells-, whole-tissue- and isolated organ-based models are routinely used to assess the effects of drugs on: 1) cell viability and related patterns of cell death, mitochondrial function, redox state, Ca2+ handling; 2) vascular responsiveness of vessel rings and single myocytes to various agents; 3) cardiac tissue electrical conduction and contractility and related biomarkers in Langendorff perfused rat heart 4) single cell ion currents through Ca2+, K+ and other channels by means of the patch-clamp technique 5) Kv11.1(hERG) channel, the target of virtually all QT interval-prolonging torsadogenic drugs in stable transfected hERG-HEK cells. Main Conclusions These approaches represent a thorough strategy to guide pharmaceutical chemists and biotechnologists and identify the ideal candidates for the discovery and development of therapeutic agents and useful tools in cell biology.

Tania Cebrero-Cangueiro (Institute of Biomedicine of Seville, Spain)
Soraya Herrera-Espejo (Institute of Biomedicine of Seville, Spain)
Ana Belén Guisado-Gil (Institute of Biomedicine of Seville, Spain)
Javier Sánchez-Céspedes (Instituto de Biomedicine de Seville, Spain)
Jerónimo Pachón (Instituto de Biomedicine de Seville, Spain)
María Eugenia Pachón-Ibáñez (Instituto de Biomedicine de Seville, Spain)
In Vitro activity of melittin from honey bee venom alone and in Combination with antibiotics against carbapenemases-producing Gram-negative bacilli

ABSTRACT. Aim To determine the synergistic activity of melittin from honey bee venom, in combination with antibiotics, against carbapenemases-producing Gram-negative bacilli (GNB).

Methods Sixteen carbapenemase-producing isolates: 6, 6, 4 of P. aeruginosa, A. baumannii and K. pneumoniae, respectively. Minimum Inhibitory Concentration (MICs) of melittin and imipenem and meropenem was determined by microdilution method (EUCAST). The cytotoxicity 50 (CC50) of melittin was determined using AlamarBlue® kit. Synergistic activity of melittin in combination was determined by time-kill curves, using the CC50 value for melittin and 1xMIC of each antibiotic.

Results Melittin MIC range was 2->256 mg/L. All P. aeruginosa and A. baumannii isolates were resistant to imipenem and meropenem. Only the K. pneumoniae KPC-3-producing strain was resistant to these carbapenems. Imipenem and meropenem were bactericidal at 8h for P. aeruginosa (GES-5) and K. pneumoniae (VIM-1/DHA-1) strains. The bactericidal and synergistic activity of the combinations is showed in Table 1.

Isolates Carbapenemases Imipenem+Melittin Meropenem+Melittin A. baumannii AbR34 NDM-1 B y S (2-24h) B (4-8h) y S (2-24h) AbR623 OXA-23 B y S (4-24h) B y S (4-24) AbR625 OXA-23 B y S (4-24h) B y S (8-24h) AbR629 OXA-23 B y S (2-24h) B y S (2-24h) AbR824 OXA-58 B y S (2-24h) B y S (2-24h) AbN690 OXA-23 S (4-8h) B y S (8-24h) P. aeruginosa PaCAT056-004 VIM-1 S (8h) - PaCLE02-006 IMP-1 - B (4-24h) y S (24h) PaMAD02-007 VIM-2 - - PaMUT01-018 IMP-33 B (8h) - PaCVA03-019 OXA-2/VIM-20 B (8h) - PaMAD02-021 GES5 - B (8h) K. pneumoniae Kpc28 OXA-48/CTX-M-15 S (8h) - Kpc29 KPC-3 - B (4-8h) Kpc07 VIM-1 - B (8h) y S (8-24h) Kpc21 VIM-1/DHA-1 B (8h) B (4h) B: Bactericidal activity, S: synergistic activity, -: without activity

Main Conclusions Melittin from honey bee venom in combination with carbapenems shows in vitro activity against carbapenemases-producing GNB infections.

Federica Cavion (Department of Life Sciences, University of Trieste, Italy)
Marco Pelin (Department of Life Sciences, University of Trieste, Italy)
Michela Carlin (Department of Life Sciences, University of Trieste, Italy)
Silvio Sosa (Department of Life Sciences, University of Trieste, Italy)
Aurelia Tubaro (Department of Life Sciences, University of Trieste, Italy)
Effects of the marine toxin palytoxin on human induced pluripotent stem cells
PRESENTER: Federica Cavion

ABSTRACT. Aim The aim of this study was to evaluate the potential teratogenic effects of a marine toxin, palytoxin (PLTX), using an in vitro model represented by human induced pluripotent stem cells (iPSC) that are able to mimic in vitro the early embryonic development. Methods Cytotoxicity of PLTX (1.0x10-17-1.0x10-7M) on human iPSC 253G1 cells was investigated using the MTT assay up to 96 h exposure. Total RNA was extracted from cells exposed to 1.0x10-11M PLTX for 24, 72 and 96 h, reverse transcribed and the expression of stemness genes (C-MYC, OCT4, NANOG, KLF4 and SOX2) and of genes markers of the three germ layers (FOXA2, SOX17, AFP for endoderm, ACTA, T, CXCR4 for mesoderm, and PAX6, SOX1 for ectoderm) was evaluated by real time PCR. Results PLTX induced a concentration- and time-dependent cytotoxic effect on iPSC with half maximal effective concentrations (EC50s) of 1.3x10-8 M, 2.0x10-10 M, 2.2x10-11 M and 2.1x10-12 M, after 4, 24, 72 and 96 h exposure, respectively; the effect was not reversed by the toxin removal (recovery condition), suggesting a non-reversible cytotoxicity. As compared to the cytotoxicity toward non-stem cells exposed to the toxin for 4 h (HaCaT keratinocytes, EC50 =7.0x10-10 M or Caco-2 enterocytes, EC50 =3.0x10-11 M), iPSC appeared to be rather resistant to PLTX effect. The expression of stemness genes was significantly changed after cells exposure to PLTX, except for OCT4. Moreover, the expression of germ layers genes was slightly perturbated by PLTX, without a clear relationship with the exposure time. Main Conclusions These preliminary results demonstrate a relative resistance of iPSCs towards PLTX and a slight, but significant, toxin ability to influence the stemness genes expression and the early cells differentiation towards cells of the three germ layers.